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Science behind Non-Specific Science

SCIENCE

BEHIND

NON-SPECIFIC

SCIENCE

Dr. Vikash Bhardwaj

Notion Press

5 Muthu Kalathy Street, Triplicane,

Chennai – 600 005

First Published by Notion Press 2014

Copyright © Dr. Vikash Bhardwaj 2014

All Rights Reserved.

ISBN: 978-93-84049-78-2

This book has been published in good faith that the work of the author is original. All efforts have been taken to make the material error-free. However, the author and the publisher disclaim the responsibility.

No part of this book may be used, reproduced in any manner whatsoever without written permission from the author, except in the case of brief quotations embodied in critical articles and reviews.

[Dedication to
loving memory of my Father]

Preface

***

“Once there was a scientist who studied frogs. One day, the scientist put the frog on the ground and told it to jump. The frog jumped four feet. So the scientist wrote in his notebook, “Frog with four feet, jumped four feet.” So the scientist cut off one of the frogs legs. The scientist told the frog to jump. The frog jumped three feet. So the scientist wrote in his notebook, “Frog with three feet, jumped three feet.” So the scientist cut of another leg. He told the frog to jump. The frog jumped two feet. So the scientist wrote in his notebook “Frog with two feet, jumped two feet.” The scientist cut off one more leg. He told the frog to jump. The frog jumped one foot. So the scientist wrote in his notebook, “Frog with one foot jumped one foot.” So the scientist cut off his last leg. “He said, “Frog jump, Frog jump, Frog jump” but the frog did not jump. So the scientist wrote in his notebook, “Frog with no feet, went deaf.”

This is one of famous joke which I heard from friends and hopefully you also. The question is what this joke wants to say? This joke says that, the scientist in this joke is not aware that legs do not have a role in the function of frog’s hearing and that’s why he comes to a wrong conclusion. Now think what will happen if scientist puts his expertise in searching what makes frog deaf after cutting the fourth leg. We know by common sense that he will never found ears within the legs.

In this book, I will introduce you a much broader definition of ‘Complementary in DNA structure’. Many molecular technologies have been developed on the basis of complementary in two strands of DNA. My sincere efforts are to make aware scientists all over the world that only half knowledge has been applied in designing, performing these molecular technologies. Hopefully after reading this book, you will have a feeling that whatever knowledge we have developed in past decades is like ‘Frog : Scientist Joke’. Many of us do not know what means ‘Complementary’ in DNA in a broader sense. Hopefully after reading this book, You yourself will have an answer, why we are not able to find treatments for many deadly diseases. On W.H.O. websites you can see, many million deaths due to ‘X’ disease in the last decades and many more millions peoples will die in coming future. So W.H.O. is also sure that we will not have any ultimate solutions for many of the disease in coming future. Applying half knowledge will not solve sufferings all over the world. Hopefully it’s time to update our knowledge so that future young scientists will have a true knowledge with them.

Hopefully you will enjoy reading this book and will update your current knowledge and will apply it to a new level. Nobody is complete so am I, all efforts are taken to avoid errors, yet some may be left out. I shall be grateful to the readers if such mistakes are highlighted to me for a better revised edition.

Dr.Vikash Bhardwaj

Foreword

***

This excellent and highly conceptual new trend book by Dr. Vikash Bhardwaj, in its first edition, will prove faithful to its central philosophy that doing research in science can not simply rely on the famously presentation of facts, but must also include the intellectual ideas that give birth to specific discoveries and results in the solution of long-standing mankind puzzles.

Deoxyribonucleic acid (DNA), is proved a central and mysterious molecule of all genetic information flow in life. Little change in the DNA sequences, can change the fate of all living creatures. In this book Dr. Vikash has written that’s all related to DNA-RNA technology based experiments and their possible flaws. He proposed ideas that can make them better.

Dr. Vikash has written a book that is neither a conventional type text book nor a reference book; even nor a protocol book. Every chapter starts with its early days of science with due credit to all significant scientific contributors. What went wrong and how it comes on the shape of today’s science. From early history; to current trends and proposal of some possible ideas from them one can able to do specific science that can convert in translational research. All in all a brief note with significant explaining and easily understandable to researcher of field.

In starting chapter; he explained about various types of available DNA structure and “Complementarity” a central feature in various DNA-RNA based tools. He proposed some of idea with explanation that are not given attention or ignored completely in past, that’s why we failed to translate many lucrative results in translational research. In subsequent chapter he explained that keeping all these ideas in mind how you can use them in many current DNA-RNA basic and important high throughput techniques e.g. Southern and Northern blotting, Microarray and siRNA technology. At the end of every chapter, he proposed some new methods, by using them one can able to design their experiment very precisely with accurate result. I hope that this book will serve sole purpose of author’s contribution towards society. I am happy and it’s my fortune to write a forwarding for this book that will serve to betterment of life sciences research.

Sita Ram Meena, PhD

Yale University, New Haven, USA.

Foreword

‘Conventions sometimes choke the new ideas’ as the author rightly discussed, especially with the development of the structure and chemical nature of DNA. This book is advocating a paradigm shift as how we look on the ‘complementarity’ question of DNA especially in the context of interpretation of some of the molecular biology problems.

The first chapter gives a nice introduction of DNA structure and chemistry. The subsequent chapter deals with the DNA based molecular biology techniques Southern and Northern blotting, microarray and siRNA technology. The author has provided valuable insights to interpret the results obtained by these techniques.

This book is interesting, refreshing and binds a reader till he finishes reading. Dr. Vikash Bhardwaj has done an excellent job and I wish him good luck .

Ram Gopal Nitharwal, PhD

Dept. of Cell and Molecular Biology

Uppsala University, Sweden

“Seven Deadly Sins
Wealth without work
Pleasure without conscience
Science without humanity
Knowledge without character
Politics without principle
Commerce without morality
Worship without sacrifice.”

Mahatma Gandhi

“Everyone knows of the drop in the ocean, the rare one knows of the ocean in the drop”

— Kabir

  • Contents *

Title

Copyright

Dedication

Preface

Foreword

1. Complementary: What It Means Broadly?

2. Southern and Northern Blotting

3. Microarray Technology

4. siRNA Technology

5. Perspective

Glossary

Chapter 1

Complementary: What It Means Broadly?

***

Introduction

In early history of modern science, proteins were considered most key target molecules to study how cell function. Much of research was focused on characterizing various protein components found in a cell. In 1868/69 a young Swiss physician and scientist Freidrich Miescher working in the laboratory of distinguished biochemist Felix Hoppe-Seyler (discoverer of hemoglobin) at University of Tubingen in Germany, planned to isolate and characterize protein components of leukocytes (white blood cells). From a local surgical clinic, Miescher used to take fresh pus-coated patient’s bandages. Once he received the bandages, he filtered out leukocytes, and used to extract and identify various biological components within white blood cells nuclei using various acidic and alcoholic solutions. He was surprised when he came across a novel substance from cell nuclei that had chemical properties unlike any protein. He found that new class of cellular substance which he has discovered consists of nitrogen, a much higher phosphorous content without sulphur and is resistant to proteolysis. Miescher coined it the term “Nuclein”. Although Miescher did most of his work in 1869, his paper on “Nuclein” wasn’t published until 1871. “Nuclein” was such a unique molecule that Hoppe-Seyler was skeptical about these findings. After a year, Hoppe-Seyler was convinced with Miescher’s results and published those in a journal edited by himself. So Mischer is being credited person who first time isolated nuclei and discovered DNA, a biological molecule which is now well considered as a key to some of the greatest mysteries of life. Later, Miescher also proposed many ideas about biological functions of this molecule. As stated by R. Dahm, Miescher was a “brilliant scientist well on his way to making one of the most fundamental discoveries in the history of science, who ultimately fell short of his potential because he clung to established theories and failed to follow through with the interpretation of his findings in a new light” (1,2,3,4,5).

Within the same time period in 1866, Gregor Mendel a member of the Augustinian monastery (what is now the current Czech Republic), through his work on garden pea plants discovered the fundamental law of inheritance. Mendel did experiment with pea plants for eight years (1856-1863) and being an amateur scientist published his findings in a relatively obscure journal. “He deduced that genes come in pairs and are inherited as distinct units, one from each parent. Mendel tracked the segregation of parental genes and their appearance in the offspring as dominant or recessive traits. He recognized the mathematical patterns of inheritance from one generation to the next” (6,7). Mendel’s work and his law of inheritance were not appreciated in his time and were rediscovered posthumously after 16 years. In 1900, three different scientists (Carl Correns, Hugo de Vries and Erich von Tschermak) from different countries rediscovered Mendel’s findings independently. Mendel’s work became famous as a result of a priority dispute between De Vries and Correns. Today Gregor Mendel (1822-1884) ranks second after Charles Darwin on most biologists’ scales of hero worship and also being credited as Father of genetics (8,9).

After Miescher’s findings about ‘Nuclein’, other scientists continued to investigate the chemical nature of it. One of these other scientists was famous Russian biochemist Phoebus Levene. A physician turned chemist, Levene was a natural product chemist, who published more than 700 papers on the chemistry of various biological molecules including nucleic acids, amino acids, proteins, lipids, carbohydrates, glycoproteins, and amino sugars. In 1919, using hydrolysis to break down yeast nucleic acids, Levene proposed that nucleic acid is composed of a series of nucleotides, and that each nucleotide was in turn composed of just one of four nitrogen-containing bases, a sugar molecule, and a phosphate group. He identified purines and pyrimidines within DNA and localized phosphodiester linkage between the sugars. He also classified the linkage as glycosidic between sugar and base within DNA and reported that D-ribose and 2-deoxy-D-ribose as the sugars occurring in RNA and in DNA, respectively. Levene proposed a tetranucleotide structure of DNA, in which the nucleotides were always linked in the same order (i.e., G-C-T-A-G-C-T-A and so on) (Figure 1). This simple tetranucleotide structure of DNA makes it unlikely candidates as carriers of the large information needed to act as genes. As stated by G. Semenza “The treacherous ‘beautiful simplicity’ of the tetranucleotide structure and Levene’s authority effectively stopped the search for alternative structures for the nucleic acids and, what is worse, proved to be an additional hurdle to the (now ‘classical’) work of Avery, MacLeod and McCarty (1944) to rapidly and universally be accepted.” (1,9,10,11)

Fig.1 Representation of proposed Levene tetranucleotide DNA structure, later shown to be incorrect.

Many other scientists eventually realized that Levene’s proposed tetranucleotide structure was overly simplistic and order of nucleotides along a stretch of DNA should be highly variable in order to act as a carrier of genetic information. In 1950, Erwin Chargaff expanded on Levene’s work by uncovering additional details of DNA structure. Chargaff set out to see whether there were any differences in DNA among different species. First, he noted that base composition of DNA varies from one species to another and DNA specimens isolated from different tissues of same species have equimolar composition. Second, Chargaff concluded that almost all DNA regardless of species maintains certain properties, even as its composition varies. In particular, the amount of adenine (A) is equal to the amount of thymine (T), and the amount of guanine (G) equal to amount of cytosine ©. In other words, the total amount of purines (A + G) and the total amount of pyrimidines (C + T) are usually nearly equal. These conclusions are now known as “Chargaff’s rule.” The work of Chargaff dealt a death blow to the tetranucleotide hypothesis proposed by Levene. Later this work was found important in establishing three dimensional structure of DNA and yielded clues how genetic information is encoded and passed from one generation to another (1,9,12,13) (Figure 2).

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Fig.2 Chargaff rule: The total amount of purines (A + G) and the total amount of pyrimidines (C + T) are usually nearly equal within a DNA sample.

In 1928 Fredrick Griffith observed a spectacular behavior on bacterium Pneumococcus virulent and nonvirulent strains. Virulent strain of bacterium contains a capsular polysaccharide which allows bacterium to escape destruction by host immune system. Virulent strains of Pneumococcus have different capsular polysaccharides and have a smooth (S) appearance. The variants of Pneumoccocus which fail to produce capsular polysaccharide have a rough ® surface and are avirulent. Avirulent bacteria did not kill mice, because absence of polysaccharides allows host to destroy bacteria. By injecting a mixture of heat killed virulent bacterium and live nonvirulent bacterium into mice, Fredrick Griffith recovered live virulent bacterium from the heart of dead mice. This process is now known as “Transformation”. In this experiment, the dead virulent bacteria were of type III capsular polysaccharide and live R bacteria were of Type II capsular polysaccharide. The virulent bacteria recovered from the mixed infection had the smooth coat of Type III. So some property of the dead type IIIS bacteria transformed the live R bacteria such that they make type III capsular polysaccharide and as a result become virulent. This raised the possibility that when virulent cells are killed by heat, their genetic components remain undamaged. Moreover, once liberated from heat killed cells, these components passed through the cell wall of living avirulent bacteria making them virulent one (Figure 3). Unfortunately, Griffith was not able to identify the chemical nature of this heat resistant “transforming principle” (14,15,16)

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Fig.3 Griffith’s experiments on Pneumococci. The scientist Frederick Griffith observed that a living nonvirulent bacterial strain could be transformed into a virulent strain after it was mixed with a dead virulent strain. Reprinted by permission from Macmillan Publishers Ltd: Nature Education, O’Connor, C. 2008, © 2013. (15)

In 1944, O. Avery, C. M. Macleod, and M. McCarty recognized the importance of Griffith’s findings, and decided to use their expertise to identify the specific molecules that could transform an avirulent bacteria into a virulent one. They separated different biomolecules of heat killed virulent bacterium and tested them for their transforming ability one at a time. They found that DNA of heat killed virulent bacterium was responsible as a transforming agent which converted avirulent bacteria into virulent one. Supporting their conclusion were key experiments showing that the transforming activity of their highly purified active fractions was destroyed by pancreatic deoxyribonuclease which degrades DNA molecules without any effect on the integrity of protein molecules or RNA. The addition of either pancreatic ribonuclease (which degrades RNA) or various proteolytic enzymes (which degrades Proteins) had no influence on the transforming activity. The experiments conducted by Oswald Avery and his colleagues were definitive, but many scientists were still reluctant to accept DNA as the genetic material (15,17) (Figure 4).

It was in 1952, A. Hershey and M. Chase incorporated the radioisotope of phosphorus (32 P) into the phage T2 virus DNA and radioactive sulfur (35S) into proteins of a separate T2 phage culture. It was known that phosphorus is not found in proteins but is an integral part of DNA, while sulfur is present in proteins but never in DNA. They used each differentially radiolabelled phage culture independently and infected E.Coli. The infected bacteria were agitated in a blender and two fractions were separated by centrifugation. One fraction contained the empty phage coats that were released from the surface of bacteria, while other consisted of the infected bacteria themselves. When 32P-labeled phages were used, indicating that phage DNA entered into cells. 32P was also recovered from phage progeny. When 35S-labeled phages were used, most of the radioactive material ended up in the empty phage coats, indicating that phage protein never entered the bacterial cell. This experiment therefore showed directly that

Fig.4 Oswald Avery, C. M. Macleod, and M. McCarty Experiment. Avery and colleagues identified the transforming molecule as DNA using a process of elimination. In lab experiments, extracts from a virulent bacterial strain were treated with enzymes that destroyed proteins, RNA, or DNA. The treated extracts were then mixed with non-virulent bacterial cultures to determine whether transformation occurred. When proteins or RNA were destroyed in the virulent bacterial extracts, transformation still occurred, and virulent cells appeared in non-virulent bacterial cultures. However, when DNA was destroyed in the virulent bacterial extracts, transformation did not occur, and non-virulent cultures remained non-virulent. Reprinted by permission from Macmillan Publishers Ltd: Nature Education, O’Connor, C. 2008, © 2013. (15)

only the DNA of parent phages enters the bacteria and then becomes part of progeny phages. This confirmed DNA to be as genetic material (15,16,18) (Figure 5).

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Fig.5 Overview of the experiment performed by Hershey & Chase, showing DNA to be the genetic material for phage. (© Adenosine at en.wikipedia CC-BY-SA-2.5 ) (19).

Earlier proteins were considered as genetic material and work was proceeding on the X-ray analysis of protein structures but a smaller number of scientists were trying to solve the X-ray diffraction pattern of DNA. The first X-ray diffraction analysis of DNA was done by William Astbury (Pioneer of protein structure) and by his PhD student Florence Bell in 1938. Bell took X-ray photographs of DNA from yeast, pancreas and Tobacco Mosaic Virus etc. It was her work with fibres of sodium thymonucleate (now known as DNA) which yielded the most informations. They proposed a ‘piles of pennies’ like DNA structure. They also deduced distance between individual nucleotide bases in the molecule is 3.3 Å.

In 1951, Elwyn Beighton, a research fellow at Leeds University working under supervision of William Astbury take X-Ray photographs of DNA, however Beighton’s work was apparently neglected by William Astbury and was not published. The apparent neglect of Beighton’s work by William Astbury has been subject of debate among historians of molecular biology. As by different historians, Astbury’s incapability to appreciate on Beighton’s work to solve DNA’s structure “was due either to his being too much of a physicist, with too little interest in or knowledge of biology, or to his being misled by an erroneous theoretical model of the gene” (20,21,22).

In December 1952, Linus Pauling and Robert B. Corey proposed a triple-stranded helix structure of DNA based on photographs taken by them and photographs published by Astbury and Bel. Quality of photographs taken by Linus Pauling and Robert B. Corey were some what inferior to Astbury and Bel one’s. They proposed DNA structure to be tri-helical structure, which are coiled about one another. Each chain being a helix with fundamental translation equal to 3.4 Å, and the three chains being related to one another by operations of a threefold axis. They proposed phosphate groups of each DNA strand closely packed about the axis of column, with nitrogenous bases facing out radially. They might have forgotten that facing towards the middle and stacked on top of each other, negative charges in each phosphate group would repel one another, making it impossible for a stable DNA structure (9,23,24).

The modern era of molecular biology began after the discovery of double helical structure of B-form DNA by James Watson, Francis Crick, Maurice Wilkin and Rosalind Franklin in 1953. In 1962, the Nobel Prize in Physiology or Medicine was awarded to James Watson, Francis Crick, and Maurice Wilkins for solving the structure of DNA.

