Deoxyribonucleic acid

Deoxyribonucleic acid (DNA) is a nucleic acid that contains the geneticinstructions used in the development and functioning of all known living organisms (with the exception of RNA viruses). The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in regulating the use of this genetic information.Sony VAIO VPCS117GG Battery

Along with RNA andproteins, DNA is one of the three major macromolecules that are essential for all known forms of life.

DNA consists of two long polymers of simple units called nucleotides, with backbones made of sugars andphosphate groups joined by ester bonds. These two strands run in opposite directions to each other and are therefore anti-parallel. Sony VAIO VPCS117GGB Battery

Attached to each sugar is one of four types of molecules called nucleobases(informally, bases). It is the sequence of these four nucleobases along the backbone that encodes information. This information is read using the genetic code, which specifies the sequence of the amino acids within proteins. The code is read by copying stretches of DNA into the related nucleic acid RNA in a process calledtranscription.Sony VAIO VPCS118EC Battery

Within cells DNA is organized into long structures called chromosomes. During cell division these chromosomes are duplicated in the process of DNA replication, providing each cell its own complete set of chromosomes.Eukaryotic organisms (animals, plants, fungi, and protists) store most of their DNA inside the cell nucleus and some of their DNA in organelles, such as mitochondria or chloroplasts.Sony VAIO VPCS119FJ/B Battery

In contrast, prokaryotes (bacteria andarchaea) store their DNA only in the cytoplasm. Within the chromosomes, chromatin proteins such as histonescompact and organize DNA. These compact structures guide the interactions between DNA and other proteins, helping control which parts of the DNA are transcribed.Sony VAIO VPCS119GC Battery

Properties

DNA is a long polymer made from repeating units called nucleotides. As first discovered by James D. Watson andFrancis Crick, the structure of DNA of all species comprises two helical chains each coiled round the same axis, and each with a pitch of 34 Ångströms (3.4 nanometres) and a radius of 10 Ångströms (1.0 nanometres).Sony VAIO VPCS11AFJ Battery

According to another study, when measured in a particular solution, the DNA chain measured 22 to 26 Ångströms wide (2.2 to 2.6 nanometres), and one nucleotide unit measured 3.3 Å (0.33 nm) long.Although each individual repeating unit is very small, DNA polymers can be very large molecules containing millions of nucleotides. Sony VAIO VPCS11AGJ Battery

For instance, the largesthuman chromosome, chromosome number 1, is approximately 220 million base pairs long.

In living organisms DNA does not usually exist as a single molecule, but instead as a pair of molecules that are held tightly together.These two long strands entwine like vines, in the shape of a double helix.Sony VAIO VPCS11AHJ Battery

The nucleotide repeats contain both the segment of the backbone of the molecule, which holds the chain together, and a nucleobase, which interacts with the other DNA strand in the helix. A nucleobase linked to a sugar is called a nucleoside and a base linked to a sugar and one or more phosphate groups is called a nucleotide. Sony VAIO VPCS11AVJ Battery

Polymers comprising multiple linked nucleotides (as in DNA) are called a polynucleotide.

The backbone of the DNA strand is made from alternating phosphate and sugar residues. The sugar in DNA is 2-deoxyribose, which is a pentose (five-carbon) sugar. The sugars are joined together by phosphate groups that formphosphodiester bonds between the third and fifth carbon atoms of adjacent sugar rings. Sony VAIO VPCS11J7E/B Battery

These asymmetric bonds mean a strand of DNA has a direction. In a double helix the direction of the nucleotides in one strand is opposite to their direction in the other strand: the strands are antiparallel. The asymmetric ends of DNA strands are called the 5?(five prime) and 3? (three prime) ends, with the 5' end having a terminal phosphate group and the 3' end a terminal hydroxyl group.Sony VAIO VPCS11M1E/W Battery

One major difference between DNA and RNA is the sugar, with the 2-deoxyribose in DNA being replaced by the alternative pentose sugar ribose in RNA.

The DNA double helix is stabilized primarily by two forces: hydrogen bonds between nucleotides and base-stacking interactions among thearomatic nucleobases.Sony VAIO VPCS11V9E Battery

In the aqueous environment of the cell, the conjugated ? bonds of nucleotide bases align perpendicular to the axis of the DNA molecule, minimizing their interaction with the solvation shell and therefore, the Gibbs free energy. The four bases found in DNA are adenine (abbreviated A), cytosine (C), guanine (G) and thymine (T).Sony VAIO VPCS11V9E/B Battery

These four bases are attached to the sugar/phosphate to form the complete nucleotide, as shown for adenosine monophosphate.

