RNA Comparison with DNA

RNA and DNA differ in three main ways. First, unlike DNA which is double-stranded, RNA is a single-stranded molecule in most of its biological roles and has a much shorter chain of nucleotides. Second, while DNA contains deoxyribose, RNA contains ribose, (there is no hydroxyl group attached to the pentose ring in the 2' position in DNA). These hydroxyl groups make RNA less stable than DNA because it is more prone to hydrolysis. Third, the complementary nucleotide to adenine is not thymine, as it is in DNA, but rather uracil, which is an unmethylated form of thymine.^[13]

The 50S ribosomal subunit. RNA is in orange, protein in blue. The active site is in the middle (red).

Like DNA, most biologically active RNAs including tRNA, rRNA, snRNAs and other, non-coding, RNAs are extensively base paired to form double stranded helices. Structural analysis of these RNAs have revealed that they are highly structured. Unlike DNA, this structure is not long double-stranded helices but rather collections of short helices packed together into structures akin to proteins. In this fashion, RNAs can achieve chemical catalysis, like enzymes.^[14] For instance, determination of the structure of the ribosome—an enzyme that catalyzes peptide bond formation—revealed that its active site is composed entirely of RNA.

http://en.wikipedia.org/wiki/RNA

RNA and DNA Editing: Molecular Mechanisms and Their Integration into Biological Systems

Latest discoveries in RNA and DNA editing research, with expert forecasts of the field's future directions

RNA and DNA Editing assembles a team of leading experts who present the latestdiscoveries in the field alongside the latest models and methodology. In addition,the authors set forth the many open questions and suggest routes for further investigation. Overall, the book serves as a practical guide for professionals in the field who need to understand the interrelationship of RNA and DNA editing with other chemical and biological processes.

This book is divided into four sections, providing a clear, contextual map of the field:

Diversification of the Proteome through RNA and DNA Editing discusses the occurrence of editing sites within transcriptomes and their distribution within individual RNAs in order to explore why nucleic acid sequences are edited instead of encoded genomically

Functional Coordination of RNA Editing with Other Cellular Mechanisms brings to the forefront why RNA and DNA editing is essential for cell survival and adaptation

Predictive Studies underscores the power of computational approaches in identifying novel editing sites and predicting the biological consequences of editing at these sites

Structural Approaches presents comparative structural analyses that suggest conserved protein folds and implicate, in some instances, their ancient phylogenic origins as components of editing machinery

This book not only discusses the current state of research in depth, it also gives new contributors an opportunity to express their vision. The perspectives voiced by these authors are provocative and intended to motivate discussion and inspire new experiments. Finally, this book will promote new hypotheses and models that can serve as springboards for the next generation of discoveries in the field.

About the Author

Harold C. Smith, PhD, is Professor in the Department of Biochemistry and Biophysics at the University of Rochester and the founder and Chief Scientific Officerof OyaGen, a biotech company that develops drugs that target editing enzymes. Dr. Smith organized the first Gordon Research Conference on RNA Editing in 1997 and holds four patents.

http://www.amazon.com/RNA-DNA-Editing-Mechanisms-Integration/dp/0470109912/ref=sr_1_3?ie=UTF8&s=books&qid=1207895995&sr=1-3

DNA-RNA-Protein

DNA carries the genetic information of a cell and consists of thousands of genes. Each gene serves as a recipe on how to build a protein molecule. Proteins perform important tasks for the cell functions or serve as building blocks. The flow of information from the genes determines the protein composition and thereby the functions of the cell.

The DNA is situated in the nucleus, organized into chromosomes. Every cell must contain the genetic information and the DNA is therefore duplicated before a cell divides (replication). When proteins are needed, the corresponding genes are transcribed into RNA (transcription). The RNA is first processed so that non-coding parts are removed (processing) and is then transported out of the nucleus (transport). Outside the nucleus, the proteins are built based upon the code in the RNA (translation).

http://nobelprize.org/educational_games/medicine/dna/index.html

Potent stimulation of transcription-coupled DNA supercoiling by sequence-specific DNA-binding proteins

Transcription by RNA polymerase can stimulate localized DNA supercoiling in Escherichia coli. In vivo, there is extensive experimental support for a "twin-domain" model in which positive DNA supercoils are generated ahead of a translocating RNA polymerase complex and negative supercoils are formed behind it. Negative supercoils accumulate in the template DNA because the positive supercoils are preferentially removed by cellular topoisomerase action. Yet, in vitro, clear and convincing support for the twin-domain mechanism has been lacking. In this article, we reconcile this inconsistency by showing that, in a defined in vitro system with plasmid DNA templates, a variety of sequence-specific DNA-binding proteins, such as the bacteriophage O replication initiator or the E. coli lactose or galactose repressors, strikingly stimulate transcription-coupled DNA supercoiling. We demonstrate further that this stimulation requires the presence in the DNA template of a recognition sequence for the relevant DNA-binding protein and depends on the production of long RNA chains by an RNA polymerase. Our data are most consistent with a model in which specific DNA-binding proteins facilitate a twin-domain mechanism to enhance DNA supercoiling during transcription. More precisely, we suggest that some nucleoprotein complexes, perhaps those that contain sharply bent DNA, can form barriers that impede the diffusion and merger of independent chromosomal supercoil domains. Localization of DNA supercoils by nucleoprotein complexes may serve as a general mechanism for modulating DNA transactions that are sensitive to DNA superhelicity.

http://www.pnas.org/cgi/content/abstract/99/14/9139

Crucial Role for DNA Supercoiling in Mu Transposition

DNA supercoiling plays an indispensable role in an early step of bacteriophage Mu transposition. This step involves formation of a nucleoprotein complex in which the Mu ends synapse and undergo two concerted single-strand cleavages. We describe a kinetic analysis of the role of supercoiling in the Mu-end synapsis reaction as measured by the cleavage assay. We observe a dependence of the reaction rate on superhelical density as well as on the length of Mu donor plasmid DNA. The reaction has a high activation enthalpy (67 kcal/mol). These results imply that the free energy of supercoiling is used directly to lower the activation barrier of the rate-limiting step of the reaction. Only the free energy of supercoiling associated with DNA outside the Mu ends appears to be utilized, implying that the Mu ends come together before the supercoiling energy is used. Our results suggest an essential function for the bacterial sequences attached to the ends of Mu virion DNA.

http://www.pnas.org/cgi/content/abstract/91/2/699

Supercoiling

What’s knotty about DNA? Under an electron microscope DNA looks like a long thin "knotted" strand that is tightly packed inside the cell nucleus. To visualize this, imagine packing 200 km of fishing line inside a basketball without tangling it! Amazingly, the cells in your body do the equivalent of this by supercoiling the DNA. Supercoiling is a very smart form of compact storage that allows for easy manipulation.