According to our current knowledge about B-form of DNA model, the two polynucleotide chains in the double helix DNA associate by hydrogen bonding between nitrogenous bases and runs in opposite direction i.e antiparallel. The bases adenine (A) and Guanine (G) are known as purines which contains a pair of fused ring and the bases Cytosine (C) and Thymine (T) are pyrimidines which contain a single ring. Guanine can hydrogen bond specifically only with cytosine, whereas adenine can bind specifically only with thymine. These associations are described as Watson-Crick base pairing and the paired bases (G with C or A with T) are said to be complementary. Adenine and thymine match up so that a hydrogen bond can form between the exocyclic amino group at C6 on adenine and the carbonyl at C4 in thymine and likewise, a hydrogen bond can form N1 of adenine and N3 of thymine. Thus an A-T base pair has two hydrogen bonds. The exocyclic NH2 at C2 on guanine lies opposite to, and can form hydrogen bond with a carbonyl at C2 on cytosine. Likewise, a hydrogen bond can form between N1 of guanine and N3 of cytosine and between the carbonyl at C6 of guanine and the exocyclic NH2 at C4 of cytosine. Thus a G-C base pairs has three hydrogen bonds (Figure 6). In B- form of DNA, nucleotides are joined to each other in polynucleotide chains through the 3’-hydroxyl of 2’-deoxyribose of one -nucleotide and the phosphate attached to the 5’-hydroxyl of another nucleotide. This is a phoshphodiester linkage in which phosphoryl group between the two nucleotides has one -sugar esterified to it through a 3’-hydroxyl and a second sugar esterified to it through 5’ hydroxyl. Thus a phosphodiester bond in actually consist of two phosphoester bonds, one on the 5’ side of phosphate and another on 3’ side of phosphate. Phosphodiester linkages create repeating sugar phosphate backbone of the polynucleotide chain. Looking along the helix, one strand runs in 5’ to 3’ direction whereas another strand runs in 3’ to 5’ direction. 5’ end has a phosphate group on the 5’ carbon of its terminal sugar and 3’ end usually has a hydroxyl group on the 3’ carbon

Fig.6 Watson Crick base pairing between Adenine : thymine and Guanine : Cytosine.

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Fig.7 Watson Crick Model of DNA structure. Reprinted by permission from Macmillan Publishers Ltd: Nature Education, Leslie A. Pray, © 2013 (1)

of its terminal sugar. The sugar phosphate backbone is on the outside and carries negative charge on the phosphate groups. Sugar is called 2’-deoxyribose because there is no hydroxyl group at position 2’. The positions on the ribose are designated with the primes to distinguish them from positions on the bases. The bases are attached to deoxyribose sugar by glycosidic linkage at N1 of the pyrimidines or at N9 of the purines. Bases are flat structures lying in pairs perpendicular to the axis of the helix. Proceeding along the helix, bases are stacked above one another like a piles of plates. Each base pair is rotated ~ 36° around the axis of the helix relative to the next base pair. So ~ 10 base pairs makes a complete turn of 360° . The twisting of two strands around one another forms a double helix with a minor groove (~ 12 Å across) and a major groove (~ 22 Å across). The order of bases along the polynucleotide chain is irregular. This irregularity is basis for the enormous information content of DNA. The double helix is right handed, the turns runs clock wise looking along helical axis. These features represent the accepted model what is known as B- form of DNA observed at high humidity (1,24,25,26,) (Figure 7,8).

Fig.8 The double-helical structure of DNA. The 3-dimensional double helix structure of DNA, correctly elucidated by James Watson and Francis Crick. Complementary bases are held together as a pair by hydrogen bonds. Reprinted by permission from Macmillan Publishers Ltd: Nature Education, Leslie A. Pray 2008, © 2013. (1)

The ‘A’ form of DNA, which is also right handed DNA, is observed when the relative humidity is reduced to less than 75%. It has 11 base pairs per turn and made up of antiparallel strands held together by Watson-Crick base pairing rules. In contrast to B –form of DNA, Base pairs in ‘A’ Form of DNA are tilted rather than perpendicular to the helix axis. Its major groove is narrower and much deeper than that of B form and its minor groove is broader and shallower. In A-form DNA sugar pucker is C3’ endo while in B-form DNA sugar pucker is C2’ endo. The vast majority of DNA in a cell is in B form, but DNA can adopt ‘A’ form in certain DNA protein complexes. The ribose configuration for double stranded RNA is C3’ endo and double stranded RNA adopts an A-like helix (28,29) (Figure 9 & 11).

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Fig.9 Two major nucleic acid sugar puckers Deoxyguanosine with attached phosphorous atoms is shown in the two major nucleic acid sugar puckers. The C2’ endopucker (left) is found in B-DNA, whereas the C3’ endo pucker (right) is found in A-DNA or in RNA. The distance between successive phosphate groups is close to 7.0 Å in C2’ endo and shortens to 5.8-6 Å in the C3> endo pucker. Nucleic acids can convert from one pucker to the other, although it takes greater energy for conversion of ribonucleotides. Carbon, large open circles; hydrogen, small open circles; nitrogen, cross hatched circles; oxygen, black circles; and phosphorous, heavily outlined open circles. Reprinted by permission from Macmillan Publishers Ltd , Alexander Rich, Nature Structural Biology. © 2003. (29)

Fig.10 Various Confirmation of deoxyadenosine and deoxycytidine in DNA. (A) Anti deoxyadenosine (B) syn deoxyadenosine © Anti deoxycytidine (D) syn deoxycytidine

Left handed Z–DNA (structure given by Alexander Rich and collegues) has been mostly found in alternating purine-pyrimidine sequences (CG)n and (TG)n. As like B–DNA, Z- DNA is also consist of two antiparallel strands held together by Watson–Crick base pairing rule. Z-DNA is thinner (18 Å) than B-DNA (20 Å ), the bases are shifted to periphery of the helix and there is only one deep, narrow groove equivalent to minor groove in B-DNA. The base pairs are sheared with respect to their stacking positions in Z- DNA. The bases which form major groove in B-form of DNA form convex outer surface in Z-form of DNA. In contrast to B-DNA where a repeating unit is 1 base pair, in Z-DNA the repeating unit is 2 base pair. The phosphate backbone follows a zigzag path as opposed to a smooth continuous coil seen in B-DNA. The helix repeat in Z-DNA is 12 base pair per turn compared with 10.5 base pair per turn in B-form DNA. Helix pitch for Z form of DNA is 44.6 Å with an average rise of 3.72 Å while helix pitch for B-form DNA is 34 Å with an average rise of 3.24 Å. The twist angles between adjacent base pairs in B-form of DNA are positive while for Z-form of DNA, the twist angle for two base pairs relative to next two base pairs are negative. The sugar and glycosidic bond conformations alternate, C2’-endo for anti dC and C3’ endo in syn dG (Figure 10). It is this syn conformation at the purines nucleotides that is responsible for the left-handedness of the helix. In solution alternating purine-pyrimidine residues assume the left–handed conformation only in the presence of high concentration of positively charged ions that shields the negatively charged phosphate groups. Further, negative supercoiling tends to unwind B-DNA while it stabilize left handed Z-DNA (25, 28, 30) (Figure 11).

Fig.11 From left to right, the structures of A-, B- and Z-DNA. © (Zephyris) at en.wikipedia (CC BY-SA 3.0) (31)

In addition to B-form of DNA, a linear duplex DNA can form X-shaped hairpin structures called cruciforms in which most bases are held together by Watson–Crick base pairing rule. Such structures tends to occur at sequence with inverted repeats and their stability is enhanced by DNA supercoiling. Cruciform DNA consist of a branch point, a stem and a loop. Size of the loop increase with increase in the gap between the mirror repeats. ‘A-T’ rich sequence within gap increases the probability of formation of cruciform DNA. Cruciform DNA play a key role in various biological process including DNA transcription, replication, recombination, regulation of gene expression, nucleosome structure and genome organization (Figure 12) (32).

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Fig.12 Changes associated with transition from the linear to cruciform state in the p53 target sequence from the p21 promoter. The promoter sequence contains a 20 bp p53 target sequence with 7 bp long inverted repeat (red), (A) as linear DNA and (B) as an inverted repeat as a cruciform structure. In the cruciform structure, the p53 target sequence is presented as stems and loops. © 2011 Brázda et al. (CC BY 2.0) (32)

In certain circumstances a DNA sequence containing oligopurine strand hydrogen bonded to oligopyrimidine strand via Watson-Crick base pairing rule can form a triple helix (also known as H-DNA) by laying a third strand into its major groove. Third strand makes hydrogen bonds to another surface of the duplex. The third strand pairs in a Hoogsteen base- pairing schemes. If the third strand is ‘G.A’ rich, it forms reverse Hoogsteen hydrogen bonds in an antiparallel orientation with the purine strand of the Watson-Crick helix. If the third strand is ‘T.C’ rich, it forms Hoogsteen bonds in a parallel orientation with the Watson–Crick paired purine strand. If the third strand is ‘G.T’ rich, It can either form Hoogsteen bonds in a parallel orientation with the Watson–Crick paired purine strand or it can form reverse Hoogsteen hydrogen bonds in an antiparallel orientation with the purine strand of the Watson-Crick helix (33,34) (Figure 13&14).

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Fig.13 Model of DNA triple helix. Image from the RCSB PDB (www.rcsb.org) of PDB ID NDB ID: 1BWG. (33a)

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Fig.14 Orientation of the three triplex motifs. Reprinted by permission of Oxford University Press. Nucleic Acids Research, Duca et al © 2008. (34)

Fig.15 G-quadruplex DNA. (A) An illustration of the interactions in a G-quartet. This quartet is represented schematically as a square in the other panels of this figure. M+ denotes a monovalent cation. b | Schematic diagrams of intramolecular (left) and intermolecular (right) G-quadruplex (G4) DNA structures. The arrowheads indicate the direction of the DNA strands. The intermolecular structures shown have two (upper) or four (lower) strands. Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Genetics. Bochman et al. ©2012. (35)

In 1910, Bang reported the fact that guanylic acid forms a gel at high concentration. Fifty years later, Gellert and collegues by X-Ray diffraction demonstrated that guanylic acids could assemble into four-stranded structures called G-quadrupulexes. These consist of a square arrangement of guanines tetrad stablilized by Hoogsteen hydrogen bonding. The formation and stability of G-quadruplexes is monovalent cation dependent, present in the centre of the tetrads. G-quadruplexes can be formed of DNA or RNA. They can be formed from one, two or four seprate strands of DNA or RNA. Depending on the direction of strands or parts of a strand that form the tetrads, structures may be described as parallel or antiparallel or hybrid thereof. Various loop structure are possible for G-quadraplexes as shown in Figure (15) (35,36,37).

Single stranded nucleic acid known as aptamers are able to fold into specific three dimensional structures and has potentiality to bind their targets with high affinity and specificity. Due to nucleic acid nature of the aptamers, they have potentialities to hybridize with complementary DNA or RNA strand as well as their specific targets. Figure (16) shows 3 dimensional structure of various aptamers. Various Watson-Crick and Non Watson-Crick base pairing rule will be responsible for these 3 dimensional structures. At slightly acidic or neutral pH, a cytosine (C) rich strand can adopt an i-motif structure. The i-motif structure consists of two parallel stranded C:C+ hemiprotanted base paired duplex that are intercalated in an antiparallel manner. Depending on the number of cytosine bases, loop length, environment, i–motif DNA is structurally dynamics and can adopt multiple conformations ranging from folded to random coil confirmations. In certain cases i-motif structures consist of symmetrical T.T pairs, Hoogsteen A.T pairs other then intercalated C:C + hemiprotanted base pairs (38,39,40,41,42). (Figure 17,18 & 21)

Fig.16 Molecular model of Aptamers showing different 3 dimensional shapes. (Image from the RCSB PDB (www.rcsb.org) of (A) PDB ID: 1AW4 (B) PDB ID: 1c38) © PDB ID: 2L5K (D) PDB ID: 1QDH (E) PDB ID: 2IDN (F) PDB ID: 148D (43,44,45,46,47,48)

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Fig.17 Left: schematic representation of the dimeric structure of d. Right: G:T:G:T minor groove tetrad showing the observed connectivities in the NMR spectra, and C:C+ base pair. Reprinted by permission of Oxford University Press. Nucleic Acids Research, Escaja N. et al ©2012 (41)

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Fig.18 C–C+ base pairs (left panel) and schematic structures of the i-motif topology from the C-rich complementary strand d(G1C2C3G4C5C6C7A8A9A10A11 C12C13C14C15C16C17G18) (middle and right panels). Reprinted by permission of Oxford University Press. Nucleic Acids Research, Xu Y. et al ©2006. (42)

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Fig.19 (A) Schematic showing poly dA15 changing between single helix to duplex conformations induced by alternate addition of acid and base respectively. (B) Shown in black is the base pairing scheme in AH+–H+A base pairs comprising protonated adenosines. Reprinted by permission of Oxford University Press. Nucleic Acids Research, Chakraborty S. et al ©2009.(49)

A-motif is found in Poly(A) tail component of mRNA or Poly (dA) stretch of single stranded DNA. At neutral or alkaline conditions, It exhibits a single stranded right handed structure stabilized by p-p stacking of adenine bases. At acidic pH it adopts a right handed helical duplex consisting of parallel chains and tilted protonated bases. Reverse Hoogsteen base pairing between two protonated adenine bases and electrostatic attraction between the positively charged protons at (N1) atoms of adenine and negatively charged phosphate groups stabilize the parallel duplex formation in A-motif (40) (Figure 19 & 20).

[]

Fig.20 (A) Equilibrium snapshot of the single-stranded dA15 after 20 ns long MD simulation using AMBER revealing highly stacked adenine nucleobases. (B) Instantaneous snapshot of N1-protonated adenosine mediated parallel duplex of dA15 after 20 ns long MD simulation revealing a ?-helical structure with tilted base. Reprinted by permission of Oxford University Press. Nucleic Acids Research, Chakraborty S. et al ©2009.(49)

[]

Fig.21 Base pairing in G-tetrad, i-motif and B-form of DNA . (A) G-tetrad (left) and schematic structure of the intramolecular Na+ G-quadruplex from 22AG (right) (B) C.C+ base pair (left) and schematic structure of the intramolecular i-motif from 22CT (right) (C) Watson- Crick A.T and G.C base pairs (left) and schematic structure of Watson –Crick duplex 22 AG-22CT (right). Reprinted by permission of Oxford University Press. Nucleic Acids Research, Phan A.T. et al ©2002. (50)

In 1986 N. Pattabiraman, asked the question. “Can the double helix be parallel with the reverse Watson and Crick base-pairing scheme for polynucleotides?” He reported a force field calculation for homopolymeric duplex d(A)6.d(T)6 indicating that a parallel right handed helix with reverse Watson-Crick base pairing could be energetically as favorable as conventional antiparallel B-DNA. He reported that one hydrogen bond between A and T (N1. . . N3) is same for both antiparallel and parallel structures. The atom N6 of adenine forms a hydrogen bond with carboxyl oxygen 04 in antiparallel structure, while with carboxyl oxygen 02 in parallel DNA structure. Thus in homopolymeric parallel duplex d(A)6.d(T)6, number and type of hydrogen bonds between A-T base pair were reported same as that of antiparallel double helix. In parallel DNA structure grooves were reported of equal sizes. The helical parameters for parallel double helix were reported same as that of the antiparallel double helix. The stacking energies between base pairs were reported similar for both antiparallel and parallel structure. He reported that parallel structure is stabilized over the antiparallel structure by 0.5 kcal and ranges of torsion angles for parallel DNA structure were in the same region as that of antiparallel DNA structure. Intrachain proton proton distance were reported same for both parallel and antiparallel DNA. For parallel DNA structure, electrostatic energy, base stacking interaction were less favorable then antiparallel structure whereas the hydrogen bond energy was found more favorable for parallel structure then antiparallel one (51). In 1988, experimental strategies were devised for demonstrating possibilities of parallel stranded DNA. In 1988 N.B. Ramsing and T.M. Jovin synthesized three linear 21 nt oligonucleotide, with different sequence of A and T residues. One pair wise combination was hybridized to form a conventional antiparallel duplex DNA (aps DNA) while another pair wise combination was hybridized to form parallel duplex DNA having A-T base pairs in a reverse Watson-Crick Configuration. Their study confirmed that DNA containing A-T base pairs can exist as a stable parallel stranded helix (ps-DNA). Further ps-DNA and corresponding aps-DNA were reported to have a similar elecrophoretic mobilities comparable to 21 base pair reference and under condition favoring duplex formation ps-DNA consistently had a 5% higher mobility than its aps-DNA counterpart. Both ps-DNA and aps-DNA were found substrate for AhaIII and DraI restriction enonuclease having recognition site ‘TTTAAA’, with a strand specific cleavage pattern. The ‘Tm’ value of both ps-DNA and aps-DNA showed a classical dependence upon salt concentration. At any given NaCl concentration, melting temperature of ps-DNA was reported 15°C lower than its aps-DNA counterpart. In 2 mM MgCl2, melting temperature for ps-DNA and aps-DNA duplex were found approximately same as those obtained in 0.2-0.3 M NaCl, demonstrating pronounced stabilization afforded by divalent cations. The ultraviolet absorption and circular dichroism spectra of the ps-DNA indicated base paired structure but systemically difference were observed as compared to aps-DNA spectra. Based on fluorescent measurement studies, it was reported that bis-benzimidazole drug BBT-258 showed a lower affinity with ps-DNA compared to aps-DNA. A similar titration experiment performed with the intercalator ethidium bromide led to a very different result. The ps-DNA produced approximately twice the specific increase in fluorescence as the aps-DNA (52). In 1988, another similar study by J.H. Sande et al on hairpin deoxyoligonucleotides having oligonucleotide sequence in parallel polarities (ps-hairpin) also confirmed existence of stable parallel stranded conformation. The electrophoretic mobilities in polyacrylamide gels of ps-hairpins and aps-hairpins were reported similar. In their study ps-hairpins were found substrates for T4 polynucleotide kinase, T4 DNA ligase and E.coli exonuclease III. The ps-hairpins formed stable duplex and denatured 10°C lower than corresponding aps oligomers. Base paired duplex in stem of ps-hairpins were confirmed by ultraviolet absorption and circular dichroism spectra but systemically difference were reported from those of the aps-hairpins counterpart. Ultraviolet spectra under denaturing condition (at 80°C) were reported virtually identical for both ps and aps hairpins. Further it was founded that bis-benzimidazole drug Hoechst-33258 (BBI-258) showed a lower affinity for ps-hairpins compared to aps-hairpins. Intercalator ethidium bromide formed a strong fluroscent complex with ps-hairpins as compared with aps-hairpins. The pattern of chemical methylation was found same for both ps and aps hairpins. By molecular mechanics calculations, it was reported that both parallel stranded and antiparallel stranded helices have a C2’ endo sugar puckering in both strands and a helical twist of about 42°. The rise per base pair and propeller twist were ~ 3.2 Å and 20° in ps-helix and the corresponding values for antiparallel helix were reported ~ 3.0 Å and 22°. Because of the symmetry in thymine bases, the hydrogen bonds between the A.T base pairs in parallel helix were of the same type as in antiparallel helix (53). So these two studies in 1988, provided evidence that DNA containing ‘A.T’ base pairs can form a stable parallel stranded duplex structure known as parallel stranded duplex DNA (ps-DNA). In 1989, N.A. Tchurikov et al reported generation of parallel duplex between two Drosophila DNA sequences. The 41 base pair nucleotide sequence of the suffix non-coding exon sequence was found to be complementary in a parallel orientation to the 42 base pair fragment of the 5’ non coding sequence of alcohol dehydrogenase gene. They proposed that these parallel DNA duplex between two short DNA sequences from the same genome might have originated as a result of molecular convergence or unusual template synthesis by a special enzyme which might be present in earlier evolution (54). In 1992, Tchurikov et al showed that parallel complementary probes of normal nucleotide content can be used for molecular hybridization experiments, suggesting the stability of ‘GC’ containing parallel DNA. The efficiency and specificity of hybridization with the parallel complementary was very close to that of antiparallel one. By adsorption isotherm study they concluded that at least 95% of bases in the parallel duplex were involved in pairing. They also confirmed that parallel complex was consisting of only two strands and was not either a triplex or a hairpin. Their results suggested that parallel complementary oligonucleotides can be used successfully as probes in molecular hybridization experiments with cloned and genomic sequences as well as for effective screening of genomic libraries. This was first report counter to the current view that molecular hybridization experiment detect only homologous antiparallel complementary sequences (55). In 1993, O.F. Borisova et al further confirmed high stability of ‘GC’ pairs in parallel stranded duplex used for hybridization experiment. They found two stage type of A-T pair melting in parallel duplex DNA. In a 40 base pair parallel duplex DNA (consisting of natural sequence), they reported that approximately 40% of AT pairs melted at Tm=14 °C and rest 60% A-T pairs melt at about Tm = 42 °C. The Tm of G-C pairs was found to be 53 °C in parallel stranded DNA. Thus G-C pairs in the 40 bp parallel duplex were shown to be more thermostable than A-T pairs. This higher stability of G-C pairs also suggested the formation of cis Watson-Crick pairs in parallel DNA (56). Further A.K. Shchyolkina reported that circular dichroism (CD) spectra of parallel stranded DNA with mixed AT/GC composition have no major distinction from those of related antiparallel DNA indicating that there are no drastic differences in nearest neighbor base pairs interactions between parallel stranded and antiparallel stranded double helices. In parallel stranded DNA, A-T pairs with two hydrogen bonds via reversed Watson-Crick base pairing and G-C pairs are somewhat shifted relative to one another and also form two hydrogen bonds via reversed Watson-Crick base pairing rule. A positive low intensity band around 290 nm is specific feature of CD spectra of parallel stranded DNA. This band is accounted for by a contribution of guanine in the transGC pair (57). Two study in 1997 and 1998 has shown that parallel stranded DNA can be constructed from oligonucleotides incorporating isoguanine–cytosine or guanine-isocytsosine base pairs instead of Watson-Crick guaninie-cytosine base pairs. The minor groove becomes shallow and wide when chain orientation changes from antiparallel to parallel. As parallel complementary DNA contains grooves of equal size in contrast to grooves (major and minor grooves) found in antiparallel B-form of DNA. Due to this transition in structure various DNA binding drugs and dyes have shown different molecular interactions with parallel stranded DNA in compare to antiparallel stranded DNA (58a and 58b). In 2006, Li Hong et al found that antibiotic actinomycin C1 (a ligand of antiparallel duplex DNA) binded with much more lower affinity to parallel stranded duplexes containing guaninine-isocytosine base pairs. The antibiotic actinomycin C1 did not bound to parallel stranded DNA which had incorporated pairs of isoguanine–cytosine residues which lacks guanine-2-amino group. They suggested that the interaction of parallel complementary DNA is strongly controlled by chiral recognition of peptide lactom rings and grooves of DNA. On contrary Actinomin (Actinomycin D analog) which contains positively charges side chains instead of chiral peptide rings were found to be strongly bound to both parallel complementary and antiparallel complementary DNA (59).