The nucleobases are classified into two types: the purines, A and G, being fused five- and six-membered heterocyclic compounds, and thepyrimidines, the six-membered rings C and T.Sony VAIO VPCS11X9E/B Battery

A fifth pyrimidine nucleobase, uracil (U), usually takes the place of thymine in RNA and differs from thymine by lacking a methyl group on its ring. Uracil is not usually found in DNA, occurring only as a breakdown product of cytosine. In addition to RNA and DNA a large number of artificial nucleic acid analogues have also been created to study the proprieties of nucleic acids, or for use in biotechnology.Sony VAIO VPCS123FGB Battery

Grooves

Twin helical strands form the DNA backbone. Another double helix may be found by tracing the spaces, or grooves, between the strands. These voids are adjacent to the base pairs and may provide a binding site. As the strands are not directly opposite each other, the grooves are unequally sized. Sony VAIO VPCS125EC Battery

One groove, the major groove, is 22 Å wide and the other, the minor groove, is 12 Å wide. The narrowness of the minor groove means that the edges of the bases are more accessible in the major groove. As a result, proteins like transcription factors that can bind to specific sequences in double-stranded DNA usually make contacts to the sides of the bases exposed in the major groove.Sony VAIO VPCS128EC Battery

This situation varies in unusual conformations of DNA within the cell (see below), but the major and minor grooves are always named to reflect the differences in size that would be seen if the DNA is twisted back into the ordinary B form.

Base pairing

In a DNA double helix, each type of nucleobase on one strand normally interacts with just one type of nucleobase on the other strand. This is called complementary base pairing.Sony VAIO VPCS129GC Battery

Here, purines form hydrogen bonds to pyrimidines, with A bonding only to T, and C bonding only to G. This arrangement of two nucleotides binding together across the double helix is called a base pair. As hydrogen bonds are not covalent, they can be broken and rejoined relatively easily. Sony VAIO VPCS12C7E/B Battery

The two strands of DNA in a double helix can therefore be pulled apart like a zipper, either by a mechanical force or high temperature.As a result of this complementarity, all the information in the double-stranded sequence of a DNA helix is duplicated on each strand, which is vital in DNA replication. Sony VAIO VPCS12L9E/B Battery

Indeed, this reversible and specific interaction between complementary base pairs is critical for all the functions of DNA in living organisms.

The two types of base pairs form different numbers of hydrogen bonds, AT forming two hydrogen bonds, and GC forming three hydrogen bonds (see figures, left). Sony VAIO VPCS12V9E/B Battery

DNA with high GC-content is more stable than DNA with low GC-content. Although it is often stated that this is due to the added stability of an additional hydrogen bond, this is incorrect.[citation needed]DNA with high GC-content is more stable due to intra-strand base stacking interactions.Sony VAIO VPCW111XX/P Battery

As noted above, most DNA molecules are actually two polymer strands, bound together in a helical fashion by noncovalent bonds; this double stranded structure (dsDNA) is maintained largely by the intrastrand base stacking interactions, which are strongest for G,C stacks. The two strands can come apart – a process known as melting – to form two ss DNA molecules.Sony VAIO VPCW111XX/PC Battery

Melting occurs when conditions favor ssDNA; such conditions are high temperature, low salt and high pH (low pH also melts DNA, but since DNA is unstable due to acid depurination, low pH is rarely used). The stability of the dsDNA form depends not only on the GC-content (% G,C basepairs) but also on sequence (since stacking is sequence specific) and also length (longer molecules are more stable). Sony VAIO VPCW111XX/T Battery

The stability can be measured in various ways; a common way is the "melting temperature", which is the temperature at which 50% of the ds molecules are converted to ss molecules; melting temperature is dependent on ionic strength and the concentration of DNA. As a result, it is both the percentage of GC base pairs and the overall length of a DNA double helix that determine the strength of the association between the two strands of DNA. Sony VAIO VPCW111XX/W Battery

Long DNA helices with a high GC-content have stronger-interacting strands, while short helices with high AT content have weaker-interacting strands. In biology, parts of the DNA double helix that need to separate easily, such as the TATAAT Pribnow box in some promoters, tend to have a high AT content, making the strands easier to pull apart. Sony VAIO VPCW111XXP Battery

In the laboratory, the strength of this interaction can be measured by finding the temperature required to break the hydrogen bonds, their melting temperature (also called Tm value). When all the base pairs in a DNA double helix melt, the strands separate and exist in solution as two entirely independent molecules. Sony VAIO VPCW111XXT Battery

These single-stranded DNA molecules (ssDNA) have no single common shape, but some conformations are more stable than others.