To illustrate supercoiling, take a long elastic band, cut it, hold one end tight and twist the other end as many times as possible (about 100 times!). Now without untwisting the elastic band, bring the ends together. You will end up with a supercoiled band, see diagram. When you bring the two end pieces together the elastic band tries to unwind, by untwisting about the centreline. However, this is not possible because you are still holding the ends, so it compromises by writhing around in space (like a well used phone cord).

The mathematical formula Lk=Tw+Wr can be used to describe this process. Lk, the linking number, represents the number of times one strand winds around the other, Tw is the twist or the amount of rotation about the centre line and Wr, the writhe, describes how hard it is to straighten out the curve. When the curve is straightened out the writhe, Wr, is zero and the twist, Tw, is high. You can feel the elastic band trying to untwist. When the elastic band is relaxed it supercoils. The twist Tw is now very small and the writhe Wr is high.

Supercoiling allows for easy manipulation and so easy access to the information coded in the DNA. When a cell is copying a DNA strand it will uncoil a strand, copy it and then recoil it. In order to obtain a more workable interpretation of the stresses in the DNA, David Stump and Peter Watson in the Mathematics Department of the University of Queensland have obtained mathematical formulas for the Twist and Writhe depending on the length of the strand and the angle beta (see figure). This then gives (through the formula above) the Linking number or the number of times one strand winds around the other.These results can then be used to explain the pictures, taken by an electron microscope, of the tiny strands of DNA coiling and uncoiling

http://www.maths.uq.edu.au/~infinity/Infinity7/supercoiling.html

DNA Supercoiling as a Pattern for Understanding Psycho-social Twistedness

The review here of twistedness in DNA provides a technical basis for the discussion in the main paper (Engaging with Questions of Higher Order: cognitive vigilance required for higher degrees of twistedness, 2004).

The insights in the main paper regarding "twistedness" reflect an intuitive understanding of complexity which calls for deeper insight to understand how twistedness works and why it may be vitally important in some psycho-social processes -- as well as being highly problematic in others. Part of the difficulty in approaching this matter is that "twistedness" is in most cases used unthinkingly as a pejorative term to characterize a pattern which is felt to inhibit right-thinking and clarity. The argument here is that, given its importance at every scale in nature, from the organization of nebula to the organization of the human cell, there is a case for distinguishing various forms of twistedness and understanding their function. This could be especially valuable to reconciling apparently irreconcilable understandings in society.

The merit of focusing on the nature and function of twisting in DNA is that it provides a rich natural template. It offers a sense of the degree of complexity that it may be required to master in order to comprehend how twistedness "works" in practice. It might also be argued that, as a process active in every human body and inherent to human life, humans may well have some kind of profound intuitive understanding of how it works and the "rightness" of such working. Some of the very explicit dynamics of this process may also offer patterns for understanding how the inhibiting effects of "twistedness" may be addressed when they are perceived to be a constraint on human development.

Understanding of how DNA works has been much enriched by concepts from topology -- as a branch of mathematics that deals with structural properties that are unchanged by deformations such as stretching and bending. This use of mathematics is especially important because there is no experimental way to observe the dynamics of enzymatic action directly, notably with respect to knotting and coiling of DNA (see De Witt Sumners. Lifting the Curtain: Using Topology to Probe the Hidden Action of Enzymes, 1995; Xiaoyan R. Bao, et al. Behavior of Complex Knots in Single DNA Molecules, 2003).

http://www.laetusinpraesens.org/docs00s/dnahelix.php are very long and thin. There is over a metre of DNA in every human cell in a space of some 0.0006 centimetres diametre. If DNA were constrained to be linear it would not fit into a cell. It must therefore fold many times to fit within the confines of a cell. The DNA is composed of 10** base pairs. This density of packing results in tangles and knots in the DNA that are essential to enable the cell to divide (involving transcription and replication).

http://www.laetusinpraesens.org/docs00s/dnahelix.php

DNA supercoil

In a "relaxed" double-helical segment of DNA, the two strands twist around the helical axis once every 10.4 base pairs of sequence. Adding or subtracting twists, as some enzymes can do, imposes strain. If a DNA segment under twist strain were to be closed into a circle by joining its two ends and then it is allowed to move freely, the circular DNA would contort into new shape, such as a simple figure-eight. Such a contortion is a supercoil.

The simple figure eight is the simplest supercoil, and is the shape a circular DNA assumes to accommodate one too many or one too few helical twists. The two lobes of the figure eight will appear rotated either clockwise or counterclockwise with respect to one another, depending on whether the helix is over or underwound. For each additional helical twist being accommodated, the lobes will show one more rotation about their axis.

The noun form "supercoil" is rarely used in the context of DNA topology. Instead, global contortions of a circular DNA, such as the rotation of the figure-eight lobes above, are referred to as writhe. The above example illustrates that twist and writhe are interconvertible. "Supercoiling" is an abstract mathematical property, and represents the sum of twist and writhe. The twist is the number of helical turns in the DNA and the writhe is the number of times the double helix crosses over on itself (these are the supercoils). The relationship of twist, writhe and supercoiling is expressed as the equation:

S = T + W.

Extra helical twists are positive and lead to positive supercoiling, while subtractive twisting causes negative supercoiling. Many topoisomerase enzymes sense supercoiling and either generate or dissipate it as they change DNA topology. DNA of most organisms is negatively supercoiled.

In part because chromosomes may be very large, segments in the middle may act as if their ends are anchored. As a result, they may be unable to distribute excess twist to the rest of the chromosome or to absorb twist to recover from underwinding--the segments may become supercoiled, in other words. In response to supercoiling, they will assume an amount of writhe, just as if their ends were joined.

Supercoiled DNA forms two structures; a plectoneme or a toroid, or a combination of both. A negatively supercoiled DNA molecule will produce either a one-start left-handed helix, the toroid, or a two-start right-handed helix with terminal loops, the plectoneme. Plectonemes are typically more common in nature, and this is the shape most bacterial plasmids will take. For larger molecules it is common for hybrid structures to form - a loop on a toroid can extend into a plectoneme. If all the loops on a toroid extend then it becomes a branch point in the plectonemic structure.

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Supercoiled structure of circular DNA molecules with low writhe. Note that the helical nature of the DNA duplex is omitted for clarity.


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Supercoiled structure of linear DNA molecules with constrained ends. Note that the helical nature of the DNA duplex is omitted for clarity


http://en.wikipedia.org/wiki/DNA_supercoil

Biotechnology a sudden new biological revolution

Biotechnology seems to be leading a sudden new biological revolution. It has brought us to the brink of a world of "engineered" products that are based in the natural world rather than on chemical and industrial processes.