Dr Kary Mullis received Nobel Prize in chemistry in 1993, for his invention of the polymerase chain reaction (PCR). The process, which Kary Mullis conceptualized in 1983, is hailed as one of the monumental scientific techniques of the twentieth century. Conventionally in a PCR reaction, Primers binds to DNA template in an antiparallel complementary manner and template DNA is amplified millions of times as it is (Figure 22).

[]

Fig.22 PCR amplification of a single stranded DNA in a conventional way. (61)

In 2000, Veitia R. and Ottolengh C. reported that several attempts to amplify L15253 by PCR using different pairs of primers which binds to template DNA in a parallel complementary manner were unsuccessful (60). In 2013 we first time reported that a PD-PCR reaction can amplify a single stranded 120 base pair template DNA using a parallel complementary primer. First primers annealed to template DNA in a parallel complementary manner and after

[]

Fig.23 PD-PCR for details see text and Bhardwaj V et al 2013 (61)

first round of synthesis, second primer annealed to new DNA in an antiparallel complementary manner. Later reaction happened in a conventional way but final PCR product was having sequence differrent from the template DNA (Parallel to template DNA) (61)(Figure 23).

In 2013, I was trying to search random sequence by BLAST (Basic Local Allignment Search Tool) which might be parallel complement of native sequence within various genome database. I did not get expected results. Due to curiosity when I aligned a parallel complement of a DNA sequence to its native DNA sequence I was surprised to note that at some part of DNA sequence shows homology to native DNA sequence. When I carefully analyzed these sequences, they were mirror sequence (Table 1). As shown in Figure (24) these mirror sequences were having same sequence when read in 5’-3’ or 3’-5’ direction. They have the potentillity to rehybridize both in parallel and antiparallel manner.

Fig.24 Denaturation/Renaturation of mirror sequence: Process of Denaturation of mirror duplex DNA results in the separation of two complementary strands of a double stranded mirror duplex DNA while renaturation may result in annealing in either antiparallel complementary manner to form a double stranded native DNA or in a parallel complementary manner to form parallel stranded duplex DNA.

| p. S. No. | p. Gene/CDS | p. No of MRs | p. Mirror repeats (MRs) | | p. 1 | p. P53 | p. 3 | p. 1.CCAAAGAAGAAACC

2.GGAACTCAAGG

3.ACCTGAAGTCCA | |
p. 2 |
p. TNF-alpha |
p. 3 |
p. 1.CCTCATCTACTCC

2.CCAGAGGGAGACC

3.CCTCATCTACTCC | |
p. 3 |
p. Tgf-beta |
p. 5 |
p. 1.TGTGAGGGGGAGTGT

2.CGCCCGCGCCCGC

3.GCTACCACCATCG

4.CCGACTTCAGCC

5.GTCCGGGCCTG | |
p. 4 |
p. MTOR |
p. 4 |
p. 1.CGCCACCACCGC

2.ACCTTCTTCTTCCA

3.ACTACAAACATCA

4.AGAAGAAGAAGA | |
p. 5 |
p. BRCA1 |
p. 5 |
p. 1.AAGAGAAGAAAAAGAAGAGAA

2.AAATGAACAGACAAGTAAA

3.TGGATTCAAACTTAGGT

4.AAAGATAATAGAAA

5.AAACCGTGCCAAA | |
p. 6 |
p. NF-kappa-B

[] |
p. 6 |
p. 1.CTGGGAGAGGGTC

2.AAAGTTATTGAAA

3.AAGAACAAGAA

4.GGAGGCGGAGG

5.AACGTATGCAA

6.GACAGTGACAG | |
p. 7 |
p. COX2 |
p. 3 |
p. 1.CGCGGTCCTGGCGC

2.ACAACTATCAACA

3.TGCAATAACGT | |
p. 8 |
p. HIF1A |
p. 7 |
p. 1.AAAACAGTGACAAAA

2.TTCAGCACGACTT

3.GAAGACACAGAAG

4.GAAAAGAAAAG

5.CAGATTTAGAC

6.CCCTATATCCC

7.ACATTATTACA |

Table (1) List of mirror repeats observed in various Human mRNA sequence. The sequence shown in red is mirror image of sequence shown in blue. At a mirror sequence, DNA has potentialities to hybridize in both parallel and antiparallel orientations. With increase in number of Mirror repeats within a gene, its potentialities to hybridize in parallel orientation also increase. For more details see Bhardwaj V. et al 2013.(62)

We designed a ‘FPCB’ strategy and find out these type of mirror sequence are also present in various human gene of biological importance. We found that CCR5 and Wnt7a genes mRNA sequence has single such mirror sequence. EIF2A, Human protein kinase C theta, FOXP2, PCK1, Ceruloplasmin and Caspase genes mRNA sequence has 2 mirror sequence. The p53, TNF-alpha, STAT3, Ubiquitin and COX2 has 3 mirror sequence. The MTOR gene mRNA sequence has 4 mirror sequence. Tgf-beta, BRCA1 and PTEN genes mRNA sequence has 5 mirror sequence. NF-kappa-B gene mRNA sequence has 6 mirror sequence and in HIF1A we found 7 mirror sequence (62).

In 2000, M.E. LeProust et al has reported unexpected formation of parallel stranded duplex DNA in GAA and TTC trinucleotide repeats within frataxin gene which is responsible for onset and progress of Friedrich Ataxia. They reported that these trinucleotide repeats form both an antiparallel and parallel DNA structure as confirmed by NMR, UV-Melting, gel electrophoresis, Chemical and enzymatic probing methods (63). When I carefully analysed these trinucleotide repeats, I found that these trinucleotide repeats have within them mirror sequence and have potentiality to hybridize both in parallel and antiparallel orientation. So mirror sequence as detected by us in various human genes will have potentialities to form two types of DNA structures. It will be highly significant to find out biological significance of these mirror sequence.

Based upon these finding we can say that “complementary” within a double stranded DNA can be classified by two ways

1. Antiparallel Complementary: Two strands of DNA runs in opposite direction with adenine base pairing via two hydrogen bonds to thymine and guanine base pairing via three hydrogen bonds to cytosine via Watson-Crick base pairing rule.

2. Parallel Complementary: Two strands of DNA runs in parallel direction with adenine base pairing via two hydrogen bonds to thymine and guanine base pairing via two hydrogen bonds to cytosine via reverse Watson-Crick base pairing rule.

[]

Fig.25 A and B model of parallel stranded DNA , C and D model of parallel DNA ( PDB ID-1JUU) and (E) diagrammatic representation of Parallel stranded DNA. (64, 65)

“DNA is like a computer program but far, far more advanced than any software ever created.”

— Bill Gates

“Volumes of history written in the ancient alphabet of G and C, A and T.”

— Sy Montgomery

“The greatest single achievement of nature to date was surely the invention of the molecule DNA.”

— Lewis Thomas

“Wanna know the truth about yourself and this universe? Just learn to understand your DNA code then you’ll see.”

— Toba Beta

“Generalizations in science are both necessary and hazardous.”

— Erwin Chargaff

“If we all did the things we are really capable of doing, we would literally astound ourselves.”

— Thomas Edison

“Science seldom proceeds in the straight forward logical manner imagined by outsiders. Instead, its steps forward (and sometimes backward) are often very human events in which personalities and cultural traditions play major roles.”

— James Watson

“Science commits suicide when it adopts a creed.”

— Thomas Henry Huxley

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Chapter 2

Southern and Northern Blotting

***

&Introduction&

One of the fundamental technique of modern molecular biology is immobilization of single stranded nucleic acids on a solid support and subsequent detection of specific sequence of interest. This technology known as Southern blot is used to detect specific DNA sequence in DNA samples. It was named after Edwin M. Southern who developed this procedure at the University of Edinburgh in 1973. Edwin M. Southern was interested in the 5S rRNA genes and was facing problem to purify it (1,2). A few years before the Southern blot was developed, Tom Kelly and Hamilton Smith, both at Johns Hopkins University, had shown that restriction endonucleases cut at specific sequence sites along DNA. Kenneth and Noreen Murray, who were leading figures in molecular biology at that time, introduced Southern to these restriction endonucleases. In 1971, Kathleen Danna and Daniel Nathans, also of Johns Hopkins University, showed that DNA fragments can be separated by gel electrophoresis. Southern wanted to exploit gel electrophoresis as a way to purify restriction fragment of 5S rRNA gene. As restriction fragments of genomic DNA formed a smear on gel electrophoresis, it was challenging for Southern to find the 5S rRNA gene. Southern was aware of Frederick Sanger’s work which involved the electrophoresis of RNA fragments on cellulose acetate strips, followed by blotting through to DEAE paper. Southern integrated these three technology—digestion of genomic DNA with restriction endonucleases, gel electrophoresis for separation of restriction fragments, and blotting methods—to create the Southern blot. Southern demonstrated that DNA could be electrophoretically fractionated, transferred to nitrocellulose, and then probed with radioactively labeled DNA sequences, which would hybridize to their cognates bound to the membrane. As reviewed in 2006 by Tofano D. et al “The story of the creation and dissemination of this technology, which was not patented and was freely distributed throughout the scientific community, stands as a case study in open science. Southern’s attempts to publish his findings were delayed. Though he invented the Southern blot sometime in 1973, his publication was not in print until 1975. His original submission to “Journal of Molecular Biology” was rejected on the grounds that it was a methods paper, which the journal would only publish if it incorporated original and interesting results. To prove that he had developed a novel technique worthy of publication, Southern needed to conduct further experiments. Southern did not wait for his work to be published to spread his findings. While he was running experiments and gathering more data for a publication, he engaged in a very liberal pre-publication sharing strategy” (3,4,5,6,7). Today Southern blotting is among the most fundamental and informative laboratory techniques which help in studying the structure, function and regulation of cellular DNA. The applications of Southern hybridization are diverse and a large number of molecular biology techniques have been developed on the principle of Southern blotting. On the basis of Southern hybridization principle, Alec Jeffreys developed “Genetic DNA fingerprinting” which is being used to distinguish every person from every other person, except an identical twin. DNA fingerprinting has been used to establish family relationship, individual identity in solving crimes and immigration disputes, in finding bases of various genetic diseases with many more other applications in biology (8). The 2005 “Albert Lasker Award” for Clinical Medical Research (one of the most respected science prize in the world) honoured E. M. Southern and Alec Jeffereys for revolutionizing human genetics and forensic diagnostics.

In 1977 Johan Alwine and George stark developed a method of transferring RNA and detection, analogous to Southern blotting and as a joke they called it “northern blotting”. Northern blotting had been widely used in molecular biology research to successfully compare the relative abundance of a particular gene expressed in cells subjected to different experimental and physiological conditions. It is also used to identify alternate RNA splicing patterns of different gene products, in the study of abnormal gene and genetic disorders. Most of the available information that is known today about gene expression and RNA function are due to northern blotting (9).

Principle

The principle behind both techniques is specific base-pairing between the DNA or RNA immobilized on blotting membrane (i.e. the target) and the labelled probe. Probe binds to target in a complementary manner. Two polynucleotides will form a stable hybrid by base-pairing if their nucleotide sequences are wholly or partly complementary. A specific nucleic acid fragment in a Southern blot / northern blot can therefore be detected if the membrane is probed with a labelled DNA or RNA probe molecule, labelled with detectable moieties including radiolabelled or enzyme colorimetric substrate or enzyme chemiluminescent substrates. Complementary sequences present at a concentration as low as one molecule per cell can be detected by hybridization reaction between probe and target sequence (10,11,12).

Steps of Southern blotting

(A) The DNA to be analyzed is digested with one or more restriction enzymes and then separated by agarose gel electrophoresis.

(B) The gel is treated with acidic solution which results in breakage of DNA into small fragments.

© The DNA fragments in the gel are denatured with alkaline solution which results in breakage of hydrogen bonds so the molecules become single stranded. Single stranded DNA molecules are transferred onto a positively charged nylon membrane by blotting.

(D) The membrane is incubated in prehybridization buffer, then in hybridization buffer with a specific probe which binds target DNA. The location of the DNA fragment that hybridizes with the probe can be displayed by suitable method which depends on the chemical properties of labelled probe (13,14,15) (Figure 1).

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Fig.1 Steps of southern blotting (A) Separation of DNA by agarose gel electrophoresis (B) and © Acid treatment and cleavage of DNA into small fragments (D) Alkali hydrolysis breaks hydrogen bonds between double stranded DNA molecules making them single stranded which are transferred to a membrane. Probe binds specifically to target DNA and nonspecifically to other sites (E) Shows specific (arrow) and nonspecific results obtained

Steps of northern blotting

(A) The first step in a northern blot is to denature or separate RNA molecules within the sample into single strands, which ensures that the strands are unfolded and that there is no bonding between strands.

(B) The RNA molecules are then separated according to their sizes by gel electrophoresis under denaturing conditions.

© Following separation, RNA molecules are transferred from the gel onto a blotting membrane either by capillary blotting or vacuum blotting or electro-blotting.

(D) After prehybridization, the membrane is treated with a small piece of DNA or RNA called a probe, which has been designed to have a sequence that is complementary to a particular RNA sequence in the sample; this allows the probe to hybridize to a specific RNA fragment on the membrane.

(E) Following hybridization, the probe permits the RNA molecule of interest to be detected from among the many different RNA molecules on the membrane (Figure 2) (13,16,17,18).

Fig.2 Steps of Northern blotting (A) Separation of RNA by agarose gel electrophoresis under denaturing condition (B) Single stranded RNA, which is transferred to a membrane, probe binds specifically to target RNA and nonspecifically to other sites © Shows specific (arrow) and nonspecific results obtained.

Discussion

When DNA is isolated from cells or tissue it usually comes in its double stranded native form. If this double stranded DNA is heated at 100°C, the non covalent interactions that normally hold two strands of the double helix together are disrupted and the double helix rapidly dissociates into two single strands, leading to denaturation of DNA. Denaturation of DNA occurs over a narrow temperature range. The midpoint of the temperature range over which two strands of DNA are 50% separated (or 50% annealed) is called melting temperature (Tm). Tm depends on proportion of the A-T and G-C base pairs. More the G-C base pairs are contained in a DNA, greater the energy is needed to separate two strands as each G-C base pair has been paired via three hydrogen bonds. The G-C base pairs are thermodynamically more stable than A-T base pairs which have only two hydrogen bonds. Thus higher the G-C content within a duplex DNA, more will be its melting temperature. The process of DNA denaturation was thought to be irreversible for a longer time. In 1961 it was discovered by J. Marmur and P. Doty that if a heat treated denatured DNA solution is permitted to cool very slowly, then reversible process called renaturation/rehybridizaion will occur leading to annealing of two denatured complementary single stranded DNA forming double helical DNA indistinguishable from the original native preparation. Renaturation depends on the specific base pairing between the complementary strands and reaction takes place in two stages. First, single strands of DNA in solution encounter one another by chance and if their sequences are complementary, the two strand base pairs to generate a short double helical region. The region of base pairing extends along molecules by a zipper like effect to form native double stranded DNA. The extent of hybridization between two single stranded nucleic acid is determined by their complementary. Two sequences need not be perfectly complementary to hybridize. If they are closely related but not identical, an imperfect duplex is formed in which base pairing is interrupted in positions where the two single strands are not perfect complementary. Hybridization reactions occur not only between any two single-stranded nucleic acid chains but can also occur between single stranded RNA/RNA, or RNA/DNA provided that they have a complementary nucleotide sequence. Disruption of base pairs is crucial aspect of the function of the double stranded molecule whereas intramolecular duplex region formed by base pairing between two complementary sequences within a single stranded DNA or RNA is essential for their activities (10,19,20,25) (Figure 3).