Sense and antisense

A DNA sequence is called "sense" if its sequence is the same as that of a messenger RNA copy that is translated into protein.Sony VAIO VPCW111XXW Battery

The sequence on the opposite strand is called the "antisense" sequence. Both sense and antisense sequences can exist on different parts of the same strand of DNA (i.e. both strands contain both sense and antisense sequences). In both prokaryotes and eukaryotes, antisense RNA sequences are produced, but the functions of these RNAs are not entirely clear.Sony VAIO VPCW115XG Battery

One proposal is that antisense RNAs are involved in regulating gene expression through RNA-RNA base pairing.

A few DNA sequences in prokaryotes and eukaryotes, and more in plasmids and viruses, blur the distinction between sense and antisense strands by having overlapping genes.Sony VAIO VPCW115XGP Battery

In these cases, some DNA sequences do double duty, encoding one protein when read along one strand, and a second protein when read in the opposite direction along the other strand. In bacteria, this overlap may be involved in the regulation of gene transcription, while in viruses, overlapping genes increase the amount of information that can be encoded within the small viral genome.Sony VAIO VPCW115XW/P Battery

Supercoiling

DNA can be twisted like a rope in a process called DNA supercoiling. With DNA in its "relaxed" state, a strand usually circles the axis of the double helix once every 10.4 base pairs, but if the DNA is twisted the strands become more tightly or more loosely wound.Sony VAIO VPCW115XW/T Battery

If the DNA is twisted in the direction of the helix, this is positive supercoiling, and the bases are held more tightly together. If they are twisted in the opposite direction, this is negative supercoiling, and the bases come apart more easily. In nature, most DNA has slight negative supercoiling that is introduced by enzymes called topoisomerases.Sony VAIO VPCW115XW/W Battery

These enzymes are also needed to relieve the twisting stresses introduced into DNA strands during processes such as transcription and DNA replication.

Alternate DNA structures

DNA exists in many possible conformations that include A-DNA, B-DNA, and Z-DNA forms, although, only B-DNA and Z-DNA have been directly observed in functional organisms.Sony VAIO VPCW117XC/P Battery

The conformation that DNA adopts depends on the hydration level, DNA sequence, the amount and direction of supercoiling, chemical modifications of the bases, the type and concentration of metal ions, as well as the presence of polyamines in solution. Sony VAIO VPCW117XC/T Battery

The first published reports of A-DNA X-ray diffraction patterns— and also B-DNA used analyses based on Patterson transforms that provided only a limited amount of structural information for oriented fibers of DNA. An alternate analysis was then proposed by Wilkins et al., in 1953, for the in vivo B-DNA X-ray diffraction/scattering patterns of highly hydrated DNA fibers in terms of squares of Bessel functions.Sony VAIO VPCW117XC/W Battery

In the same journal, James D. Watson and Francis Crick presented their molecular modeling analysis of the DNA X-ray diffraction patterns to suggest that the structure was a double-helix.

Although the `B-DNA form' is most common under the conditions found in cells, it is not a well-defined conformation but a family of related DNA conformations that occur at the high hydration levels present in living cells.Sony VAIO VPCW119XJ Battery

Their corresponding X-ray diffraction and scattering patterns are characteristic of molecularparacrystals with a significant degree of disorder.