Biotechnology has been described as "Janus-faced." This implies that there are two sides. On one, techniques allow DNA to be manipulated to move genes from one organism to another. On the other, it involves relatively new technologies whose consequences are untested and should be met with caution. The term "biotechnology" was coined in 1919 by Karl Ereky, an Hungarian engineer. At that time, the term meant all the lines of work by which products are produced from raw materials with the aid of living organisms. Ereky envisioned a biochemical age similar to the stone and iron ages.

A common misconception among teachers is the thought that biotechnology includes only DNA and genetic engineering. To keep students abreast of current knowledge, teachers sometimes have emphasized the techniques of DNA science as the "end-and-all" of biotechnology. This trend has also led to a misunderstanding in the general population. Biotechnology is NOT new. Man has been manipulating living things to solve problems and improve his way of life for millennia. Early agriculture concentrated on producing food. Plants and animals were selectively bred, and microorganisms were used to make food items such as beverages, cheese, and bread.

The late eighteenth century and the beginning of the nineteenth century saw the advent of vaccinations, crop rotation involving leguminous crops, and animal drawn machinery. The end of the nineteenth century was a milestone of biology. Microorganisms were discovered, Mendel's work on genetics was accomplished, and institutes for investigating fermentation and other microbial processes were established by Koch, Pasteur, and Lister.

Biotechnology at the beginning of the twentieth century began to bring industry and agriculture together. During World War I, fermentation processes were developed that produced acetone from starch and paint solvents for the rapidly growing automobile industry. Work in the 1930s was geared toward using surplus agricultural products to supply industry instead of imports or petrochemicals. The advent of World War II brought the manufacture of penicillin. The biotechnical focus moved to pharmaceuticals. The "cold war" years were dominated by work with microorganisms in preparation for biological warfare, as well as antibiotics and fermentation processes.

Biotechnology is currently being used in many areas including agriculture, bioremediation, food processing, and energy production. DNA fingerprinting is becoming a common practice in forensics. Similar techniques were used recently to identify the bones of the last Czar of Russia and several members of his family. Production of insulin and other medicines is accomplished through cloning of vectors that now carry the chosen gene. Immunoassays are used not only in medicine for drug level and pregnancy testing, but also by farmers to aid in detection of unsafe levels of pesticides, herbicides, and toxins on crops and in animal products. These assays also provide rapid field tests for industrial chemicals in ground water, sediment, and soil. In agriculture, genetic engineering is being used to produce plants that are resistant to insects, weeds, and plant diseases.

A current agricultural controversy involves the tomato. A recent article in the New Yorker magazine compared the discovery of the edible tomato that came about by early biotechnology with the new "Flavr-Savr" tomato brought about through modern techniques. In the very near future, you will be given the opportunity to bite into the Flavr-Savr tomato, the first food created by the use of recombinant DNA technology ever to go on sale.

What will you think as you raise the tomato to your mouth? Will you hesitate? This moment may be for you as it was for Robert Gibbon Johnson in 1820 on the steps of the courthouse in Salem, New Jersey. Prior to this moment, the tomato was widely believed to be poisonous. As a large crowd watched, Johnson consumed two tomatoes and changed forever the human-tomato relationship. Since that time, man has sought to produce the supermarket tomato with that "backyard flavor." Americans also want that tomato available year-round.

New biotechnological techniques have permitted scientists to manipulate desired traits. Prior to the advancement of the methods of recombinant DNA, scientists were limited to the techniques of their time - cross-pollination, selective breeding, pesticides, and herbicides. Today's biotechnology has its "roots" in chemistry, physics, and biology . The explosion in techniques has resulted in three major branches of biotechnology: genetic engineering, diagnostic techniques, and cell/tissue techniques.

http://www.accessexcellence.org/RC/AB/BC/Overview_and_Brief_History.html

Other DNA

Protein-coding sequences (specifically, coding exons) comprise less than 1.5% of the human genome.^[3] Aside from genes and known regulatory sequences, the human genome contains vast regions of DNA the function of which, if any, remains unknown. These regions in fact comprise the vast majority, by some estimates 97%, of the human genome size. Much of this is composed of:

repeat elements

• Tandem repeats
  • Satellite DNA
  • Minisatellite
  • Microsatellite
• Interspersed repeats
  • SINEs
  • LINEs

transposons

• Retrotransposons
  • LTR
    • Ty1-copia
    • Ty3-gypsy
  • Non-LTR
    • SINEs
    • LINEs
• DNA Transposons

pseudogenes

However, there is also a large amount of sequence that does not fall under any known classification.

Much of this sequence may be an evolutionary artifact that serves no present-day purpose, and these regions are sometimes collectively referred to as "junk" DNA. There are, however, a variety of emerging indications that many sequences within are likely to function in ways that are not fully understood. Recent experiments using microarrays have revealed that a substantial fraction of non-genic DNA is in fact transcribed into RNA,^[7] which leads to the possibility that the resulting transcripts may have some unknown function. Also, the evolutionary conservation across the mammalian genomes of much more sequence than can be explained by protein-coding regions indicates that many, and perhaps most, functional elements in the genome remain unknown.^[8] The investigation of the vast quantity of sequence information in the human genome whose function remains unknown is currently a major avenue of scientific inquiry.^[9]

http://en.wikipedia.org/wiki/Human_genome

Chemical differences between DNA & RNA

Both RNA and DNA are composed of repeated units. The repeating units of RNA are ribonucleotide monophosphates and of DNA are 2'-deoxyribonucleotide monophosphates.

Both RNA and DNA form long, unbranched polynucleotide chains in which different purine or pyrimidine bases are joined by N-glycosidic bonds to a repeating sugar-phosphate backbone.

The chains have a polarity. The sequence of a nucleic acid is customarily read from 5' to 3'. For example the sequence of the RNA molecule is AUGC and of the DNA molecule is ATGC

The base sequence carries the information, i.e. the sequence ATGC has different information that AGCT even though the same bases are involved.

Consequences of RNA/DNA chemistry

The DNA backbone is more stable, especially to alkaline conditions. The 2' OH on the RNA forms 2'3'phosphodiester intermediates under basic conditions which breaks down to a mix of 2' and 3' nucleoside monophosphates. Therefore, the RNA polynucleotide is unstable.

The 2' deoxyribose allows the sugar to assume a lower energy conformation in the backbone. This helps to increase the stability of DNA polynucleotides. The following link shows 3-D models of the DNA and RNA nucleotides.

Cytidine deamination to Uridine can be detected in DNA but not RNA because deamination of Cytidine in DNA leads to Uridine not Thymidine. Uridine bases in DNA are removed by a specific set of DNA repair enzymes and replaced with cytidine bases.

http://www.biochem.uwo.ca/meds/medna/NAchem.html

STRUCTURE AND FUNCTION OF RNA

RNA is structurally similar to DNA!

Both nucleic acids are sugar-phosphate polymers and both have nitrogen bases attached to the sugars of the backbone- but there are several important differences.