In Southern blotting, after the purified DNA has been treated with one or more restriction endonucleases, it is separated by agarose gel electrophoresis. The gel is then soaked in hydrochloric acid, which results in breaking of DNA

Fig.3 Denaturation/Renaturation: Process of denaturation results in the separation of two complementary strands of a double stranded DNA while renaturation results in annealing of complementary single strands to form double stranded native DNA.

molecules in individual bands within the gel into smaller fragments. Smaller DNA fragments will transfer to nylon or nitrocellulose membrane more quickly than larger DNA fragments. In next step gel is treated with an alkaline solution that denatures double stranded DNA molecules by breakage of their hydrogen bonds so the molecules become single stranded. Next single stranded DNA molecules are transferred to nitrocellulose or nylon membrane. The most frequent methods used for transferring are either capillary transfer or electroblotting. Capillary transfer of the nucleic acid molecules to a solid matrix involves the passive transfer of the DNA molecules to matrix membrane under the influence of capillary force while electroblotting involves the use of electric current to transfer DNA molecules from the gel to solid matrix membrane. The usual step after the completion of blotting is to ensure that the DNA molecules properly bind to solid matrix upon which it is adsorbed. This is done principally by exposing the membrane to UV or by baking it at 80°C. Exposure to UV causes the nucleic acid molecule to undergo extensive cross-linking with the membrane (13,10,17).

As most RNAs are single stranded having ability to form secondary structures by intramolecular base pairing, in northern blot analysis RNAs molecules are separated by denaturing gel electrophoresis. A number of different denaturants are employed for this purpose (formaldehyde or glyoxal/dimethyl sulfoxide or guanidine thiocyanate) to disrupt secondary structures within RNA molecules. The process is followed by transfer and cross-linking of separated single stranded RNA molecules to positively charged nylon membrane (1,18,21).

After transferring DNA or RNA to a membrane next step in both techniques is to prehybridize membrane in a prehybridization solution. Prehybridization solution is used to block unused DNA or RNA binding sites on membrane surface. It decreases any non-specific hybridization of probe to membrane surface and minimizes background and non-specific signals via increasing signal to noise ratios. If this step is omitted, then the probe will bind nonspecifically to surface of the membrane and the signal resulting from hybridization to the specific restriction fragment will be difficult to identify. Prehybrization solution contains a combination of – blocking reagents, detergent, and denatured, fragmented DNA. DNA from an organism unrelated to the one whose DNA has been blotted is also used to block unused sites on the membrane surface. Salmon sperm DNA / Herring Sperm DNA / yeast tRNAs are popular choice. Denhardt’s solution is often referred to as a blocking reagent which is a mixture of blocking reagents and volume excluder or rate accelerator. If non-specific binding is observed then it is suggested to either increase the concentration of blocking reagents or switch to a different blocking agent (10,19). As far best of my knowledge, there is no study which proves that Salmon sperm DNA or Herring sperm DNA will not cross hybridize to test samples and probe. Cross hybridization of Salmon sperm DNA with test samples nucleic acid will block the sites of targets such that it will be difficult for the probe to search its cognate part. Salmon sperm DNA is used both in prehybridization and hybridization buffer. This DNA is phenol-chloroform extracted, ethanol precipitated, and sonicated to produce single-stranded fragments which comigrate with 587 and 831 base pair marker fragments. Oncorhynchus keta (Fish) is used as a source of Salmon sperm DNA (23). BLAST alignment of various genes of Oncorhynchus keta with human genome shows significant partial homology. So there are many chances that Salmon sperm DNA can cross react with probe thus limiting binding of probe to target nucleic acid and also can mask target DNA on membrane which will further limits binding of probe to target sequence (Figure 4). Cross reactivity of Salmon sperm DNA to both target sample and probe will further limit the sensitivity of Southern hybridization. Homomix (denatured yeast tRNA) is also used as a blocking agent and is more effective in reducing background than Salmon sperm DNA (24). One of the possible reason of this could be due to lower nucleic acid sequence content in yeast tRNA than Salmon sperm DNA. Thus, I hypothesized, in place of Salmon sperm DNA or Yeast tRNA, custom synthesized single stranded DNA or RNA (Blocking probe: a known DNA or RNA sequence having no similarity to target DNA and Probe) should be used to block unused DNA or RNA binding sites on membrane surface.

Success of performing Southern and northern blotting depends upon choice of probe which is a nucleic acid molecule (Single stranded DNA, Double stranded DNA or Single stranded RNA) with a strong affinity to a specific target (DNA or RNA sequence). Probe and target base sequences must be complementary to each other but depending on conditions, they do not necessarily be exactly complementary. Gene probes are generally longer than 500 bases and comprise all or most of target genes. They can be synthesized by various ways but Polymerase Chain Reaction (PCR) is a powerful procedure for making gene probes because it makes possible to amplify and label at the same time using labelled nucleotides in extension step. Oligonucleotide probes are generally targeted to specific sequence within genes. The most common oligonucleotides probes contain 18-30 bases, but the current synthesizer allows efficient synthesis of probes containing at least 100 bases. An Oligonucleotide probe can match perfectly to target sequence and allow the use of hybridization conditions that will prevent hybridization to other closely related sequences, making it possible to identify and detect DNA with slight differences in

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Fig.4 Non-specific hybridization by pre-hybrization buffer (A) Single stranded DNA being transferred on a membrane is treated with prehybridization buffer which contain nonspecific DNA to block unused DNA binding sites (B) Nonspecific DNA may also hybridize with test DNA and probe thus limiting signal from specific target DNA. The cross hybridization at nonspecific sites can also give nonspecific signals (D).

sequence within a highly conserved sequence. In both Southern and northern hybridization, either double or single stranded DNA or single stranded RNA is used as a probe. Double stranded DNA probe is heated and quick chilled to stop rehybridization of two strands of the probe. When a double stranded DNA probe is used during hybridization steps effective probe concentration may decline due to base pairing of two complementary probe molecules to one another which may decrease signals at the last step.Thus single stranded nucleic acid probe has been found more specific and sensitive than double stranded probe (10,26).

In both techniques, formation of desired hybrid, and destabilization of nonspecific hybrids, can be achieved by utilizing an appropriate combination of buffer composition and hybridization temperature. A number of different strategies are possible. A good starting point for inorganic (nondenaturing) buffers are hybridization temperatures of 50 to 65°C for DNA applications and 55 to 70°C for RNA applications. If hybridization is carried out at a much lower temperature, it may results in high nonspecific background. If the probe is bigger in length with high Tm value, then the initial hybridization is usually carried out at high temperature in a high-salt buffer. High stringent conditions will allow only stable hybrids to form, with very little if any nonspecific hybridization. Sometime background reduction using high hybridization temperature may also result in decrease of specific hybridization signal as well. To increase the specificity of hybridization reaction, probe DNA should be chosen such that it contains a region that is completely complementary to all or a part of the blotted nucleic acid sequence to be detected. The probe also has the potential to hybridize to any other blotted nucleic acid fragments with which it has partial complementarity. The hybridization experiment will give different signal if the probe is more similar to a second restriction fragment than it is to the one being sought. “Many factors may contribute to non-specific nucleic acid hybridizations. These factors include method of transferring DNA/RNA to membnrane, UV crosslinking and baking of membrane, presence of solvent (formamide), salts, pH hybridization temperature, duration of prehybridization and hybridization, volume of hybridization solution, degree and method of agitation, concentration and specificity of the probe, use of blocking reagents, use of molecular agents to increase the rate of nucleic acid reassociation, and the degree of stringency used during the washing of the membrane” (10,22,23). The most critical is hybridization step which must be carried out at a ‘stringency’ that results in the specific hybrids remaining stable while all other non-specific hybrids to be unstable. The stringency being determined by the composition of the hybridization buffer and the temperature at which the experiment is carried out. Specificity of Southern hybridization reaction can also be increased during post-hybridization washes. It depends on the ionic strength of the final wash solution and the temperature at which this wash is carried out. Post hybridization washing steps are designed to remove nonhybridized probe and non-specifically hybridized probe. First low stringent condition are used for short oligonucleotide probe so that all potential hybrids, including nonspecific ones are able to form. By a series of post hybridization washes via increasing temperatures and lowering salt concentration within buffer, specificity is achieved such that only the desired hybrid remains at the end. Sequential lowering of salt concentrations in wash buffers will wash non-specific signals, but may also wash away specific signal if washing is too stringent. By controlling various stringency conditions, membrane is washed so that any nonspecifically bound probe is removed, leaving only probe that is base paired to target DNA. The hybridized membrane is then viewed using a suitable detector system based on the type of probe used in hybridization reaction. Various troubleshooting factors which can enhance the specificity and sensitivity for DNA blotting and hybridization analysis has been discussed in details by Terry Brown (10,22).

Now one of the critical factors which can influence specific and nonspecific binding of the probe is “Complementary” which is not broadly considered for Southern and northern hybridization. As discussed in the first chapter of this book, two DNA strands of a duplex DNA can bind to each other either by antiparallel complementary or parallel complementary. Specific binding in Southern blot hybridization experiments of both complementary antiparallel and parallel 40 bp synthetic DNA probes, corresponding to a cloned Drosophila DNA fragment has been observed by N. A Tchurikov et al in 1992. The highly cooperative annealing and melting were observed in solution probes, which were complementary in the same direction and possess 17 G-C pairs. The efficiency and specificity of hybridization with the parallel complementary was very close to that of antiparallel one. The same results were obtained with parallel 45 base pairs and 40 base pairs synthetic probes corresponding to the cloned sequence of Drosophila melanogaster suffix element and E. coli lon gene. By adsorption isotherm study N. A. Tchurikov et al concluded that at least 95% of the bases in the parallel duplex were involved in pairing. They also confirmed that the parallel complex was consisting of only two strands and was not either a triplex or a hairpin. Their results suggested that parallel complementary oligonucleotides can be used successfully as probes in molecular hybridization experiments with cloned and genomic sequences as well as for effective screening of genomic libraries. Their study shows that molecular hybridization experiments detects both parallel and antiparallel complementary sequences having specificity and backgrounds satisfying all classical criteria. This was first report counter to the current view that molecular hybridization experiments detect only homologous antiparallel complementary sequences (27). Until now non-specific binding of probe to different test samples was attributed due to potentiality of a probe to hybridize with other DNA sequences which are partially complementary in antiparallel direction to the probe. But a probe has potentiality to bind specifically to its target sequence in antiparallel manner and it can also bind non-specifically to other sequence both in parallel and antiparallel orientation. Double stranded DNA probes will have more chances of binding to nonspecific sites in test samples than single stranded DNA or RNA probes. This could be one of the additional reasons why single stranded nucleic acid probes are more specific and sensitive than double stranded probe. I further hypothesize that if a single stranded probe having mirror sequence (which has potentialities to bind similarly in an antiparallel and parallel manner) is used as a probe it may increase the chances of specific hybridization.

Thus, in conclusion, Southern and northern hybridization techniques can be improved by using a blocking probe in place of Salmon sperm DNA/yeast tRNA. Blocking probe must be designed in such a way that it does not have any binding potentialities to test samples and probe both in parallel and antiparallel orientation. Probe to be used for detection of specific target DNA or RNA must be checked for not having any complementary both in parallel and antiparallel orientation to nonspecific target A single stranded probe containing mirror repeat sequence may have a higher specificity than conventionally designed probes. Various Bioinformatics tools used for designing probes can be improved by considering parallel and antiparallel complimentary binding of two strands of duplex DNA.

“The good thing about science is that it’s true whether or not you believe in it.”

— Neil deGrasse Tyson

“Science moves with the spirit of an adventure characterized both by youthful arrogance and by the belief that the truth, once found, would be simple as well as pretty.”

— James Watson

“If an elderly but distinguished scientist says that something is possible, he is almost certainly right; but if he says that it is impossible, he is very probably wrong.”

— Arthur C. Clarke

“Science is a beautiful gift to humanity; we should not distort it”.

— A. P. J. Abdul Kalam

“New scientific ideas never spring from a communal body, however organized, but rather from the head of an individually inspired researcher who struggles with his problems in lonely thought and unites all his thought on one single point which is his whole world for the moment.”

— Max Planck

Bibliography

1. Southern, E. M. Blotting at 25. TIBS 25 585–588 (2000).

2. Southern, E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503–517 (1975).

3. Kelly, T. J. & Smith, H. O. A restriction enzyme from Hemophilus influenzae II. Base sequence of the recognition site J. Mol. Biol. 51, 393 (1970).

4. Danna, K. & Nathans, D. Specific cleavage of simian virus 40 DNA by restriction endonuclease of Hemophilus influenza Proc. Natl. Acad. Sci. USA 68, 2913 (1971).

5. Sanger, F., Brownlee, G.G. and Barrell, B.G. A two-dimensional fractionation procedure for radioactive nucleotides. J. Mol. Biol. 13,373–398 (1965).

6. An Overview of Northern and Southern Blotting https://www.nationaldiagnostics.com/electrophoresis/article/overview-northern-and-southern-blotting

7. Tofano, D., Wiechers, I. R. & Cook-deegan, R. Edwin Southern DNA blotting , and microarray technology : A case study of the shifting role of patents in academic molecular biology. Genomics,society and policy 2, 50–61 (2006).

8. Jeffreys, A.; Wilson, V.; Thein, S. “Individual-specific ‘fingerprints’ of human DNA”.Nature 316 (6023):76–79 (1985).

9. Alwine, J.C., Kemp., D.J., and Stark, G.R. Method for detection of specific RNAs in aga- rose gels by transfer to diazobenzyloxymethyl- paper and hybridization with DNA probes. Proc. Natl. Acad. Sci. U.S.A. 74:5350-5354 (1977).

10. Brown, T. A. Southern Blotting and Related DNA Detection Techniques. Encycl. Life Sci., Nature Publ. Gr. 1–6 (2001).

11. Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter. Molecular Biology of the Cell, 4th edition New York: Garland Science; (2002)

12. Umeoguaju, F. U. Principles and procedures of the Northern blot technique. Ufumes Sch. Niger. Online Publ. Press 1–6 (2009).

13. Hybridisation Guide. Thermo electron Corp. (2003).

14. Southern blotting: Capillary transfer of DNA to membranes Nature methods vol.1 no.1,91 (2004).

15. Weiss, A. S. Southern Transfer and Hybridization : A Class Experiment. Biochem. Educ. 20, 231–233 (1992).

16. Northern blotting: transfer of denatured RNA to membranes Nature methods, vol.2 no.12, 997, (2005).

17. Hayes, P. C., Wolf, C. R. & Hayes, J. D. Blotting techniques for the study of DNA, RNA, and proteins. Br. Med. J. 299, 965–968 (1989).

18. Brown, T. Analysis of RNA by Northern and Slot-Blot PROTOCOL. Curr. Protoc. Immunol. (1993).

19. Lewin, Benjamin, Genes IX, Jones & Bartlett Learning Edition:9 (2007).

20. Marmur J. and Doty P. Thermal renaturation of deoxyribonucleic acids J. Mol. Biol. 3, 585–594 (1961)

21. Kroczek, R. Southern and Northern analysis. J. Chromatogr. 618, 133–145 (1993).

22. Herzer, S. & Englert, D. F. Nucleic Acid Hybridization. Molecular Biology Problem Solver: A Laboratory Guide. Edited by Alan S. Gerstein Copyright © 2001 by Wiley-Liss, Inc.

23. Deoxyribonucleic acid (DNA), Sodium salt, from Salmon testes. Sigma Product Information. D1626

24. Mitchell, T. & Morley, B. J. Isolation of RNA and analysis by Northern blotting and primer extension. Tech. Tips Online 3, 20–22 (1998).

25. Craig, R. K. Methods in molecular medicine. Br. Med. J. 295, 646–649 (1987).

26. Muro, M. A. de. Probe Design, Production, and Applications. Med. Biomethods Handb. (2005).

27. Tchurikov, N. A, Shchyolkina, a K., Borissova, O. F. & Chernov, B. K. Southern molecular hybridization experiments with parallel complementary DNA probes. FEBS Lett. 297, 233–6 (1992).

Chapter 3

Microarray Technology

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Introduction

In 1988, Zaffaroni approached Lubert Stryer, requesting him to become Chief Scientific Officer of the new company (Affymax) which Zaffaroni had founded along with J. Leighton Read and Peter Schultz. The goal was to develop novel chemical approaches to automated drug discovery. Using photolithography they developed peptide microarrays. While work continued on peptide microarrays, Stryer and the Affymax Scientific Board recognized a much more immediate opportunity in the development of nucleic acid microarrays (1). On the other side, Pat Brown at Stanford University was working on a scheme that had the ultimate aim of determining whole–genome genotypes of millions of peoples for linkage and association studies. It involved a biochemical method that he called “genomic mismatch scanning” for isolating the sequences that were identical between two genomes and then mapping them by hybridizing to a physically ordered arrangement of the human genome. Pat Brown had a small pilot grant from the NHGRI (National Human Genome Research Institute) to develop the genomic mismatch scanning method and once the method was worked out with Stan Nelson, Pat Brown submitted a renewal application that also included the “Microarrays”. He got little note-card back from NIH (National Institute of Health) with a worst priority score. He was so much deflated that he literally lay down on his office floor for ten minutes to regain his composure. He again got back in touch with NHGRI and resubmitted the grant proposal. He received a much smaller grant by which he recruited Dari to start the microarray work. Later, microarray grew out of the cumulative efforts of many of scientists from academia and industry. Microarray today provides powerful tools for global characterization of gene expression (2).

A DNA microarray (DNA chip or biochip) is a collection of microscopic DNA spots attached to a solid surface. Microarrays can be categorized as either cDNA arrays or oligonucleotide arrays. cDNA microarrays are with probes of up to few hundred base pairs while oligonucleotide arrays use short probes. Both types of arrays allow measuring mRNA expression of thousand genes at a time. It makes possible to relate physiological cell states to gene expression patterns for studying tumors, disease progression, cellular response to different stimuli, and drug target identification. More recent uses of DNA microarrays are not limited to gene expression analysis. DNA microarrays are being used to detect single nucleotide polymorphism, genomic mutation, aberration in methylation patterns, alteration in gene copy numbers, alternative splicing, DNA sequence analysis, immunology and pathogen detection etc. (3).