• They differ in composition:

1. The sugar in RNA is ribose, not the deoxyribose in DNA (as we previously learned).
2. The base uracil is present in RNA instead of thymine.
   

• They also differ in size and structure:

1. RNA molecules are smaller (shorter) than DNA molecules,
2. RNA is single-stranded, not double-stranded like DNA.
   

• Another difference between RNA and DNA is in function. DNA has only one function-STORING GENETIC INFORMATION in its sequence of nucleotide bases. But there are three main kinds of ribonucleic acid, each of which has a specific job to do.

1. Ribosomal RNAs-exist outside the nucleus in the cytoplasm of a cell in structures called ribosomes. Ribosomes are small, granular structures where protein synthesis takes place. Each ribosome is a complex consisting of about 60% ribosomal RNA (rRNA) and 40% protein.
2. Messenger RNAs-are the nucleic acids that "record" information from DNA in the cell nucleus and carry it to the ribosomes and are known as messenger RNAs (mRNA).
3. Transfer RNAs-The function of transfer RNAs (tRNA) is to deliver amino acids one by one to protein chains growing at ribosomes.

http://ncc.gmu.edu/dna/rna.htm

What is the difference between DNA and RNA?

DNA and RNA Introduction

Introduction:

The nucleic acids are informational molecules because their primary structure contains a code or set of directions by which they can duplicate themselves and guide the synthesis of proteins. The synthesis of proteins - most of which are enzymes - ultimately governs the metabolic activities of the cell. In 1953, Watson, an American biologist, and Crick, an English biologist, proposed the double helix structure for DNA. This development set the stage for a new and continuing era of chemical and biological investigation. The two main events in the life of a cell - dividing to make exact copies of themselves, and manufacturing proteins - both rely on blueprints coded in our genes.

There are two types of nucleic acids which are polymers found in all living cells. Deoxyribonucleic Acid (DNA) is found mainly in the nucleus of the cell, while Ribonucleic Acid (RNA) is found mainly in the cytoplasm of the cell although it is usually synthesized in the nucleus. DNA contains the genetic codes to make RNA and the RNA in turn then contains the codes for the primary sequence of amino acids to make proteins.

Nucleic Acid Parts List:

The best way to understand the structures of DNA and RNA is to identify and examine individual parts of the structures first. The complete hydrolysis of nucleic acids yields three major classes of compounds: pentose sugars, phosphates, and heterocyclic amines (or bases).

Phosphate: A major requirement of all living things is a suitable source of phosphorus. One of the major uses for phosphorus is as the phosphate ion which is incorporated into DNA and RNA.

Pentose Sugars:

There are two types of pentose sugars found in nucleic acids. This difference is reflected in their names--deoxyribonucleic acid indicates the presence of deoxyribose; while ribonucleic acid indicates the presence of ribose.

In the graphic on the left, the structures of both ribose and deoxyribose are shown. Note the red -OH on one and the red -H on the other are the only differences. The alpha and beta designations are interchangeable and are not a significant difference between the two.

Heterocyclic Amines:

Heterocyclic amines are sometimes called nitrogen bases or simply bases. The heterocyclic amines are derived from two root structures: purines or pyrimidines. The purine root has both a six and a five member ring; the pyrimidine has a single six member ring.

There are two major purines, adenine (A) and guanine (G), and three major pyrimidines, cytosine (C), uracil (U), and thymine (T). The structures are shown in the graphic on the left. As you can see, these structures are called "bases" because the amine groups as part of the ring or as a side chain have a basic property in water.

A major difference between DNA and RNA is that DNA contains thymine, but not uracil, while RNA contains uracil but not thymine. The other three heterocyclic amines, adenine, guanine, and cytosine are found in both DNA and RNA. For convenience, you may remember, the list of heterocyclic amines in DNA by the words: The Amazing Gene Code (TAGC).

http://www.elmhurst.edu/~chm/vchembook/580DNA.html

DNA vs. RNA

What's the difference between DNA and RNA?

Both DNA and RNA are composed of repeating units of nucleotides. Each
nucleotide consists of a sugar, a phosphate and a nucleic acid base.
The sugar in DNA is deoxyribose. The sugar in RNA is ribose, the same
as deoxyribose but with one more OH (oxygen-hydrogen atom combination
called a hydroxyl). This is the biggest difference between DNA and RNA.
Another difference is that RNA molecules can have a much greater variety
of nucleic acid bases. DNA has mostly just 4 different bases with a few
extra occasionally.  The difference in these bases (between DNA and RNA)
allows RNA molecules to assume a wide variety of shapes and also  many
different functions. DNA, on the other hand, serves as a set of directions
and that's about all (but that's absolutely necessary!). ---DrPam

http://www.newton.dep.anl.gov/askasci/bio99/bio99410.htm

DNA fingerprinting methods

DNA fingerprinting begins by extracting DNA from the cells in a sample of blood, saliva, semen, or other appropriate fluid or tissue.

RFLP analysis

Main article: Restriction fragment length polymorphism

The first methods used for DNA fingerprinting involved restriction enzyme digestion, followed by Southern blot analysis. Although polymorphisms can exist in the restriction enzyme cleavage sites, more commonly the enzymes and DNA probes were used to analyze VNTR loci. However, the Southern blot technique is laborious, and requires large amounts of undegraded sample DNA. Also, Jeffreys' original technique looked at many minisatellite loci at the same time, increasing the observed variablitiy, but making it hard to discern individual alleles (and thereby precluding parental testing). These early techniques have been supplanted by PCR-based assays.

PCR analysis

Main article: Polymerase chain reaction

With the invention of the polymerase chain reaction (PCR), DNA fingerprinting took huge strides forward in both discriminating power and the ability to recover information from very small (or degraded) starting samples. PCR greatly amplifies the amounts of a specific region of DNA, using oligonucleotide primers and a thermostable DNA polymerase. Early assays such as the HLA-DQ alpha reverse dot blot strips grew to be very popular due to their ease of use, and the speed with which a result could be obtained. However they were not as discriminating as RFLP. It was also difficult to determine a DNA profile for mixed samples, such as a vaginal swab from a sexual assault victim.

Fortunately, the PCR method is readily adaptable for analyzing VNTR loci. In the U.S.A the FBI has standardized a set of 13 VNTR assays for DNA typing, and has organized the CODIS database for forensic identification in criminal cases. Similar assays and databases have been set up in other countries. Also, commercial kits are available that analyze Single Nucleotide Polymorphisms (SNPs). These kits use PCR to amplify specific regions with known variations and hybridize them to probes anchored on cards, which results in a colored spot corresponding to the particular sequence variation.

STR analysis

Main article: Short tandem repeats

The most prevalent method of DNA fingerprinting used today is based on PCR and uses short tandem repeats (STR). This method uses highly polymorphic regions that have short repeated sequences of DNA (the most common is 4 bases repeated, but there are other lengths in use, including 3 and 5 bases). Because different people have different numbers of repeat units, these regions of DNA can be used to discriminate between individuals. These STR loci (locations) are targeted with sequence-specific primers and are amplified using PCR. The DNA fragments that result are then separated and detected using electrophoresis. There are two common methods of separation and detection, capillary electrophoresis (CE) and gel electrophoresis.

The polymorphisms displayed at each STR region are by themselves very common, typically each polymorphism will be shared by around 5 - 20% of individuals. When looking at multiple loci, it is the unique combinations of these polymorphisms to an individual that makes this method discriminating as an identification tool. The more STR regions that are tested in an individual the more discriminating the test becomes.

From country to country different STR based DNA profiling systems are in use. In North America systems which amplify the CODIS 13 core loci are almost universal, while in the UK the SGM+ system, which is compatible with The National DNA Database in use. Whichever system is used, many of the STR regions under test are the same. These DNA profiling systems are based around multiplex reactions, whereby many STR regions will be under test at the same time.

Capillary electrophoresis works by electrokinetically (movement through the application of an electric field) injecting the DNA fragments into a thin glass tube (the capillary) filled with polymer. The DNA is pulled through the tube by the application of an electric field, separating the fragments such that the smaller fragments travel faster through the capillary. The fragments are then detected using fluorescent dyes that were attached to the primers used in PCR. This allows multiple fragments to be amplified and run simultaneously, something known as multiplexing. Sizes are assigned using labeled DNA size standards that are added to each sample, and the number of repeats are determined by comparing the size to an allelic ladder, a sample that contains all of the common possible repeat sizes. Although this method is expensive, larger capacity machines with higher throughput are being used to lower the cost/sample and reduce backlogs that exist in many government crime facilities.

Gel electrophoresis acts using similar principles as CE, but instead of using a capillary, a large polyacrylamide gel is used to separate the DNA fragments. An electric field is applied, as in CE, but instead of running all of the samples by a detector, the smallest fragments are run close to the bottom of the gel and the entire gel is scanned into a computer. This produces an image showing all of the bands corresponding to different repeat sizes and the allelic ladder. This approach does not require the use of size standards, since the allelic ladder is run alongside the samples and serves this purpose. Visualization can either be through the use of fluorescently tagged dyes in the primers or by silver staining the gel prior to scanning. Although it is cost effective and can be rather high throughput, silver staining kits for STRs are being discontinued. In addition, many labs are phasing out gels in favor of CE as the cost of machines becomes more manageable.

The true power of STR analysis is in its statistical power of discrimination. In the U.S.A., there are 13 core loci (DNA locations) that are currently used for discrimination in CODIS. Because these loci are independently assorted (having a certain number of repeats at one locus doesn't change the likelihood of having any number of repeats at any other locus), the product rule for probabilities can be applied. This means that if someone has the DNA type of ABC, where the three loci were independent, we can say that the probability of having that DNA type is the probability of having type A times the probability of having type B times the probability of having type C. This has resulted in the ability to generate match probabilities of 1 in a quintillion (1 with 18 zeros after it) or more.

AmpFLP

Main article: Amplified fragment length polymorphism

Another technique, AmpFLP, or amplified fragment length polymorphism was also put into practice during the early 1990s. This technique was also faster than RFLP analysis and used PCR to amplify DNA samples. It relied on variable number tandem repeat (VNTR) polymorphisms to distinguish various alleles, which were separated on a polyacrylamide gel using an allelic ladder (as opposed to a molecular weight ladder). Bands could be visualized by silver staining the gel. One popular locus for fingerprinting was the D1S80 locus. As with all PCR based methods, highly degraded DNA or very small amounts of DNA may cause allelic dropout (causing a mistake in thinking a heterozygote is a homozygote) or other stochastic effects. In addition, because the analysis is done on a gel, very high number repeats may bunch together at the top of the gel, making it difficult to resolve. AmpFLP analysis can be highly automated, and allows for easy creation of phylogenetic trees based on comparing individual samples of DNA. Due to its relatively low cost and ease of set-up and operation, AmpFLP remains popular in lower income countries.

Y-chromosome analysis

Recent innovations have included the creation of primers targeting polymorphic regions on the Y-chromosome (Y-STR), which allows resolution of multiple male profiles, or cases in which a differential extraction is not possible. Y-chromosomes are paternally inherited, so Y-STR analysis can help in the identification of paternally related males. Y-STR analysis was performed in the Sally Hemings controversy to determine if Thomas Jefferson had sired a son with one of his slaves.

Mitochondrial analysis

Main article: Mitochondrial DNA

For highly degraded samples, it is sometimes impossible to get a complete profile of the 13 CODIS STRs. In these situations, mitochondrial DNA (mtDNA) is sometimes typed due to there being many copies of mtDNA in a cell, while there may only be 1-2 copies of the nuclear DNA. Forensic scientists amplify the HV1 and HV2 regions of the mtDNA, then sequence each region and compare single nucleotide differences to a reference. Because mtDNA is maternally inherited, directly linked maternal relatives can be used as match references, such as one's maternal grandmother's sister's son. A difference of two or more nucleotides is generally considered to be an exclusion. Heteroplasmy and poly-C differences may throw off straight sequence comparisons, so some expertise on the part of the analyst is required. mtDNA is useful in determining unclear identities, such as those of missing persons when a maternally linked relative can be found. mtDNA testing was used in determining that Anna Anderson was not the Russian princess she had claimed to be, Anastasia Romanov.

mtDNA can be obtained from such material as hair shafts and old bones/teeth.

National DNA databases

The United States maintains the largest DNA database in the world: The Combined DNA Index System, with over 4.5 million records as of 2007. The United Kingdom maintains the National DNA Database (NDNAD), which is of similar size. The size of this database, and its rate of growth, is giving concern to civil liberties groups in the UKSDLF, where police have wide-ranging powers to take samples and retain them even in the event of acquittal.^[1]

The U.S. Patriot Act of the United States provides a means for the U.S. government to get DNA samples from other countires if they are either a division of , or a head office of, a a company operating in the U.S.A. Under the act, the American offices of the compnay can't divulge to their subsidiaries/offices in other countries the reasons that these dna samples are sought or by whom

The Future of Genetic Fingerprinting

The Future of Genetic Fingerprinting - An Interview with Professor Sir Alec Jeffreys

Hotspots, Minisatellites and a Short Cut Through the Human Genome

On Monday morning at 9.05 am on 10 September 1984, Alec Jeffreys, now Professor Sir Alec Jeffreys, FRS, and the Royal Society Wolfson Research Professor in the University of Leicester Department of Genetics, discovered the world's first genetic fingerprint.