Principle

Microarray is a reverse dot blot based on same principle of complementary hybridization as in Southern and northern blots. DNA microarrays are typically composed of DNA “probes” that are bound to a solid substrate such as glass. Each spot in the array lattice is composed of many identical probes that are complementary to gene of interest. The principle behind microarrays is complementary hybridization between the two nucleic acid strands. A high number of complementary base pairs in a nucleotide sequence means tighter bonding between two strands. During hybridization, DNA “targets” diffuse passively across the glass surface, and sequences complementary to a probe anneal and form a DNA duplex. After washing off non-specific bonding sequences, only strongly paired strands remain hybridized. Hybridized targets are then detected using one of many reporter molecule system (4).

Steps of Microarray technology

1. DNA microarrays are created by spotting every probe for genes in a genome onto a glass microscope slide.

2. Total RNA is isolated from the samples to be tested.

3. cDNA is synthesized from mRNA using reverse transcriptase. Labelling is achieved by including in the reaction modified fluorescent nucleotide. Two mRNA samples which are to be compared are differently labelled. Differently labelled cDNAs of both the samples is mixed.

4. DNA microarrays are prehybridized using blocking solution which contains Saline Sodium Citrate, SDS, Nonspecific DNA such as yeast DNA, Salmon Sperm DNA or repetitive sequences, BSA etc.

5. cDNAs samples are hybridized to the microarray. Hybridization is carried out according to conventional protocols. Hybridization is usually performed at 42-45 °C for approx 16 hrs.

6. Microarray slides are washed with washing solution and slide are scanned (5,6) (Figure 1).

Discussion

During the years from 1992 to 1998, less than 100 reports were published using microarray technology. In last decades, thousands of articles have been published using microarray technology and functions have been assigned to previously un-annotated genes. Putting successful outcomes of all these researches is out of scopes of this book. In a similar trend, many publications come out, which have questioned validation of data obtained through microarray technology. In 2007 Yauk, C. L. & Berndt, M. L. has reviewed both type of investigations which have shown a low level of correlation and high level of correlation by microarray analysis (8).

In 2002, W. P. Kuo et al using two high-throughput DNA microarray technologies, (Stanford type cDNA microarrays and Affymetrix oligonucleotide microarrays) compared mRNA

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Fig.1 Steps of Microarray technology (7).

measurements of 2895 sequence-matched genes in 56 cell lines from the standard panel of 60 cancer cell lines from the National Cancer Institute (NCI 60). Their results suggested that data from spotted cDNA microarrays could not be directly combined with data from synthesized oligonucleotide arrays and corresponding measurements from the two platforms showed poor correlation. They concluded that gene-specific, or more correctly probe-specific, factors have influenced measurements differently in the two platforms, suggesting that more research on the hybridization dynamics on both platforms are needed before measurements are sufficiently well understood to be utilized across technologies (9). Similarly, in 2002 R. Kothapalli et al found a 3.8 fold differential expression of perforin gene by cDNA microarray whereas the oligonucleotide microarray indicated a 103 fold increase. By northern blot analysis they found that upregulation of perforin gene was neither 103 fold as indicated by oligonucleotide array nor 3.8 fold as determined by the cDNA microarray data. Instead, the actual value was determined by them to fall between these two extreme values (10). In June 2003, Nick Fischer who was president of the Statistical Society of Australia, feared that sloppy statistics could undermine the revolution promised by genomics and biotechnology. He issued a dire-sounding warning stating “If the collection, analysis and interpretation of the data are flawed then it may not only be a waste of a valuable resource — we could draw faulty conclusions and potentially risk our health and environment” (11). To demonstrate that the variation observed in various microarray platforms might be due to lab-to-lab variability (lab effect), a consortium of ten labs from the Washington, DC–Baltimore, USA, area was formed to compare the performance of three leading microarray platforms (Affymetrix GeneChips, two-color spotted cDNA arrays, two-color long oligonucleotide arrays). Exact copy of four samples were hybridized by ten labs using their platform of choice. In general, 5 labs which used Affymatrix microarray platform achieved better accuracy and precision with correlation ranging from 0.48 to 0.79. Two-color cDNA labs 1 and 3 outperformed most Affymetrix oligo labs in some categories. Best performance was by Lab number 2 which have used ‘Two-color’ oligo microarray platform (Correlation 0.90 and s.d. 0.10) while the worst performance was by Lab number 1 which also have used same Two-color oligo microarray platform (correlation 0.68 and s.d. 0.51). Thus the best and worst overall performance was achieved using the same microarray platform by two different labs. Thus, this study by R.A. Irizarry et al demonstrated that there are relatively large differences in data obtained in different labs using the same platform (12).

Fig.2 Microarray technology: mRNA is isolated from two samples and cDNA is synthesized with incorporation of fluorescent labels (Cy3 for Red and Cy5 for Green). Labelled cDNAs are mixed and hybridized to microarray slides. Red spot represents gene which are expressed in control sample while green spots represents genes expressed in test sample. Yellow spots gives information about genes which are expressed in both samples.

Fig.3 Biological Variation: mRNA is isolated from two control samples and cDNA is synthesized with incorporation of fluorescent labels (Cy3 for Red and Cy5 for Green). Labelled cDNA are mixed and hybridized to microarray slides. Yellow spots gives information about the genes which are expressed in both control samples. Red and green spot indicates biological variation in these two control samples.

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Fig.4 Technical Variation mRNA is isolated from one control sample and cDNA is synthesized in duplicates with incorporation of fluorescent labels (Cy3 for Red and Cy5 for Green). Labelled cDNA samples are mixed and hybridized to microarray slides. Red and green spot indicates technical variation. The similar experiment when compared with another microarray slides can give more information about technical variation.

DNA microarray experiments are supposed to be affected by numerous variables, including experiment design, sample preparation, chip manufacturing, etc. In general speaking two major sources of variation are proposed to be involved in microarray experiment: technical variation and biological variation (Figure 2,3,4). Technical variations may occur during manufacturing of microarray processing, sample processing which includes mRNA isolation, cDNA synthesis, labelling, chip hybridization, washing and chip scanning. Biological variation is the intrinsic differences of gene expression profiles of various test samples in nature due to genetic or epigenetic factors. Technical variation due to defects in manufacturing of chip has also been reported (13). In 2002 R. Kothapalli et al purchased 20 clones representing the differentially expressed genes and verified that only 70% genes spotted on microarray matched the correct sequence of the clones. So error in preparing cDNA microarray must be checked as mistakes made at this stage will not be corrected then later by using these microarray (10).

In 2007, J. Xu et al has evaluated technical variation between two cRNA samples prepared from two parallel RNA samples derived from the same animal and hepatocytes cultures at different time points. Their studies showed that gene expression profiles of two replicates sample at all time points for all six animals were highly correlated to each other with correlation coefficients ranging from 0.922 to 0.990. But the correlation coefficient of gene expression profiles among six animals which were identically fed, grown up showed a range from 0.699 to 0.96. This change in correlation coefficient has been described due to biological variation (13).

Similarly, in 2003, S.D. Jenson et al has validated cDNA microarrays profiles data obtained from linearly amplified RNA using in vitro transcription based protocol. Comparing Non-amplified mRNA samples with each other in replicates, the range of correlation coefficient was from 0.8080 to 0.8242 indicating each sample differed from one another. The range of correlation coefficient for replicates cDNA microarray hybridization using SUDHL-6 mRNA and Tonsillar B cell mRNA was 0.7008 to 0.7982. These variations were improved when they used linearly amplified RNA (aRNA). When Cy5 labelled aRNA prepared from SUDHL-6 were compared with Cy3 labelled aRNA prepared from the same sample in replicates, a high range of correlation coefficient was obtained from 0.9781 to 0.9803. Similarly, they found correlation coefficient for replicate cDNA microarray hybridization using SUDHL-6 aRNA and tonsillar B cell aRNA was 0.9484 to 0.958. The decrease in dynamic range of expression in aRNA generated microrrays data was found to be less among over-expressed genes than under-expressed genes. Their studies showed that only 88 genes were found to be over-expressed and 72 genes were found to be under-expressed two fold or more using mRNA generated microarrays while aRNA generated microarrays showed that 12 genes were over expressed and 22 were underexpressed - two fold or more. Overall agreement of quantitative RT-PCR data with mRNA generated data was approximately 75% (14). Recently H. Sudo et al reported different results comparing aRNA and non-amplified RNA samples using a novel 3D-GENE TM microarray which features a micro-columnar structure composed of black resin substrate and a bead agitation technique for low background noise, enhanced signal intensity and high reproducibility. Using RNA derived from human brain, breast and colon, they demonstrated that non-amplification method could be transformed to a highly quantitative method with a dynamic range of five orders of magnitudes. They found that microarray data processed using non- amplification method had the highest correlation (0.84 to 0.93 ) with qRT PCR. The correlation decreased to (0.44 to 0.74) with one round of linear amplification and for 2 nd round of amplification, it was further decreased to (0.29 to 0.62) indicating that amplification process during sample preparation reduced the accuracy of microarray data. They also discussed that amplification bias could be generated during in-vitro transcription steps (15). Gilbert et al has investigated T7 in vitro transcription kinetics and discovered that aRNA production was linear only for 40 minutes for the first round while for a second round of amplification it was for 50 minutes. This was followed by a non-linear phase, which introduces the bias and leads to inaccuracies in transcript abundance. Most of commercial platforms have recommended 4-14 hours incubation time for aRNA production thus providing subsequent bias (16).

Microarray hybridization specificity [“the ability of a probe to provide a signal that is influenced only by the presence of target molecule”] is one of the main effectors of microarray result quality. It is meaningless to have a technology which shows reproducibility, sensitivity but low specificity. Low specificity is being suggested to be one of the prime measures affecting discrepancies in gene expression profiles between different probes targeting the same region of a given transcript and if this factor is once improved, it may increase the validity of microarray results. The probe has been designed to bind target nucleotide sequence in a complementary manner according to Watson-Crick rules of base pairing. As by H. Koltai et al, hybridization specificity can be defined across four levels of hybridization: the single probe, the single set, spot set and microarray platform (Figure 5) (17). Here hybridization specificity due to the single probe will be discussed and the same principle can be applied to other 3 level of hybridization specificity. Two molecules can have either perfect hybridization, partial or mismatch hybridization or no hybridization. “No hybridization” means target molecules are not hybridized to any probe or probe did not hybridize to target molecules. No hybridization, can happen due to less concentration of target sequence within test sample or due to less probe density, i.e. the number of different probes that are fabricated on the microarray chip in a given area. Earlier condition can be improved using protocols which can increase the concentration of test samples by linear amplification while latter condition can be improved by the manufacturer of microarrays chips. The linear amplification of DNA within test samples increase concentration of both specific and nonspecific targets, but as specific targets have more chance to competitively bind in a microarray, more specific results are obtained after linear amplification. It is being further suggested that if a probe is

Fig.5 Illustration of the different levels of specificity of microarray hybridization. (A) Specificity at the probe level; matching between a single probe molecule and a single target molecule: perfect match (i), low match (ii), no match (iii). (B) Specificity at the spot level; matching between multiple probe molecules that make up one spot and multiple different (i.e. derived from more than one reference sequence) target molecules: perfect match (i, red spot), low match (ii, pink spot), no match (iii, gray spot) or cross-hybridization (iv, yellow spot). (C) Specificity at the spot-set level; matching between multiple spots that represent the same reference sequence, but through its different segments, and multiple different target molecules: perfect match (i), low match (ii), no match (iii) or cross-hybridization (iv). Black lines denote probe molecules, red lines denote target molecules of a single reference sequence that perfectly match the spot probes, pink lines denote target molecules of a single reference sequence with lower sequence similarity to the spot probes, green and blue lines denote target molecules of other reference sequences. (D) Specificity at the microarray level; matching between multiple spot-sets and multiple different target molecules: perfect match (red spot), low match (pink spot), no match (gray spot) or cross-hybridization (yellow spot). Reprinted by permission of Oxford University Press. Nucleic Acids Research, H. Koltai et al 2008 (17).

hybridized by nonspecific cross hybridization by nonspecific targets, a competition may occur which may not allow binding of specific hybridization of probes with its target molecule (17). In many of microarray experiments, Non-specific DNA (e.g. Salmon sperm DNA or Yeast tRNA) has been used during prehybridization steps (5,6). I hypothesized that some of the nucleic acid sequence within nonspecific DNA may cross hybridize to probe sequence thus limiting the binding of test sample sequences to probe sequence on microarray platform. It will be ideal to use a blocking probe (as dicussed in 2nd chapter of this book) in place of non-specific DNA sequence during prehybrization/hybridization steps. It is further suggested that “No hybridization” can also be due to 3 dimensional structure of the target or probe. A single stranded nucleic acid sequence may adopt a 3 dimensional structure based on complementary sequence present within a sequence. There are bioinformatics tools available which can predict the tertiary structure of single stranded RNA molecules. These tools detect base pairing that occurs in RNA through region of self complementary due to Watson-Crick base pairing. To best of my knowledge, there is no bioinformatics tool which can predict tertiary structure of single stranded DNA. When DNA is present in single stranded form, it has same chance to adopt different tertiary structure as like single stranded RNA. If a probe or the target sequence adopts a complex tertiary structure, there may be a chance, these 3 D structures might block binding of target and probe to each other. The chances of having a complex 3 D structure will increase with increasing the length of the probe. So it might be one of the reasons why cDNA microarray did not produce same reproducible specific results on different or same platforms. cDNA microarrays consist single stranded DNA molecules on a chip are of few hundred base pair lengths, so chances of a cDNA probe to adopt a complex 3 dimensional structure is more as it may contain more region of the self complementary sequence. In case of small oligonculeotide arrays which have probe length of 25 mer, the chances of probe to adopt complex 3 dimensional structure is less and this could be one of the reason of getting more specific, reproducible results using Affymetrix oligonucleotide array. It is further suggested that taking hybridization condition which can minimize effects due to adoption of tertiary structure state within a single stranded DNA sample will also increase specificity of microarray hybridization.

Now two molecules can have either perfect hybridization or partial or mismatch hybridization. Perfect hybridization will be due to binding of two nucleotide sequences i.e. both probe and target in an antiparallel complementary manner by Watson- crick base pairing rule. Here it is again emphasized that two nucleotide sequences can be complementary in two ways. In antiparallel complementary, both strand runs in opposite directions while in parallel complementary, both strands will run in the same direction. Microarray Chips have been manufactured in keeping mind only antiparallel complementary. No study has been done on the binding of parallel complementary probes but it does not rule out this possibility as DNA has both possibilities of adopting a duplex which can be either parallel complementary or antiparallel complementary. Out of two types of microarray platforms, oligonucleotide arrays have been found to be more reproducible and sensitive than cDNA arrays. As oligonucleotide arrays are generally of 25 mer length, it reduces the chance of hybridizing a parallel complementary target because smaller the probe length there will be also less chances of having a region which can hybridize non-specifically to other sequences in a parallel complementary manner. Also increasing the probe length to few hundred base pairs also increase chances of nonspecific cross hybridization of different sequences other than the target sequence in a partial manner. Thus most of the time parallel complementary probe - target hybridization will not be perfect if probe length is smaller as in oligonucleotide probe, but as the length of probe increase in few hundred base pairs as in cDNA arrays chances of both specific binding (antiparallel binding of target and probe) and Nonspecific binding (due to cross hybridization in antiparallel and parallel manner) will also increase. Thus specificity of oligonucleotide probe can be further increased, in keeping in mind that DNA can hybridize both in parallel and antiparallel complementary manner. Probe sequence should be selected in such a manner that it will hybridize only to target specific sequence without any hybridization to other sequence in a parallel complementary manner. This can be tested using BLAST (Basic Local Alignment Server Tool) for each probe such that it has only binding to specific sequence and there should be no binding in a parallel complementary way. Further It is required to develop new bioinformatics tool which can design specific probe in keeping mind that DNA hybridization can happen both in parallel and antiparallel manner. It is further emphasized that probe sequence which contains a mirror repeat sequence within them can hybridize a target sequence both in parallel and antiparallel manner. There are chances that these mirror sequences may be present in other sequence, thus favouring binding to nonspecific target both in parallel and antiparallel complementary manner. The probe sequence consisting of unique mirror sequence specific to one target sequence will have potentiality to bind a single target both in parallel and antiparallel manner may enhance the specificity of microarray hybridization. So in general a higher level of matching between probe and target molecule can be improved in taking consideration length of probe, hybridization in parallel and antiparallel manner, GC content, mirror sequence and other thermodynamic parameters.

Fig.6: Different possibilities of complementary hybridization of a target sequence to immobilized probes on a microarray slides. (1) shows ‘No-hybridization’ (2) shows hybridization may be blocked due to binding of Nonspecific DNA sample to probe during prehybridization step. (3) Perfect antiparallel complementary Watson-Crick base pairing and hybridization of specific target DNA sequence to specific probe. (4) antiparallel complementary mismatch hybridization of target sample to nonspecific probe (5) Partial antiparallel complementary hybridization of target sequence to a nonspecific probe (6) Parallel complementary mismatch hybridization of target sequence to nonspecific probe (7) Partial Parallel complementary hybridization of test sequence to a nonspecific probe (8) Perfect parallel complementary hybridization of target sequence to a nonspecific probe (9) Perfect parallel complementary hybridization of target sequence to a specific probe. It can happen when probe sequence consist of unique mirror sequence.

In Affymetrix microarrays, each probe pair consists of perfect match (PM) and mismatch (MM) probe. ‘PM’ probe is designed to be antiparallel complementary to target sequence while ‘MM’ probe is designed to be antiparallel complementary to target sequence except for one base mismatch. This system is intended to discriminate in between true signal and nonspecific hybridization. In 2002 R. Kothapalli et al showed that perforin, PAC-1, Human autoantigen, carboxyl ester lipase like protein was binding strongly to both ‘PM’ and ‘MM’ probe sets. The strong ‘MM’ signal has masked ‘PM’ signal indicating a lower expression or absence of these genes, even though these genes have been present normally in the test sample. They also found that it was very difficult to distinguish between two similar genes (granzyme B and granzyme H) expression which share approximately 80% similarity at DNA level but have different enzymatic activities (10).