As the technique began to solve paternity and immigration cases and to revolutionise criminal investigations, he refined and simplified the process, turning it into what became known as "genetic profiling", producing a pattern of DNA unique to a particular person.

Instrumental to these techniques were ‘minisatellites’, short sequences of chemical building blocks used to chart human DNA instability. Minisatellites show greater variation from one person to the next than most of other DNA material, exhibiting this variation in the numbers of repeat units or stutters.

Now, twenty years on, Sir Alec has moved away from minisatellites, using them as a platform to think about alternative ways of detecting inherited rearrangements in our DNA. He is looking at two genetic processes, mutation (change) and recombination (reshuffling) and their impact on human DNA diversity.

"We appreciated early on that minisatellites were variable because they were unstable," he said, adding: "We faced two problems:

"The first is that DNA changes at extremely low rates. Minisatellites have allowed us to get round that. The second is the smallness of family sizes.

"Even if you have ten children, you will only get at most only one or two minisatellite mutants. We needed families of millions of children, particularly to study other modes of DNA instability, so we started to use minisatellites to find an alternative way of research. For us, a child is simply a complex and expensive way of amplifying DNA from a single sperm and egg. However, using the most sophisticated methods, we found we could type the DNA of a single molecule or cell as an alternative approach to studying inherited DNA rearrangements."

Once able to dispense with children in favour of cells, Sir Alec turned to the study of sperm. “A single ejaculation will produce one hundred million sperm, equivalent to one hundred million offspring. This gives us numbers of progeny that go way beyond mouse or fruit fly production and well into the realm enjoyed by microbial geneticists."

What came as a surprise was that the mutation in minisatellites comes about by abnormal recombination and that mutation and recombination are not different processes in these stuttered regions of DNA. Minisatellites, it seems evolve as parasites in hotspots of crossover activity, propagating themselves through recombination.

Sir Alec said: "That then led us to ask a very simple set of questions:

How recombination events are distributed along human chromosomes;

What sort of processes are going on during human recombination;

How these patterns and processes impact on human diversity.

"Over the past few years we have developed a whole range of technologies to look at the DNA in sperm, searching for ones that show crossover in a given region. Our findings have transformed how people view human recombination. Crossovers are far from randomly formed, but are concentrated in hotspots, in between which are areas dead of activity. It is as if you shuffle a pack of cards with some blocks of cards stuck together so that they don't get reshuffled."

This turned out to be crucial in sparking off a major international project, known as the HapMap Project, to investigate how human DNA diversity is organised in human chromosomes. The project is specifically aimed at identifying the "blocks of cards" that are stuck together and not reshuffled by recombination from generation to generation. These so-called haplotype blocks and their associated recombination hotspots are not just of academic interest - they also hold considerable promise for disease analysis.

"Our recombination work is important for understanding how human DNA diversity is organised, and underpins international efforts into trying to analyse common human diseases," Sir Alec said. "To find a disease gene then you have to find the mutation that predisposes people to that condition. However, if the mutation resides in one of these haplotype blocks then it will tend to follow markers in the block in patients, and thus the problem can be reduced first to finding the relevant block, then to searching within the block for the real mutation. This has changed the way people think about genetic association studies and reduced the cost of scanning the whole human genome. It is a very exciting time."

In trying to understand hotspots, Sir Alec is investigating why they occur where they occur, and to grasp the rules that appear to prevent crossover hotspots from triggering dangerous rearrangements in the genome. "There is something going on in humans that prevents this," he said. "We are also trying to extend the whole concept of using single sperm analysis to look at other processes of DNA instability, for example jumping DNA and single base changes in our chromosomes. We understand very little about these processes and how and where they occur."

In developing new systems to chart the alteration of the DNA sequence, Sir Alec is quietly optimistic that they will work. So far, he says, things are looking good, and there are few else in the world following the same challenging avenues of enquiry.

While the research done by his laboratory team feeds into medical genetics, as well as studies of genome diversity, the genetic analysis of human origins and the work done by Professor Yuri Dubrova on radiation and DNA, it is, in itself, pure research. "We are not specifically looking at applications, though almost inevitably applications will arise" Professor Sir Alec Jeffreys said. "I feel I've done my bit on research application." Police officers and those fighting immigration and paternity cases must surely agree.

AN AWARD WINNER

Professor Sir Alec Jeffreys has been acclaimed across the world for his discovery of DNA profiling and for his continuing work in the field of genetics. Among his most recent awards are:

March 2004: Lifetime Achievement award at the Daily Mirror's Pride of Britain Awards. "This was fantastic because it was public recognition in the broadest sense," Sir Alec said. "It is the nearest I will ever get to an Oscar."

April 2004: Louis-Jeantet Prize for Medicine, awarded to scientists who are distinguished for the highest quality of biomedical research in Europe.

July 2004: Honorary doctorate from the University of Leicester, about which Sir Alec said: "An honorary degree from your own university is something extremely special. It is important because it is recognition from your own community, as with the award of Honorary Freeman of the City of Leicester, which I was awarded in 1993."

http://www2.le.ac.uk/offices/press/media-centre/dna-fingerprinting/geneticsfuture

Genetic fingerprinting

Genetic Fingerprinting (also called DNA testing, DNA typing, or DNA profiling) is a technique used to distinguish between individuals of the same species using only samples of their DNA. Although two individuals will have the vast majority of their DNA sequence in common, DNA profiling exploits highly variable repeat sequences called VNTRs. These loci are variable enough that two unrelated humans are unlikely to have the same alleles. The technique was first reported in 1984 by Dr. Alec Jeffreys at the University of Leicester, and is now the basis of several national DNA identification databases.

Reference samples

DNA identification must be done by an extraction of DNA from substances such as:

• Personal items (e.g. toothbrush, razor, ...)
• Banked samples (e.g. banked sperm or biopsy tissue)
• Blood kin (biological relative)
• Human remains previously identified

Reference samples are often collected using buccal swab.

Variations of VNTR allele lengths in 6 individuals.


http://images.google.com/imgres?imgurl=http://upload.wikimedia.org/wikipedia/en/thumb/2/23/Phylogenetic-Groups.svg/400px-Phylogenetic-Groups.svg.png&imgrefurl=http://tech1207.blogspot.com/2007_12_28_archive.html&h=341&w=400&sz=38&hl=en&start=10&um=1&tbnid

Telomeres

Telomeres, together with the enzyme Telomerase are mentioned a lot lately in association with both aging and with cancer. It seems that their gradual shortening during the lifetime of a cell eventually results in damage to DNA and thus the genes of which they are a part. This has led to a great deal of research into Telomeres and Telomerase, some of which will be outlined in this section of the site. First of all however, let us get some of the basic definitions out of the way.