Affymetrix microarray can be improved using a ‘PM’ probe and ‘PP’ (Parallel Probe). First ‘PM’ probe may be designed in taking consideration length of probe, hybridization in parallel and antiparallel manner, G-C content, mirror sequence, finally in tacking a portion of gene which binds only to unique portion of a gene. It will be highly appreciable if probe designed can compare between alternative spliced form of a gene and two isoform of a gene. If a unique ‘PM’ probe can not be generated using mirror sequence then introduction of a new type of probe, i.e. parallel probe can give certain advantage. The difference between a ‘PM’ probe and ‘PP’ probe is that ‘PP’ probe will have a reverse polarity than ‘PM’ probe. ‘PP’ probe must be present as a neighboring spot near the ‘PM’ probe. Now, as discussed nonspecific binding can happen by binding of nonspecific sequence in a parallel orientation to ‘PM’ probe. Due to presence of nearby ‘PP’ probe, nucleotide sequence which were being cross hybridized in a parallel complementary manner to the ‘PM’ probe will be having more chances to hybridize to ‘PP’ probe in an antiparallel manner. Further nonspecific hybridization at ‘PM’ probe can be minimized using more stringent hybridization, washing conditions.

“The aim of science is not to open the door to infinite wisdom, but to set a limit to infinite error.”

— Bertolt Brecht

“He who knows nothing is closer to the truth than he whose mind is filled with falsehoods and errors.”

— Thomas Jefferson

“An expert is a person who has made all the mistakes that can be made in a very narrow field.”

— Niels Bohr

“In all science, error precedes the truth, and it is better it should go first than last.”

— Hugh Walpole

“To raise new questions, new possibilities, to regard old problems from a new angle, requires creative imagination and marks real advance in science.”

— Albert Einstein

“Formula for breakthroughs in research: Take young researchers, put them together in virtual seclusion, give them an unprecedented degree of freedom and turn up the pressure by fostering competitiveness.”

— James Watson

“Science is a self-correcting discipline that can, in subsequent generations, show that previous ideas were not correct”.

—Brian Greene

Bibliography

1. Lenoir, T. & Giannella, E. The emergence and diffusion of DNA microarray technology. J. Biomed. Discov. Collab. 1, (2006).

2. Gitschier, J. You Say You Want a Revolution : An Interview with Pat Brown. PLoS Genet. 5, e1000560 (2009).

3. Trevino, V., Falciani, F. & Barrera-saldaña, H. A. DNA Microarrays : a Powerful Genomic Tool for Biomedical and Clinical Research. Mol. Med. 13, 527–541 (2007).

4. Call, D. DNA microarrays – their mode of action and possible applications in molecular diagnostics. Vet. Sci. Tomorrow 1–9 (2001).

5. http://www.cvmbs.colostate.edu/mip/tb/pdf/arrays.pdf

6. https://cbse.soe.ucsc.edu/sites/default/files/affymetrix_protocol050404.pdf

7. http://en.wikipedia.org/wiki/File:DNA_microarray_experiment.svg

8. Yauk, C. L. & Berndt, M. L. Review of the Literature Examining the Correlation Among DNA Microarray Technologies Carole. Enviornmental Mol. Mutagen. 48, 380–394 (2007).

9. Kuo, W. P., Jenssen, T., Butte, A. J., Ohno-machado, L. & Kohane, I. S. Analysis of matched mRNA measurements from two different microarray technologies. Bionformatics (Oxford Univ. Press. 18, 405–412 (2002).

10. Kothapalli, R., Yoder, S. J., Mane, S. & Loughran, T. P. Microarray results: how accurate are they? BMC Bioinformatics 3, 22 (2002).

11. Tilstone, C. Vital statistics. Nature 424, 610–612 (2003).

12. Irizarry, R. A. et al. Multiple-laboratory comparison of microarray platforms. Nat. Methods 2, 345–349 (2005).

13. Xu, J. Deng, X. Chan, V. Kelley-Loughnane, N. Harker, B. W. Shi, L. Hussain, S. M. Frazier, J.M. Wang, C. Variability of DNA microarray gene expression profiles in cultured rat primary hepatocytes. Gene Regul. Syst. Bio. 1, 235–49 (2007).

14. Jenson, S. D*.], Robetorye, R. S.[,_] Bohling, S. D. _,_ Schumacher, J. A. _,_ Morgan, J. W. _,_ Lim, M. S.[, Elenitoba-Johnson, K. S. J.*]_] Validation of cDNA microarray gene expression data obtained from linearly amplified RNA. Mol. Pathol. 56, 307–12 (2003).

15. Sudo, H., Mizoguchi, A., Kawauchi, J., Akiyama, H. & Takizawa, S. Use of non-amplified RNA samples for microarray analysis of gene expression. PLoS One 7, e31397 (2012).

16. Gilbert I, Scantland S, Dufort I, Gordynska O, Labbe A, Real-time monitoring of aRNA production during T7 amplification to prevent the loss of sample representation during microarray hybridization sample preparation. Nucleic Acids Res 37(8): e65. (2009).

17. Koltai, H. & Weingarten-Baror, C. Specificity of DNA microarray hybridization: characterization, effectors and approaches for data correction. Nucleic Acids Res. 36, 2395–405 (2008).

Chapter 4

siRNA Technology

***

Introduction

Historically studying loss of function, phenotypes in cell culture or whole organism has been a critical aspect in determining the function of a gene. RNA interference is biological mechanism by which double-stranded RNA (dsRNA) induces gene silencing by targeting complementary mRNA. siRNA technology has revolutionized the way researchers find out the function of an unknown gene. By introducing double stranded RNA homologous to a particular mRNA, scientists can quickly and easily reduce the expression of a particular gene in nearly all organisms/cells. As compared to other conventional gene knockout strategies it allows them to quickly analyze the function of a particular gene (1,2).

In 1990, while working for a biotechnology company C. Napoli et al targeted to make petunias flower more purple than normal. Chalcone synthase is the key enzyme of flavonoid synthesis responsible for deep violet coloration in petunias flower. They overexpressed chimeric Chalcone synthase gene into petunia with a prediction that increasing the copy number of CHS genes would increase CHS protein levels, which would result in production of very purple flowers than wild types. Unexpectedly overexpression of chalcone synthase did not result in purple flower but leads to production of white petunia flowers. They found that the levels of CHS messenger RNA in white petunia flowers were 50 times lower than wild type purple petunia flowers. Somehow, the overexpression of CHS gene (the transgene) lowered expression of both the transgene and the endogenous gene. It was a mystery for almost 10 years that how overexpression of a gene can result in downregulation of its own mRNA. One theory suggested that the silencing was caused by endogenous anti-sense RNA (single-stranded RNA that is the reverse complement of mRNA). The idea was that endogenous anti-sense RNA would have base paired to the transgene mRNA and inhibited protein production (2,3,4).

In 1998, Andrew Z. Fire and Craig C. Mello, working with C. elegans stated that trigger for gene silencing was not single stranded RNA but was double stranded RNA and double stranded RNAs can trigger potent silencing of complementary messenger RNA sequences. They found that although both sense and antisense single strands produced modest RNA interference, double stranded mixtures produced potent and specific interference. They further reported that these potent and specific effects were also evident in both the injected animals and their progeny (5). The Nobel Prize in Physiology or Medicine (2006) was awarded jointly to Andrew Z. Fire and Craig C. Mello “for their discovery of RNA interference – gene silencing by double-stranded RNA”. It was initially thought that this approach would not be applicable in mammals as dsRNA molecules that were longer than 30 bp when used as siRNA in mammals resulted in the global shutdown of protein synthesis. In 2001, it was shown by S.M. Elbashir et al and Caplen and colleagues that chemically synthesized short dsRNA molecules of 21–22 nucleotides (siRNAs) could be used to silence genes in mammalian system without global shutdown of protein synthesis (6,7). Today RNAi (by siRNAs) is one of the fastest advancing fields in molecular biology research with flow of discoveries giving true meaning to the expression ‘from the workbench to the bedside’. It is being described as a powerful and promising technology for both basic research and therapeutic intervention.

Principle

In many non-mammalian systems, introduction of long double stranded RNA (dsRNA) can triggers the RNAi pathway. Dicer (a cytoplasmic nuclease) first cleaves these long dsRNA into 21–23 bp small interfering RNAs (siRNAs), and then unwind these siRNAs and assemble into RNA-induced silencing complexes (RISCs). The antisense strand of siRNA guides RISC to complementary RNA molecules, and activated RISC cleaves targeted mRNA, leading to specific gene silencing. The strand of the siRNA that is complementary to the target mRNA sequence(s) is known as “Guide strand” while the other strand is known as sense strand or “Passenger strand”. The 5’ region of the guide strand of an siRNA, extending from nucleotides 2-7 (Hexamer) or 2-8 (heptamer) is known as “seed” sequence. In most mammalian cells, RNA interference can be induced by transfecting cells with siRNAs (typically 21 bp RNA molecules with 3’ dinucleotide overhangs) or by using DNA-based vectors to express short hairpin RNAs, (shRNAs), that are processed by Dicer into siRNA molecules. Processed siRNAs are then incorporated into cellular protein complex known as the RNA-induced silencing complex (RISC). RISC by its helicase activity unwind siRNA duplex in an ATP dependent reaction. Activated RISC-siRNA complex recognizes targets mRNAs leading to specific gene silencing. The activated RISC-siRNA complex can silence gene expression either via post-transcriptional gene silencing (PTGS) or transcriptional gene silencing (TGS). PTGS is being categorized in two primary mechanisms: direct sequence-specific cleavage, and translational repression and RNA degradation. When the guide strand of activated RISC-siRNA is perfectly complementary to targeted mRNA, it results into direct sequence specific cleavage while translational repression and RNA degradation occur when the guide strand of activated RISC-siRNA has only limited complementarity to the target in the ‘seed’ region resulting in base pairing of activated siRNA at 3’UTR region of targeted mRNA. Activated siRNAs which are complementary to promoter regions, when present within nucleus can trigger chromatin remodelling and histone modifications resulting in transcriptional gene silencing. In mammalian cells, the details of transcriptional gene silencing via siRNAs are still under investigation. Thus RNAi acts at the level of mRNA synthesis, decreasing mRNA levels and the ability of the mRNA to be translated (8,9). (Figure 1,2).

Fig.1 Mechanisms of cellular gene silencing Primary microRNAs (pri-miRNAs) are, in plants and animals, processed by Drosha and its partner DGCR8 into precursor miRNAs (pre-miRNAs) and then transported to the cytoplasm by exportin 5 (XPO5). In the cytoplasm, they are bound by a Dicer-containing pre-RISC and processed to yield the guide sequence that is loaded into the holo-RISC, which contains all the components required for gene silencing. AGO2 is the catalytic core of the RISC (present but not shown in the schematically drawn holo-RISC). The guide sequence binds to the corresponding target sequences in the 3' UTRs of cellular mRNAs. If the miRNA guide sequence is fully complementary to its target site (left pathway), it triggers site-specific cleavage and degradation of the mRNA through the catalytic domain of AGO2. If the base-pairing is incomplete (right pathway) but includes pairing of the seed region (nucleotides 2–8 of the miRNA) with the target, translational inhibition occurs, and this can be accompanied by non-sequence-specific degradation of the mRNA in P bodies. b, Similarly to miRNAs, artificially transcribed shRNAs (in this case from a plasmid) are transported to the cytoplasm by XPO5. The dsRNA in the cytoplasm is recognized and processed by Dicer into ~21–25-nucleotide siRNA fragments that are loaded into the RISC. The siRNAs can target complementary sequences of cellular mRNAs and trigger their degradation through AGO2-mediated cleavage. c, When siRNAs are present in the nucleus and are complementary to promoter regions, they can trigger chromatin remodelling and histone modifications that result in transcriptional gene silencing. In mammalian cells, the details of this mechanism are still under investigation but are known to include Argonaute-family proteins. Accessory proteins indicated in the figure are TRBP (HIV tar-RNA-binding protein; also known as TRBP2P) and PACT (activator of protein kinase PKR; also known as PRKRA). m7G, 7-methylguanosine. Reprinted by permission from Macmillan Publishers Ltd, Daniela Castanotto and John J. Rossi Nature.; © 2009 (10)

Steps of siRNA technology

1. Trypsinize and count cells. Dilute cells in antibiotic-free complete medium to achieve the appropriate plating density. Plate cells into each well of a 96-well plate. Incubate cells at 37°C with 5% CO 2 overnight.

2. Remove culture medium from the wells of the 96-well plate and add the appropriate transfection medium (optimal concentration of siRNA duplex + antibiotic free complete medium + recommended transfection reagents) to each well.

3. Incubate cells at 37°C in 5% CO 2 for 24–48 hours (for mRNA analysis) or 48–96 hours (for protein analysis)

Fig.2 The processing of long dsRNAs, hairpin microRNAs (miRNAs) or plasmid-synthesized short hairpin RNAs (shRNAs) by Dicer (an RNAse III family member) leads to the formation of small interfering RNAs (siRNAs) — 21–23-nucleotide (nt) RNA duplexes with symmetric 2–3-nt 3’ overhangs and 5’- phosphate groups. Exogenously provided synthetic siRNAs are converted into active functional siRNAs by an endogenous kinase that provides 5’-phosphate groups in the presence of adenosine triphosphate (ATP). siRNAs associate with cellular proteins to form an RNA-induced silencing complex (RISC), which contains a helicase that unwinds the duplex siRNA in an ATP-dependent reaction. In an ideal situation the antisense strand guides RISC to the target mRNA for endonucleolytic cleavage. In theory, each of the siRNA strands can be incorporated into RISC and direct RNA interference (RNAi). The antisense strand of an siRNA can direct the cleavage of a corresponding sense RNA target, whereas the sense strand of an siRNA can direct the cleavage of an antisense target. An RNA with a perfect match to a target mRNA behaves like an siRNA and results in mRNA degradation, whereas an RNA with a partial match functions as an miRNA and causes translational repression. Interestingly, recent data show that miRNAs can induce the degradation of fully complementary mRNA targets. ORF, open reading frame. Reprinted by permission from Macmillan Publishers Ltd, V.Mittal, Nat. Rev. Genet.© (2004)(8).

Discussion

RNAi is induced in mammalian cell either by the introduction of synthetic double stranded small interfering RNAs (siRNAs) or by plasmid and viral vector system that express double stranded short hairpin RNAs (shRNAs). These RNAs are subsequently processed to activated siRNA-RISC complex by the cellular machinery. A vital assumption in this approach is that the knockdown of a targeted gene is specific both at mRNA and protein level. Specific gene silencing promises the potential to harness human genome data to elucidate gene function, identify drug targets and develop more specific therapeutics (11,14).

As reviewed by R.K.M. Leung and P.A. Whittaker in 2005, a careful selection of sequences is needed to maximize gene silencing and minimize off target and nonspecific effect. An siRNA duplex (thoughts to have laser like specificity requiring near identity between the siRNA and the target mRNA) may target more than one mRNA molecules because of sequence homologies and mismatches (11). Off-target effects were first described by Jackson and co-workers in 2003. As stated by them in 2004 “The current challenge facing the use of RNA interference (RNAi) to define gene function is the need to combine high specificity with high efficacy. Ineffective siRNAs can lead to false negative conclusions, whereas non-specific siRNAs can produce false-positive conclusions regarding the role of the target gene in functional assays”. Thus RNAi based experiments can have less sensitivity due to partial suppression of gene expression or a lack of specificity due to suppression of nonspecific targets (12). Using genome-wide microarray, A.L. Jackson et al identified modest, 1.5 to 4 fold changes in the expression of dozens of genes following transfection of individual siRNA specifically designed to target two different genes, MAPK14 and IGF1R. Transcriptional profling revealed that each of the siRNAs produced a distinct pattern of effects on transcription. 8 siRNAs duplex which were designed to target MAPK14 produced a distinct expression pattern, likewise each of the 16 siRNAs duplex designed to target IGF1R produced a unique expression pattern. They found that number of off-target genes did not correlate with the extent of target silencing, and the off-target effects could not be eliminated by decreasing the concentration of siRNAs. Further, no single siRNA concentration could be found that maintained full target gene silencing while reducing off-target silencing. They reasoned that the expressions of nontarget genes were suppressed due to cross-hybridization of transcripts containing regions of partial homology with the siRNA sequence. They also reported that many of the genes which lacked any substantial sequence similarity to the siRNA were also regulated. First time they found that, the off-target gene silencing was directed by the antisense strand of the siRNA and also for few siRNA off-target gene silencing appeared to be directed by the sense strand, suggesting that both the sense and antisense strands (Guide strand) of siRNA duplex can contribute to transcript silencing (13). It was in contradiction to earlier views that the antisense strand (Guide strand) only directs RISC to complementary mRNA while the second strand (sense strand) is degraded. In 2004 V. Mittal has likewise stated that “The antisense strand of an siRNA can direct the cleavage of a corresponding sense RNA target, whereas the sense strand of an siRNA can direct the cleavage of an antisense target”(8).

Similarly, in 2003 P.C. Scacheri et al investigated the specificity of siRNA–mediated gene silencing by transfecting 10 different siRNAs corresponding to a single gene (MEN1). Unexpectedly, they detected significant and divergent changes in the level of p53 and p21 after transfecting with 10 different MEN1 siRNA in parallel. They reported that the observed effect on p53 and p21 were not related to MEN1 silencing and did not define a functional relationship between MEN1 and either p53 or p21. They further found that titration of the siRNA reduced the silencing of the MEN1 target, but did not completely abolished the effect on p53 and p21 induced by some of siRNAs. These off-target effects were also independent of lipid agent used for transfecting siRNA. They further reported that significant changes in p53/p21 were not limited to HeLa cells only and these changes in expression were also observed in CaSki, SiHa, and MCF7 cells. Together they suggested that siRNA can induce nonspecific but sequence dependent effects by acting on other unknown targets (14).

In 2004, S.P. Persengiev et al reported that after transfection with 200 nm luciferase siRNA under standard conditions, out of the 33,000 genes represented on an Affymetrix U133 chip, the expression of 1154 genes increased and the expression of 689 genes decreased by = 2.5-fold compared with untreated cells. By RT-PCR analysis, they further confirmed expression level of twelve genes which were either increased or decreased by luciferase siRNA and reported that treatment of cells with the transfection reagent alone did not affect expression of any of the 12 genes analyzed, indicating the effect were attributable to luciferase siRNA sequence. Further, they reported that the nonspecific effects on gene expression were dependent upon siRNA concentration in a gene-specific manner (15). In a similar study by C. Tschuch et al in 2008 showed that even an siRNA (distributed by several companies and widely used for RNAi experiments as negative control) directed against exogenous GFP, resulted in deregulation of 397 genes as compared to mock transfection. They reported, 48% (190 genes) were upregulated and 52% (207 genes) downregulated stating that a large number of transcripts were deregulated due to secondary indirect effects. In out of 207 downregulated transcripts, they found only 50 mRNAs with an 8 mer homology to sense and 88 to the antisensnse GFP siRNA. Thus 33.33% of downregulated transcripts did not show any homology to both sense and antisense transcripts. They also found that these off-target effects specifically increased with increasing amounts of transfected GFP siRNA in different cell lines. Thus, their studies showed that siRNA molecules that are commercially distributed and widely used as negative controls actually can target various endogenous genes which have important roles in several biological pathways (16). The off-target effects observed by C. Tschuch et al and S.P. Persengiev et al, can not be explained by off-target regulation because the siRNAs used in their experiments lacked significant sequence similarity to any human gene.

As stated by A.L. Jackson et al in 2006, “A high proportion of unintended transcripts silenced by siRNAs showed 3’ UTR sequence complementarity to the seed region of the siRNA. Base mismatches within the siRNA seed region reduced the set of original off-target transcripts but generated new sets of silenced transcripts with sequence complementarity to the mismatched seed sequence”(17). In 2006, the work of A. Birmingham et al has shed more light on off targeting. They suggested that a majority of experimentally verified off targets have a 6–7 nucleotide match to the siRNA in the so-called “seed” region, thus the overall identity makes little or no contribution to determining whether the expression of a particular gene will be affected by a given siRNA and off-targeting is associated with the presence of one or more perfect 3’UTR matches within the hexamer or a heptamer seed region. Using microarray technology, they generated a database of experimentally validated off-targeted genes from the expression signatures of HeLa cells transfected with one of twelve different siRNAs targeting three different genes. They reported 347 off-targeted genes after transfection of cells with 12 different siRNA. In silico methods predicted off-target typically exceeded the number identified by microarray analysis resulting in a false positive rate of over 99% at the 79% identity cutoff. In their study the number of predicted off-targets represent more than one third of number of mRNAs in the human genome. Further comparison shows only 23 of the 347 experimentally validated off-targets were identified by in silico methods (at 79% identity cut off) which represented a false negative rate of approximately 93%. Higher cutoffs produced similarly poor overlap between experimental and in silico target prediction. Finally author has concluded that “as sheer numbers of genes that contains matches with any given siRNA seed region is very large in comparison to number of actual off-targets for that siRNA, the value of the identified parameter (by itself) is limited. The identification of additional factors that have roles in off-targeting will likely lead to development of predictive algorithms that minimize off-targets and enhance siRNA design.” (18)

For identification of additional factor which may have roles in off-targeting, I will first discuss the work of N.A. Tchurikov et al and Yi Shuang Liu et al. In 2000, N.A. Tchurikov first time reported that in contrast to gene-specific silencing observed by either expression or transfection of antiparallel double stranded RNA, potent and specific gene silencing in bacteria can be induced by expression of parallel RNA that is complementary in parallel orientation to target mRNA. Further, they reported that expression of parallel RNA was found to be more effective at producing interference than an expression of antisense RNA corresponding to the same mRNA region. Their findings strongly suggested that both parallel and antiparallel RNAs are mediators of a potent and specific repression of the target gene expression (19). In 2000, N.A. Tchurikov published an article entitled “Generation of Kruppel phenocopies by injecting into Drosophila embryos RNA complementary to mRNA in parallel orientation” in Russian language, English translation of abstract available on NCBI Pubmed is as follow “RNA preparations synthesized in vitro were used to study the influence of RNA interference on the Kruppel gene activity in Drosophila embryos. RNA complementary in parallel orientation to the mRNA fragment proved to induce the development of Kruppel phenocopies. The data obtained indicate that mechanisms of specific regulation of gene activity exist in Drosophila cells, which are sensitive to the formation of both parallel and antiparallel RNA-RNA duplexes that include mRNA of the corresponding gene” (20). Similarly, In 2009 Liu Y. S. et al has shown that parallel complementary RNA could induce gene specific silencing in Pseudomonas aeruginosa. They found that the efflux function of Mex AB-OprM was inhibited in Pseudomonas aeruginosa cell expressing pRNA (parallel RNA) of mexA gene (21).

Fig. 3 Broader picture of siRNA technology: see text for details.

So considering work of N.A. Tchurikov et al and Y.S. Liu et al, we now have a broader picture of siRNA technology. Both the strands of siRNA duplex have the possibility to be incorporated in RISC complex. Both of the strands of siRNA duplex have the possibility to bind to different mRNA

(A)      (B)

Table 1 (A & B) shows an example showing BLAST alignment results obtained after taking two guide strand sequence specific for BRCA1 and p53 gene. We can see the ratio of Target versus Off Target mRNA in case of ‘Guide RNA sequence 1’ is 1:21 while the ratio of Target versus Off-Target mRNA in case of ‘Guide RNA sequence 2’ is 1:15.

sequence in parallel and antiparallel orientation (Figure 3). Thus, I hypothesize that one of the possible additional behind off-targeting might be binding of guide RNA sequence in parallel orientation to any off-target mRNA sequence and similarly off-targeting might be possible due to binding of sense strand to off-target mRNA sequence in a parallel orientation. Mismatch binding both in parallel and antiparallel orientation, mediated by both ‘Guide’ RNA and ‘Sense’ RNA will also lead to off-targeting by RISC (Figure 2). Table 1 (A & B) shows two hypothetical examples that how many genes will be targeted by two different siRNA due to parallel and antiparallel binding of both sense and guide RNA mediated gene silencing by RISC as detected by the BLAST tool available at the NCBI website.

Now a further question arises, can we theoretically minimize nonspecific binding of siRNA duplex so that we can use it for therapeutic purpose? I hypothesize it can be done by following ways

1. We know that a mirror repeat sequence as discussed in the first chapter of this book has an equal possibility to bind in both parallel and antiparallel orientations. An siRNA duplex containing a mirror repeat sequence will have lesser chance of, off-targeting as both the sense and guide strand will recognize the same target sequence both in parallel and antiparallel orientation, (Figure 4, Table 2). It will minimize off-targeting which might have happened due to parallel binding of sense and guide RNA in other cases. The mirror repeat sequence can be searched easily in any gene using FPCB strategy as developed by us in 2013 (22). Further, if we can find out technologies which allows specific degradation of sense strand of siRNA duplex after RISC/RNA complex formation it will further reduce nonspecific binding by siRNA duplex.

2. An siRNA duplex containing palindrome sequence formed by intermolecular annealing of one RNA sequence will also reduce off-targeting. These palindromic siRNA duplex has full dyad symmetry so that both strands will enter with equal probability in RISC/RNA complex formation. After unwinding of palindromic siRNA duplex only one type of RISC/RNA complex will be generated (Figure 5). In 2006, M. Hossbach et al has observed specific inhibition of lamin A/C mRNA and protein synthesis using partially palindromic siRNAs against lamin A/C mRNA (23).

Fig.4 siRNA having mirror repeat sequence: An siRNA duplex having mirror repeat sequence will give rise to the RNA / RISC complex, which will have equal probabilities in binding to same target sequence both in parallel and antiparallel orientation. It will minimize off-targeting effects due to binding of any RNA/RISC complex in parallel orientation.

(A)        (B)

Table 2 (A & B) shows an example showing BLAST alignment results obtained after taking two guide strand sequence (having mirror repeat sequence) specific for BRCA1 and p53 gene. We can see the ratio of ‘Target’ versus ‘Off Target’ mRNA in case of ‘Guide RNA sequence 3’ is 1:9 while the ratio of Target versus Off-Target mRNA in case of ‘Guide RNA sequence 4’ is 1:4.

Fig.5 Palindromic siRNA: An siRNA duplex having palindromic sequence will give rise to only one type of active RISC/RNA complex. These types of siRNA will also reduce cost of experiment as palindromic siRNA would only require the synthesis of a single siRNA strand.

3. An siRNA duplex containing both palindrome sequence and mirror repeat sequence (PM siRNA) will also further minimize nonspecific priming as it will generate only one type of RISC/RNA complex, which will have possibility to bind a target sequence, once both in parallel and antiparallel orientation. These types of siRNA will have a much higher potentialities to be used for therapeutic purpose (Figure 6).

Fig.6 Palindromic mirror siRNA (PM siRNA) A hypothetical example showing siRNA duplex having both palindromic and mirror repeat sequence. ‘PM’ siRNA will give rise to only one type of active RISC/RNA complex, which will have an equal probability in binding a target sequence both in parallel and antiparallel orientation.

“The scientist is not a person who gives the right answers, he’s one who asks the right questions.”

— C.L.Strauss

“The best scientist is open to experience and begins with romance – the idea that anything is possible”.

— Ray Bradbury

“It is a good morning exercise for a research scientist to discard a pet hypothesis every day before breakfast. It keeps him young”.

— Konrad Lorenz

“In science the important thing is to modify and change one’s ideas as science advances”.

— Herbert Spencer

“The history of science shows that theories are perishable. With every new truth that is revealed we get a better understanding of Nature and our conceptions and views are modified.”

— Nikola Tesla

“No amount of experimentation can ever prove me right; a single experiment can prove me wrong.”

— Albert Einstein

“Science, like life, feeds on its own decay. New facts burst old rules; then newly divined conceptions bind old and new together into a reconciling law.”

— William James

Bibliography

1. Overview of RNAi & requirements for a typical RNAi experiment(http://www.lifetechnologies.com/in/en/home/references/ambion-tech-support/rnai-sirna/tech-notes/rnai-how-to-for-new-users.html.)

2. Sen, G. L. & Blau, H. M. A brief history of RNAi : the silence of the genes. FASEB J. 20, 1293–99 (2006).

3. Napoli, C., Lemieux, C. & Jorgensen, R. Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans. Plant Cell 2, 279–289 (1990).

4. Bruce Nash, RNA Interference: Turning Genes Off at Will Part I DNA Learning Center, Cold Spring Harbor Laboratory.

5. Fire, A., Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811 (1998).

6. Elbashir, S. M., Lendeckel, W. & Tuschl, T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15, 188–200 (2001).

7. Caplen, N. J., Parrish, S., Imani, F., Fire, A. & Morgan, R. A. Specific inhibition of gene expression by small double- stranded RNAs in invertebrate and vertebrate systems . Proc. Natl Acad. Sci. USA. 98, 9742–9747 (2001)

8. Mittal, V. Improving the efficiency of RNA interference in mammals. Nat. Rev. Genet. 5, 355–65 (2004).

9. Castanotto, D. & Rossi, J. J. The promises and pitfalls of RNA-interference-based therapeutics. Nature 457, 426–33 (2009).

11. Leung, R. K. M. & Whittaker, P. A. RNA interference : from gene silencing to gene-specific therapeutics. Pharmocology Ther. 107, 222–239 (2005).

12. Jackson, A. L. & Linsley, P. S. Noise amidst the silence: off-target effects of siRNAs? Trends Genet. 20, 521–4 (2004).

13. Jackson, A. L., Bartz, S. R., Schelter, J., Kobayashi, S. V., Julja, B., Mao, M., Li, B., Cavet, G., Linsley, P. S. off-target gene regulation by RNAi. Nat. Biotechnol. 21, 635–638 (2003).

14. Scacheri, P. C., Rozenblatt-Rosen, O., Caplen, N. J., Wolfsberg, T. G., Umayam, L., Lee, J.C. Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells. Proc. Natl. Acad. Sci. U. S. A. 101, 1892–7 (2004).

15. Persengiev, S. P., Zhu, X. & Green, M. R. Nonspecific, concentration-dependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs). RNA 10, 12–18 (2004).

16. Tschuch, C., Schulz, A., Pscherer, A., Werft, W., Benner, A., Hotz-Wagenblatt, A., Barrionuevo, L. S. Lichter, P., Mertens, D., Off-target effects of siRNA specific for GFP. BMC Mol. Biol. 9, 60 (2008).

17. Jackson, A. L., Burchard, J., Schelter, J., Chau, B. N., Cleary, M., Lim, L., Linsley, P. S. Widespread siRNA ‘off-target ’ transcript silencing mediated by seed region sequence complementarity. RNA 12, 1179–1187 (2006).

18. Birmingham, A., Anderson, E. M., Reynolds, A., Ilsley-tyree, D., Leake, D., Fedorov, Y., Baskerville, S., Maksimova, E., Robinson, K., Karpilow, J., Marshall, W. S., Khvorova, A. 3’ UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nat. Methods 3, 199–204 (2006).

19. Tchurikov, N. A., Chistyakova, L. G., Zavilgelsky, G. B., Manukhov, I. V., Chernov, B. K., Golova, Y. B. Gene-specific silencing by expression of parallel complementary RNA in Escherichia coli. J. Biol. Chem. 275, 26523–9 (2000).

20. Tchurikov N. A., Shostak N.G., Okladnova O.V., Chernov B.K. Generation of Kruppel phenocopies by injecting into Drosophila embryos RNA complementary to mRNA in parallel orientation Genetika. 36(2):149-54 (2000).

21. Liu, Y. S., Zhang, Y. Q., Yang, L. X., Yao, T. J. & Xiao, C. L. Gene-specific silencing induced by parallel complementary RNA in Pseudomonas aeruginosa. Biotechnol. Lett. 31, 1571–1575 (2009).

22. Bhardwaj V.; Gupta S.; Meena S.; Sharma K. FPCB : a simple and swift strategy for mirror repeat identification. eprint arXiv:1312.3869 (2013).

23. Hossbach, M., Gruber, J., Osborn, M., Weber, K. & Tuschl, T. Gene Silencing with siRNA Duplexes Composed of Target-mRNA-Complementary and Partially Palindromic or Partially Complementary Single-Stranded siRNAs. Landes Biosci. 82–89 (2006).

Chapter 5

Perspective

***

Our fundamental knowledge of various reactions happening in biological system is largely based on B-form of DNA. This structure has been successfully employed in describing replication, transcription and other biochemical process occurring within a cell. Many of molecular biology tools has been developed based on the information about B-form of antiparallel complementary DNA. These molecular biology tools has their own limitations and various scientist has described various reasons behind these errors. In this book, I have put hypothesis that one of the additional reason behind these errors is parallel complementary pairing of two strands of DNA. Even though I have explained only four preliminary techniques, but this principle can be applied to other molecular biology techniques also. The principle of antiparallel complementary binding of oligonucleotide primers is used to amplify a double stranded DNA in PCR. To increase the specificity of a PCR reaction, oligonucleotide primers pairs should not be complementary (antiparallel) otherwise it may lead to formation of primer dimer. The annealing of oligonucleotide primers at nonspecific position within a DNA sequence can also lead amplification of wrong product. I suggest that this mispriming and primer dimer formation may also happen due to binding of oligonucleotide primers in a parallel complementary manner also. So its a requirement to develop bioinformatics tool which can design and check oligonucleotide primers based on both parallel and antiparallel complementary DNA structure. One may simply suggest that these mispriming reaction due to parallel complementary binding in PCR and other hybridization reaction [ Southern, Northern and colony hybridization] can be simply omitted by increasing the hybridization/annealing temperature. Its true, but we can’t increase the specificity of Antisense RNA technology, SiRNA technology just by putting the transfected cells or animals at higher temperature. After transfection cells are generally put at 37°C and at this lower temperature complementary RNA can target binding in both parallel and antiparallel manner to specific and nonspecific sites. So its a requirement to develop bioinformatics tools which can design specific probes/primers based on parallel and antiparallel hybridization of DNA which can hybridize to specific targets at lower temperature.

Another molecular biology tool is generation of knockout mouse or knockout cell which is helpful in studying the function of various genes. The knockout technology is also based on homologus base pairing. Few of literature has shown off targeting effect of knock out technology. Various knock out technologies claims specific knock out of a single gene but in best of my knowledge there is no genome sequence available of any knockout animals. Only whole genome sequenicing can confirms that only single gene has been taken out and others are not. Various knock out technology can be improved by designing ‘homologus sequence’ in considering parallel and antiparallel complementary binding within DNA.

Another commonly used tool is deduction of nucleotide sequence of DNA based on Sanger method. This technology also use a specific primer which binds to template DNA in antiparallel complementary manner. The mispriming by primer in parallel complementary manner to template DNA can also lead to wrong results. As this method has been used in deduction of various genomes, so it is necessary to recheck if any errors has been incorporated in deducing nucleotide sequence of these genomes due to parallel complementary binding of primers.

In conclusion, I can say that various molecular biology techniques which have been developed and used for collecting data based on complementary binding of either probe or primer should be readdressed so that if by chance an error has been incorporated due to parallel complementary binding must be resolved.

“We know very little, and yet it is astonishing that we know so much, and still more astonishing that so little knowledge can give us so much power.”

— Bertrand Russell

“Science is based on experiment, on a willingness to challenge old dogma, on an openness to see the universe as it really is. Accordingly, science sometimes requires courage – at the very least the courage to question the conventional wisdom.”

— Carl Sagan

“As long as scientists are free to pursue the truth wherever it may lead, there will be a flow of new scientific knowledge to those who can apply it to practical problems.”

— Vannevar Bush

As long as men are free to ask what they must, free to say what they think, free to think what they will, freedom can never be lost and science can never regress.

— Marcel Proust

“Science, my lad, is made up of mistakes, but they are mistakes which it is useful to make, because they lead little by little to the truth.”

— Jules Verne

“There are three stages in scientific discovery. First, people deny that it is true, then they deny that it is important; finally they credit the wrong person.”

— Bill Bryso

Glossary

***

Acid: A substance that can donate proton.

Agrose: Linear carbohydrates polymers made by red algae, that fprm a loose mesh.

Alllele: One member of a pair (or any of the series) of genes occupying a specific spot on a chromosome (called locus) that controls the same trait.

Alternative splicing: The tissue specific pattern of splicing of a given pre-mRNA that result in variations in the excision and retention of exons and introns.

Antiparallel: Running in opposite direction.

Antisense: The non-coding DNA strand of a gene. A cell uses antisense DNA strand as a template for producing messenger RNA (mRNA) that directs the synthesis of a protein. Antisense can also refer to a method for silencing genes. To silence a target gene, a second gene is introduced that produces an mRNA complementary to that produced from the target gene. These two mRNAs can interact to form a double-stranded structure that can not be used to direct protein synthesis.

Autoradiography: The process where by the radioactivity of isotopes is detected by photographic film applied on to the isotope labelled probe.

B-form DNA (B-DNA): The canonical right-handed double helical secondary structure assumed by bulk DNA

Bioinformatics: The study of biological information in the form of molecular sequences and structures.

C2’-endo: A ribose confirmation in which C2’ is displaced toward the same side of the ring as C5’.

C3’-endo: A ribose conformation in which C3’ is displaced towards the same side of the ring as C5’.

Cell: It is the basic building block of living things. All cells can be sorted into one of two groups: eukaryotes and prokaryotes. A eukaryote has a nucleus and membrane-bound organelles, while a prokaryote does not. Plants and animals are made of numerous eukaryotic cells, while many microbes, such as bacteria, consist of single cells. An adult human body is estimated to contain between 10 and 100 trillion cells.