Definition of a Telomere

A telomere is a region of highly repetitive DNA at the end of a linear chromosome that functions as a disposable buffer. Every time linear eukaryotic chromosomes are replicated during late S-phase the DNA polymerase complex is incapable of replicating all the way to the end of the chromosome; if it were not for telomeres, this would quickly result in the loss of useful genetic information, which is needed to sustain a cell’s activities. http://en.wikipedia.org/wiki/Telomere

Definition of Telomerase

Telomerase is the reverse transcriptase responsible for the extension of telomeric repeat sequences in most species studied. If telomerase activity is diminished or absent, telomeres will shorten. Shortened telomeres appear to lead to cell senescence. Eventually telomeric sequences can shorten to the point where they are not long enough to support the telomere-protein complex protecting the ends and the chromosomes become unstable. These shortened ends become 'sticky' and promote chromosome rearrangements. Some rearrangements may contribute to the development of cancers. http://www.genlink.wustl.edu/teldb/tel.html

Role, Function and Compesition of Telomeres

In the majority of Prokaryotes, the chromosomes are circular. This means that they do not have ends that are prone to damage or ‘premature replication termination.’ Note that a small number of bacteria, Borrelia and Streptomyces for example, do possess linear chromosomes (together with circular plasmids). These chromosomes are very different however, from those which are found in Eukaryotic cells (such as those in humans.

A telomere consists of repeating base sequences (in humans this is a repeating string of TTAGGG bases between 3 to 20 kilobases in length). In addition to the telomere itself, there is a 100-300 kilobase sequence associated with the telomere, which is located between the telomere and the rest of the chromosome.

As replication of DNA occurs during the lifetime of an organism, the telomeres gradually shorten. This shortening eventually results in damage to the chromosomes themselves and to the genes that they contain. The results of this are either a reduction in the cells ability to express its genes and a descent into cellular senescence (cellular dormancy) or in some cases the over or under-expression of specific genes. When this chromosomal damage leads to either an over or under-expression of certain genes, then cellular functionality can be compromised. In some cases the organisms survival can also be compromised as cellular replication can become unchecked. These cells thus become cancerous and can lead to the death of the organism.

Role, Function and Compesition of Telomeres

During DNA replication, The DNA unzips and a complimentary strand is formed against the unzipped sections. Telomeres shorten during this process due to the ‘lagging strand’ phenomenon.

Basically DNA replication does not begin at the end of the DNA, but in the centre. DNA Helicase unzips the DNA forming Replication bubbles. An RNA Primer or Primase then attaches to each DNA strand and replication begins in the 3-5 direction, thus forming a new strand in the 5 -3 direction.

DNA polymerases move and replicate the DNA in the 3 to the 5 direction (thus making a replica strand in the 5 to 3 direction). Note that 3 refers to the 3 OH group of the sugar and the 5 refers to the 5 phosphate group of the nucleotide.

The leading strand of DNA is the strand that is oriented in the 3-5 direction. The primer of complementary molecules that binds to the first few of the exposed bases ends with a 3 sugar group. The phosphate of a new nucleotide can be attached here by DNA polymerase. DNA polymerase then continues along the strand, synthesising a new strand as it goes. An animation of this process can be seen below, provided by the Nobel e-museum.

http://www.nobel.se/medicine/educational/dna/a/replication/replication_ani.html.

The lagging strand however, faces more problems when it comes to replication. Because DNA polymerase can only attach to the 3 sugar group and thus move in the 3 to the 5 direction, it needs therefore to move in small jumps (in the opposite direction to overall replication) to replicate the lagging strand. See below animation. This is again provided by the Nobel e-museum.

http://www.nobel.se/medicine/educational/dna/a/replication/lagging_ani.html

Note that the small segments of DNA and primers which are produced on the lagging strand are called Okazaki fragments. Another DNA polymerase enzyme is then recruited to remove the primers and to replace them with DNA. Finally DNA Ligase seals the gaps between the Okazaki fragments.

DNA Replication

The problem with Telomere shortening occurs because, in order to change the RNA primers into DNA, there must be another DNA segment in front of the primer. There is a DNA segment ahead of the primer at every section of the strand, except where the last primer attaches (the end of the telomere). This means that this final primer cannot be replaced with DNA. It does get removed by various enzymes however, but in the process, the telomere shortens.

In human blood cells, the range of lengths of the telomeres varies between 8000 base pairs at birth, to 1,500 base pairs in the elderly. During cellular division, an average of 30 to 200 base pairs are removed from the ends of the telomeres.

In normal cases, the cells of a human can divide between 50 to 70 times, with the telomeres shortening with each division, until the cells either commit suicide through a process known as Apoptosis, become Senescent (dormant) or transform to cancerous cells due to genetic damage.

The process of Cellular senescence due to Telomere Shortening

As a cells telomeres shorten during multiple cellular divisions, DNA damage occurs and the cell, recognising this damage shuts itself down. Below is a simplified diagram of how this occurs.

Telomere shortening and aging

DNA damage results in an activation of the p53 gene. p53 then activates p21, which blocks the actions of a number of CDK’s (Cycline Dependent Kinases). Note that CDK’s are involved in the regulation of the cell cycle, transcription and mRNA processing. The blocking of certain CDK’s prevents the phosphorylation of pRb. This lack of hyperphosphorylated pRb results in a failure of expression of several critical genes, which are involved in cellular division. Cellular division then stops.

Telomerase and Telomere extension

A study was conducted (as so many aging studies are) using the nematode Caenorhabditis elegans (Joeng KS, Song EJ, Lee KJ, Lee J (2004). "Long lifespan in worms with long telomeric DNA". Nature Genetics 36 (6): 607-11.). This study indicates that by lengthening the Telomere, longevity can be increased.

Two distinct groups of Nematodes were engineered. The only difference between the two groups, was the length of the Telomere. The group with the longer telomere’s lived approximately 20 percent longer than the group with the shorter telomeres. Also, it was observed that the Nematode’s with the longer telomeres possessed a greater resistance to the effects of heat exposure.

Telomerase is the natural enzyme which promotes telomere repair. It is however not active in most cells. It certainly is active though in stem cells, germ cells, hair follicles and (worryingly) in 90 percent of cancer cells. Telomerase functions by adding bases to the ends of the telomeres. As a result of this telomerase activity, these cells seem to possess a kind of immortality.

In 1990 a team at Geron Corp in Menlo Park, California led by Serge Lichtsteiner and Andrea Bodnar,in association with the University of texas Southwestern Medical center, managed to activate the production of telomerase in cells that do not usually produce this enzyme. The results were that the telomeres started to lengthen. By the time that they released the results of their study, their cells had divided 20 or more times than would normally be expected before senescence would set in. These cells also seemed to retain their normal gene expression and showed no signs of becoming cancerous.

I will now try to explain in more detail the details and results of the teams study.

Basically, telomerase is present in all cell types, however the human gene for the catalytic protein telomerase transcriptase (hTRT) is present only in immortal cells (such as stem cells or cancerous cells). Scientists from the teams observed that lengthening of telomeres in retinal pigment epithelial cells, foreskin fibroblasts and vascular endothelial cells by introducing the hTRT gene into these cells, results in a resumption of telomerase activity. This resumption resulted in the extended longevity of the cultured cells, as discussed previously.

Varying Telomere decline

One interesting study by Peter Lansdorp of the Terry Fox Laboratory, Canada, observed in human fibroblasts that the rate of telomere shortening as cells divide varies between different telomeres. This variation is between 50 to 150 base pairs per cell division. It is worth noting that the telomeres that are shorter initially. For example in humans the 17p telomere, are not necessarily the ones to be destroyed first! It does seem that it is the shortening of specific telomeres that are linked to a cells decline into senescence, apoptosis or its transformation into a cancer. Martens et al, 1998, observed that the shortening of telomeres 1p, 5p and 22p, but not that of 17p, showed a statistical correlation with the descent into cellular senescence.

Despite all of the above outlined observations, it is a statement of fact that the mean telomere length of a species does not always relate to the longevity of that species. For example Katuo et al, 1999, noted that of all studied primates, humans seem to have both the shortest telomeres and the longest lifespan! Bassham et al, 1998, also observed that the long lived frog Xenopus Laevis displayed a great variation in telomere length and that telomere length could even diminish between parent and offspring, with no detectable consequences.

To summarise, it seems that it has not yet been determined whether the instability of a chromosome and its eventual deterioration is a result of general telomere shortening or the shortening of specific telomeres. It has however been found that by artificially lengthening the telomeres of cells, which do not normally have a mechanism to lengthen their own telomeres, the cells longevity and ability to function normally for longer, does increase. More research therefore needs to be conducted in order to determine the exact significance of telomere length in the aging of an organism and the exact consequences of artificially stimulating telomere repair.

http://www.whyweage.com/node/11

Chromosomes

Eukaryotic DNA is arranged into chromosomes. It has long been assumed that each chromosome contains a single very long, linear DNA molecule though this has been demonstrated in only a few species. For example in yeast pulsed field gel electrophoresis (which is a technique for separating very long pieces of DNA) shows that the number of individual DNA molecules in the nucleus is approximately the same as the number of chromosomes.

These chromosomes have three functions:

1. they replicate once per cell cycle - during the S phase of interphase

2. they segregate to daughter cells at mitosis

3. they package DNA into managable units

Chromosomes possess within their DNA, specialised nucleotide sequences which are necessary for these functions.

Replication Sequences

Origins of replication

These are the sites where replication begins and there are usually a large number of these on each eukaryotic chromosome.

The DNA sequences which comprise origins of replication have been isolated from yeast. These have been called autonomously replicating sequences or ARSs. An ARS is defined as a region of DNA which is necessary and sufficient to ensure replication of a circular piece of DNA once per S phase.

ARS sequences were identified by cloning random fragments of yeast chromosomal DNA into a plasmid vector and then introducing them into yeast cells. If this plasmid contains a drug resistance marker and the yeast cells are plated on a medium containing the drug - only those cells containing a replicating plasmid will be able to form a colony. About a dozen different ARSs have been isolated in this way and it has been calculated that there are approximately 400 ARSs spread over the 17 chromosomes of yeast.

Mutational analysis has identified a region of about 50 bp that is required for proper ARS function. When the nucleotide sequence of different ARSs was compared it was found that there is an 11 bp sequence found in all of them - this suggests that this sequence is important, and indeed mutations in this region (region A) abolish ARS activity. However, the 11 bp sequence on its own cannot act as an ARS. Additional DNA sequences (B1-B3) are necessary for its function - this DNA shows no similarity between different ARSs but it often contains imperfect matches to the consensus sequence.

A complex of 6 proteins, called the origin recognition complex or ORC, binds to the ARS. The function of these proteins has yet to be determined. One important function must be the initial unwinding of the two strands of the DNA helix. It may be significant that the conserved core sequence is AT rich - AT base pairs are weaker than GC base pairs and so are more easily broken.

http://bssv01.lancs.ac.uk/ADS/BIOS336/336L5.html

History of DNA research

Further information: History of molecular biology

DNA was first isolated by the Swiss physician Friedrich Miescher who, in 1869, discovered a microscopic substance in the pus of discarded surgical bandages. As it resided in the nuclei of cells, he called it "nuclein".^[117] In 1919 this discovery was followed by Phoebus Levene's identification of the base, sugar and phosphate nucleotide unit.^[118] Levene suggested that DNA consisted of a string of nucleotide units linked together through the phosphate groups. However, Levene thought the chain was short and the bases repeated in a fixed order. In 1937 William Astbury produced the first X-ray diffraction patterns that showed that DNA had a regular structure.^[119]

In 1928, Frederick Griffith discovered that traits of the "smooth" form of the Pneumococcus could be transferred to the "rough" form of the same bacteria by mixing killed "smooth" bacteria with the live "rough" form.^[120] This system provided the first clear suggestion that DNA carried genetic information, when Oswald Avery, along with coworkers Colin MacLeod and Maclyn McCarty, identified DNA as the transforming principle in 1943.^[121] DNA's role in heredity was confirmed in 1952, when Alfred Hershey and Martha Chase in the Hershey-Chase experiment showed that DNA is the genetic material of the T2 phage.^[122]

In 1953, based on X-ray diffraction images^[123] taken by Rosalind Franklin and the information that the bases were paired, James D. Watson and Francis Crick suggested^[123] what is now accepted as the first accurate model of DNA structure in the journal Nature.^[5] Experimental evidence for Watson and Crick's model were published in a series of five articles in the same issue of Nature.^[124] Of these, Franklin and Raymond Gosling's paper was the first publication of X-ray diffraction data that supported the Watson and Crick model,^[125][126] this issue also contained an article on DNA structure by Maurice Wilkins and his colleagues.^[127] In 1962, after Franklin's death, Watson, Crick, and Wilkins jointly received the Nobel Prize in Physiology or Medicine.^[128] However, debate continues on who should receive credit for the discovery, as the Watson and Crick article in Nature was based on Franklin's data without either acknowledgment or her knowledge.^[129]

In an influential presentation in 1957, Crick laid out the "Central Dogma" of molecular biology, which foretold the relationship between DNA, RNA, and proteins, and articulated the "adaptor hypothesis".^[130] Final confirmation of the replication mechanism that was implied by the double-helical structure followed in 1958 through the Meselson-Stahl experiment.^[131] Further work by Crick and coworkers showed that the genetic code was based on non-overlapping triplets of bases, called codons, allowing Har Gobind Khorana, Robert W. Holley and Marshall Warren Nirenberg to decipher the genetic code.^[132] These findings represent the birth of molecular biology.

http://en.wikipedia.org/wiki/DNA