Chiral centre: An atom whose substitution are arranged such that is not superimposable on its mirror image.

Chromatography: A technique for separating the components of a mixture of molecules based on their partition between a mobile solvent phase and a stationary phase.

Circular dichroism: This method measures the difference in absorption of right and left circularly polarized light as it passes through a solution containing molecules that absorb at that wavelength. The circular-dichroism spectrum is plotted as a function of wavelength.

Complementary base pairs: These are defined by the pairing reactions in double helical nucleic acids (A with T in DNA or with U in mRNA and C with G).

Complementary DNA (cDNA): A DNA molecule usually synthesized by the action of reverse transcriptase, that is complementary to an mRNA molecule.

Correlation Coefficient: A correlation coefficient is a number between -1 and 1 which measures the degree to which two variables are linearly related. If there is perfect linear relationship with positive slope between the two variables, we have a correlation coefficient of 1; if there is positive correlation, whenever one variable has a high (low) value, so does the other. If there is a perfect linear relationship with negative slope between the two variables, we have a correlation coefficient of -1; if there is negative correlation, whenever one variable has a high (low) value, the other has a low (high) value. A correlation coefficient of 0 means that there is no linear relationship between the variables.

Cruciforms DNA: Four-armed DNA secondary structures, similar to Holliday junctions, that can form at inverted repeat sequences and are stabilized by DNA supercoiling.

DNA fingerprinting: It is a laboratory technique used to establish a link between biological evidence and a suspect in a criminal investigation. A DNA sample taken from a crime scene is compared with a DNA sample from a suspect. If the two DNA profiles are a match, then the evidence came from that suspect. Conversely, if the two DNA profiles do not match, then the evidence cannot have come from the suspect. DNA fingerprinting is also used to establish paternity.

Electrophoresis: It is a laboratory technique used to separate DNA, RNA, or protein molecules based on their size and electrical charge. An electric current is used to move molecules to be separated through a gel. Pores in the gel work like a sieve, allowing smaller molecules to move faster than larger molecules. The conditions used during electrophoresis can be adjusted to separate molecules in a desired size range.

Epigenetics: It is an emerging field of science that studies heritable changes caused by the activation and deactivation of genes without any change in the underlying DNA sequence of the organism. The word epigenetics is of Greek origin and literally means over and above (epi) the genome.

Gene expression: The decoding, via transcription and translation of the information contained in a gene to yield a functional RNA or protein product.

Gene knockout: A genetic engineering process that deletes or inactivates a specific gene in an animal.

Gene: The basic physical unit of inheritance is gene. Genes are passed from parents to offspring and contain the information needed to specify traits. Genes are arranged, one after another, on structures called chromosomes. A chromosome contains a single, long DNA molecule, only a portion of which corresponds to a single gene. Humans have approximately 20,000 genes arranged on their chromosomes.

Genome: All of the DNA found within each of the cells of an organism. Eukaryotic genomes can be subdivided into their nuclear genome (chromosomes found within the nucleus) and their mitochondrial genome.

Genomic DNA: DNA derived from chromosomes.

Guide strand: The strand of the siRNA or microRNA that is complementary to the target mRNA sequence(s).

Hoogsteen base pair: It is a variation of base-pairing in nucleic acids such as the A·T pair. In this manner, two nucleobases, one on each strand, can be held together by hydrogen bonds in the major groove. A Hoogsteen base pair applies the N7 position of the purine base (as a hydrogen bond acceptor) and C6 amino group (as a donor), which bind the Watson-Crick (N3–N4) face of the pyrimidine base.

Human Genome Project: This was an international project that mapped and sequenced the entire human genome. Completed in April 2003, data from the project are freely available to researchers and others interested in genetics and human health.

Hybridization: The process of combining two complementary single-stranded DNA or RNA molecules and allowing them to form a single double-stranded molecule through base pairing. In a reversal of this process, a double-stranded DNA (or RNA, or DNA/RNA) molecule can be heated to break the base pairing and separate the two strands. Hybridization is a part of many important laboratory techniques such as polymerase chain reaction and Southern blotting.

Hydrogen bond : A largely electrostatic interactions between a weekly acidic donor group such as O-H or N-H and a weekly basic acceptor atom such as O or N.

i-motif: A structure composed of two parallel stranded duplexes held together in an antiparallel orientation. The structure is stabilized by hemiprotonated C:C+ base pairs.

In situ: In place

In vitro: in the laboratory

In vivo: In a living organism

Knockout: Typically refers to an organism that has been genetically engineered to lack one or more specific genes. Scientists create knockouts (often in mice) so that they can study the impact of the missing genes and learn something about the genes’ function.

Lymphocyte: A type of white blood cell that is part of the immune system. There are two main types of lymphocytes: B cells and T cells. The B cells produce antibodies that are used to attack invading bacteria, viruses, and toxins. The T cells destroy the body’s own cells that have themselves been taken over by viruses or become cancerous

Major groove: The groove on a DNA double helix onto which the glycosidic bonds of a base pair form an angle of > 180°. In B-DNA , this groove is wider than the minor groove.

Microarray technology: It is a developing technology used to study the expression of many genes at once. It involves placing thousands of gene sequences in known locations on a glass slide called a gene chip. A sample containing DNA or RNA is placed in contact with the gene chip. Complementary base pairing between the sample and the gene sequences on the chip produces light that is measured. Areas on the chip producing light identify genes that are expressed in the sample.

Minor groove: The groove on a DNA double helix onto which the glycosidic bonds of a base pair form an angle of < 180°. In B-DNA , this groove is narrower than the minor groove..

miRNA (Micro RNA): A mature endogenous small double stranded RNA , often with imperfect pairing of the two strand, originating from processing of a precursor RNA molecule by cellular RNases. miRNAs target RISC complex to sites that have imperfect sequence complementarity and that are typically located in the 3’unttranslated region of the mRNA

Mutation: It is a change in a DNA sequence. Mutations can result from DNA copying mistakes made during cell division, exposure to ionizing radiation, exposure to chemicals called mutagens, or infection by viruses. Germ line mutations occur in the eggs and sperm and can be passed on to offspring, while somatic mutations occur in body cells and are not passed on.

Northern blot: is a laboratory technique used to detect a specific RNA sequence in a blood or tissue sample. The sample RNA molecules are separated by size using gel electrophoresis. The RNA fragments are transferred out of the gel to the surface of a membrane. The membrane is exposed to a DNA probe labeled with a radioactive or chemical tag. If the probe binds to the membrane, then the complementary RNA sequence is present in the sample.

Nuclease: An enzyme that hydrolytically degrades nucleic acids.

Nucleic acid: It is an important class of macromolecules found in all cells and viruses. The functions of nucleic acids have to do with the storage and expression of genetic information. Deoxyribonucleic acid (DNA) encodes the information the cell needs to make proteins. A related type of nucleic acid, called ribonucleic acid (RNA), comes in different molecular forms that participate in protein synthesis.

Nucleotide: The basic building block of nucleic acids. RNA and DNA are polymers made of long chains of nucleotides. A nucleotide consists of a sugar molecule (either ribose in RNA or deoxyribose in DNA) attached to a phosphate group and a nitrogen-containing base. The bases used in DNA are adenine (A), cytosine ©, guanine (G), and thymine (T). In RNA, the base uracil (U) takes the place of thymine.

Nucleus: It is a membrane-bound organelle that contains the cell’s chromosomes. Pores in the nuclear membrane allow for the passage of molecules in and out of the nucleus.

Palindrome: A sequence of double stranded DNA that is same when one strand is read left to right or its complement is read right to left; consists of adjacent inverted repeats.

Parallel: Running in same direction.

Peptide: One or more amino acids linked by chemical bonds. The term also refers to the type of chemical bond that joins the amino acids together. A series of linked amino acids is a polypeptide. The cell’s proteins are made from one or more polypeptides.

Phenotype: An individual’s observable traits, such as height, eye color, and blood type. The genetic contribution to the phenotype is called the genotype. Some traits are largely determined by the genotype, while other traits are largely determined by environmental factors.

Pitch: The number of base pairs per complete turn of the DNA helix.

Polymerase chain reaction (PCR): It is a laboratory technique used to amplify DNA sequences. The method involves using short DNA sequences called primers to select the portion of the genome to be amplified. The temperature of the sample is repeatedly raised and lowered to help a DNA replication enzyme copy the target DNA sequence. The technique can produce a billion copies of the target sequence in just a few hours.

Polymorphism: It involves one of two or more variants of a particular DNA sequence. The most common type of polymorphism involves variation at a single base pair. Polymorphisms can also be much larger in size and involve long stretches of DNA. Called a single nucleotide polymorphism, or SNP (pronounced snip), scientists are studying how SNPs in the human genome correlate with disease, drug response, and other phenotypes.

Primer: It is a short, single-stranded DNA sequence used in the polymerase chain reaction (PCR) technique. In the PCR method, a pair of primers is used to hybridize with the sample DNA and define the region of the DNA that will be amplified. Primers are also referred to as oligonucleotides.

Probe: A single-stranded sequence of DNA or RNA used to search for its complementary sequence in a sample genome. The probe is placed into contact with the sample under conditions that allow the probe sequence to hybridize with its complementary sequence. The probe is labeled with a radioactive or chemical tag that allows its binding to be visualized. In a similar way, labeled antibodies are used to probe a sample for the presence of a specific protein.

Propeller twist: Rotation of one base with respect to the other in the same base pair.

Restriction enzyme: It is an enzyme isolated from bacteria that cuts DNA molecules at specific sequences. The isolation of these enzymes was critical to the development of recombinant DNA (rDNA) technology and genetic engineering.

Ribonucleic acid (RNA): It is a molecule similar to DNA. Unlike DNA, RNA is single-stranded. An RNA strand has a backbone made of alternating sugar (ribose) and phosphate groups. Attached to each sugar is one of four bases—adenine (A), uracil (U), cytosine ©, or guanine (G). Different types of RNA exist in the cell: messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA). More recently, some small RNAs have been found to be involved in regulating gene expression.

RISC: The RNA Induced Silencing complexes are multiprotein complexes containing an Argonaute protein , a small single stranded RNA and additional regulatory proteins.

Rise: Displacement along the DNA helix axis

RNA interference : A cellular process that downregulates gene expression at a post- transcriptional step. One strand of a small double stranded RNA ( Si RNA or miRNA ) integrates in a protein complex called RISC and targets an mRNA with sequence complementarity for cleavage, translation inhibition and/or mRNA decay.

RNAi off-target effect: Any effect of an RNAi based treatment that is a consequence of reducing the expression of an unintended target.

Short interfering RNA (siRNA): An endogenous or artificial(synthetic) small double-strand RNA with perfect pairing of the two strands. One strand (called the guide strand) targets RISC to perfectly complementary mRNAs, resulting in endonucleolytic cleavage of the mRNA by the RISC component Ago2.

Single Nucleotide Polymorphism: A single base difference in the genomes of two individuals, such differences occur every 1250 base pairs on average in human genome.

siRNA or miRNA seed sequence: The 5= region of the guide strand of an siRNA or miRNA sequence, extending from nucleotides 2-7 (hexamer) or 2-8 (heptamer).

Southern blotting: It is a laboratory technique used to detect a specific DNA sequence in a blood or tissue sample. A restriction enzyme is used to cut a sample of DNA into fragments that are separated using gel electrophoresis. The DNA fragments are transferred out of the gel to the surface of a membrane. The membrane is exposed to a DNA probe labeled with a radioactive or chemical tag. If the probe binds to the membrane, then the probe sequence is present in the sample.

Standard Deviation: Standard deviation is a measure of the spread or dispersion of a set of data. The more widely the values are spread out, the larger the standard deviation

Tertiary structure: The entire three dimensional structure of a single chain polymer, including that of its side chain.

Transfer RNA (tRNA): is a small RNA molecule that participates in protein synthesis. Each tRNA molecule has two important areas: a trinucleotide region called the anticodon and a region for attaching a specific amino acid. During translation, each time an amino acid is added to the growing chain, a tRNA molecule forms base pairs with its complementary sequence on the messenger RNA (mRNA) molecule, ensuring that the appropriate amino acid is inserted into the protein.

Triplex DNA: Three-stranded DNA in which single-stranded DNA hydrogen bonds into the major groove of purine-rich standard B-form DNA

Twist: Rotation around the DNA helix axis.

Vector: It is any vehicle, often a virus or a plasmid that is used to ferry a desired DNA sequence into a host cell as part of a molecular cloning procedure. Depending on the purpose of the cloning procedure, the vector may assist in multiplying, isolating, or expressing the foreign DNA insert

Fig.23 PD-PCR for details see text and Bhardwaj V et al 2013 (61)

| p. S.No. | p. Gene/CDS | p. No of MRs | p. Mirror repeats (MRs) | | p. 1 | p. P53 | p. 3 | p. 1.CCAAAGAAGAAACC

2.GGAACTCAAGG

3.ACCTGAAGTCCA | |
p. 2 |
p. TNF-alpha |
p. 3 |
p. 1.CCTCATCTACTCC

2.CCAGAGGGAGACC

3.CCTCATCTACTCC | |
p. 3 |
p. Tgf-beta |
p. 5 |
p. 1.TGTGAGGGGGAGTGT

2.CGCCCGCGCCCGC

3.GCTACCACCATCG

4.CCGACTTCAGCC

5.GTCCGGGCCTG | |
p. 4 |
p. MTOR |
p. 4 |
p. 1.CGCCACCACCGC

2.ACCTTCTTCTTCCA

3.ACTACAAACATCA

4.AGAAGAAGAAGA | |
p. 5 |
p. BRCA1 |
p. 5 |
p. 1.AAGAGAAGAAAAAGAAGAGAA

2.AAATGAACAGACAAGTAAA

3.TGGATTCAAACTTAGGT

4.AAAGATAATAGAAA

5.AAACCGTGCCAAA | |
p. 6 |
p. NF-kappa-B

[] |
p. 6 |
p. 1.CTGGGAGAGGGTC

2.AAAGTTATTGAAA

3.AAGAACAAGAA

4.GGAGGCGGAGG

5.AACGTATGCAA

6.GACAGTGACAG | |
p. 7 |
p. COX2 |
p. 3 |
p. 1.CGCGGTCCTGGCGC

2.ACAACTATCAACA

3.TGCAATAACGT | |
p. 8 |
p. HIF1A |
p. 7 |
p. 1.AAAACAGTGACAAAA

2.TTCAGCACGACTT

3.GAAGACACAGAAG

4.GAAAAGAAAAG

5.CAGATTTAGAC

6.CCCTATATCCC

7.ACATTATTACA |

Table (1) List of mirror repeats observed in various Human mRNA sequence. The sequence shown in red is mirror image of sequence shown in blue. At a mirror sequence, DNA has potentialities to hybridize in both parallel and antiparallel orientations. With increase in number of Mirror repeats within a gene, its potentialities to hybridize in parallel orientation also increase. For more details see Bhardwaj V. et al 2013.(62)

Fig.25 A and B model of parallel stranded DNA , C and D model of parallel DNA ( PDB ID-1JUU) and (E) diagrammatic representation of Parallel stranded DNA. (64, 65)

Fig.4 Non-specific hybridization by pre-hybrization buffer (A) Single stranded DNA being transferred on a membrane is treated with prehybridization buffer which contain nonspecific DNA to block unused DNA binding sites (B) Nonspecific DNA may also hybridize with test DNA and probe thus limiting signal from specific target DNA. The cross hybridization at nonspecific sites can also give nonspecific signals (D).

Fig.6 Different possibilities of complementary hybridization of a target sequence to immobilized probes on a microarray slides. (1) shows ‘No-hybridization’ (2) shows hybridization may be blocked due to binding of Nonspecific DNA sample to probe during prehybridization step. (3) Perfect antiparallel complementary Watson-Crick base pairing and hybridization of specific target DNA sequence to specific probe. (4) antiparallel complementary mismatch hybridization of target sample to nonspecific probe (5) Partial antiparallel complementary hybridization of target sequence to a nonspecific probe (6) Parallel complementary mismatch hybridization of target sequence to nonspecific probe (7) Partial Parallel complementary hybridization of test sequence to a nonspecific probe (8) Perfect parallel complementary hybridization of target sequence to a nonspecific probe (9) Perfect parallel complementary hybridization of target sequence to a specific probe. It can happen when probe sequence consist of unique mirror sequence.

Fig.2 Microarray technology: mRNA is isolated from two samples and cDNA is synthesized with incorporation of fluorescent labels (Cy3 for Red and Cy5 for Green). Labelled cDNAs are mixed and hybridized to microarray slides. Red spot represents gene which are expressed in control sample while green spots represents genes expressed in test sample. Yellow spots gives information about genes which are expressed in both samples.

Fig.3 Biological Variation: mRNA is isolated from two control samples and cDNA is synthesized with incorporation of fluorescent labels (Cy3 for Red and Cy5 for Green). Labelled cDNA are mixed and hybridized to microarray slides. Yellow spots gives information about the genes which are expressed in both control samples. Red and green spot indicates biological variation in these two control samples.

Fig.4 Technical Variation mRNA is isolated from one control sample and cDNA is synthesized in duplicates with incorporation of fluorescent labels (Cy3 for Red and Cy5 for Green). Labelled cDNA samples are mixed and hybridized to microarray slides. Red and green spot indicates technical variation. The similar experiment when compared with another microarray slides can give more information about technical variation.


Science behind Non-Specific Science

The cover page of my book shows factsheets data available on the W.H.O. website. We can see, millions of people have died and many millions more will die in the coming future due to various diseases. Throughout the world, trillions of dollars are being invested to find solutions to various diseases and many more trillions of dollars will be invested in the coming future. All over the world scientists do perform experiments using well established protocols with or without minor modification as per their experimental approach. In this book, I have discussed a possible hypothesis behind ‘Non-specific results’ obtained by four techniques (Southern blotting, Northern blotting, Microarray, siRNA technology) which are widely used in molecular biology research. I have also tried to give a better hypothetical solution which can minimize experimental errors. I will introduce you to a broader definition of “Complementary” in DNA structure (which has never been discussed in standard biology books), a novel PD-PCR technology developed by me and presence of novel mirror repeat sequences within most of human genes. Hopefully knowledge within this book will be helpful in developing various molecular biology techniques which will not show any experimental error. Doing scientific experiments without any error will only solve the sufferings of mankind. After reading my book, feel to answer my only question, “Over a period of time, have we collected valid scientific data to be used by coming generation of young scientists in molecular biology research?”

  • ISBN: 9781370314447
  • Author: Vikash Bhardwaj
  • Published: 2016-08-13 08:35:20
  • Words: 28073
Science behind Non-Specific Science Science behind Non-Specific Science