Compared to B-DNA, the A-DNA form is a wider right-handed spiral, with a shallow, wide minor groove and a narrower, deeper major groove. Sony VAIO VPCW119XJ/P Battery

The A form occurs under non-physiological conditions in partially dehydrated samples of DNA, while in the cell it may be produced in hybrid pairings of DNA and RNA strands, as well as in enzyme-DNA complexes.Segments of DNA where the bases have been chemically modified by methylation may undergo a larger change in conformation and adopt the Z form. Sony VAIO VPCW119XJ/W Battery

Here, the strands turn about the helical axis in a left-handed spiral, the opposite of the more common B form. These unusual structures can be recognized by specific Z-DNA binding proteins and may be involved in the regulation of transcription. Sony VAIO VPCW11AXJ Battery

Alternate DNA chemistry

For a number of years exobiologists have proposed the existence of a shadow biosphere, a postulated microbial biosphere of Earth that uses radically different biochemical and molecular processes than currently known life. One of the proposals was the existence of lifeforms that use arsenic instead of phosphorus in DNA.Sony VAIO VPCW11S1E/P Battery

A December 2010 NASA press conference stated that the bacterium GFAJ-1, which has evolved in an arsenic-rich environment, is the first terrestrial lifeform found which may have this ability. The bacterium was found in Mono Lake, east of Yosemite National Park. Sony VAIO VPCW11S1E/T Battery

GFAJ-1 is a rod-shaped extremophile bacterium in the familyHalomonadaceae that, when starved of phosphorus, may be capable of incorporating the usually poisonous element arsenic in its DNA.This discovery may lend weight to the long-standing idea that extraterrestrial life could have a different chemical makeup from life on Earth.Sony VAIO VPCW11S1E/W Battery

The research was carried out by a team led byFelisa Wolfe-Simon, a geomicrobiologist and geobiochemist, a Postdoctoral Fellow of the NASA Astrobiology Institute with Arizona State University. This finding has, however, faced strong criticism from the scientific community; scientists have argued that there is no evidence that arsenic is actually incorporated into biomolecules. Independent conformation of this finding has also not yet been possible.Sony VAIO VPCW121AX Battery

Quadruplex structures

At the ends of the linear chromosomes are specialized regions of DNA called telomeres. The main function of these regions is to allow the cell to replicate chromosome ends using the enzyme telomerase, as the enzymes that normally replicate DNA cannot copy the extreme 3? ends of chromosomes.Sony VAIO VPCW126AG Battery

These specialized chromosome caps also help protect the DNA ends, and stop the DNA repair systems in the cell from treating them as damage to be corrected. In human cells, telomeres are usually lengths of single-stranded DNA containing several thousand repeats of a simple TTAGGG sequence.Sony VAIO VPCW127JC/P Battery

These guanine-rich sequences may stabilize chromosome ends by forming structures of stacked sets of four-base units, rather than the usual base pairs found in other DNA molecules. Here, four guanine bases form a flat plate and these flat four-base units then stack on top of each other, to form a stable G-quadruplex structure.Sony VAIO VPCW127JC/T Battery

These structures are stabilized by hydrogen bonding between the edges of the bases and chelation of a metal ion in the centre of each four-base unit.Other structures can also be formed, with the central set of four bases coming from either a single strand folded around the bases, or several different parallel strands, each contributing one base to the central structure.Sony VAIO VPCW127JC/W Battery

In addition to these stacked structures, telomeres also form large loop structures called telomere loops, or T-loops. Here, the single-stranded DNA curls around in a long circle stabilized by telomere-binding proteins. At the very end of the T-loop, the single-stranded telomere DNA is held onto a region of double-stranded DNA by the telomere strand disrupting the double-helical DNA and base pairing to one of the two strands.Sony VAIO VPCW127JC/WZ Battery

This triple-stranded structure is called a displacement loop or D-loop.

Branched DNA

In DNA fraying occurs when non-complementary regions exist at the end of an otherwise complementary double-strand of DNA. However, branched DNA can occur if a third strand of DNA is introduced and contains adjoining regions able to hybridize with the frayed regions of the pre-existing double-strand. Sony VAIO VPCW12AAJ Battery

Although the simplest example of branched DNA involves only three strands of DNA, complexes involving additional strands and multiple branches are also possible. Branched DNA can be used in nanotechnology to construct geometric shapes, see the section on uses in technology below.Sony VAIO VPCW12AKJ Battery

Vibration

DNA may carry out low-frequency collective motion as observed by the Raman spectroscopy and analyzed with a quasi-continuum model.

Base modifications

The expression of genes is influenced by how the DNA is packaged in chromosomes, in a structure called chromatin.Sony VAIO VPCW12AVJ Battery

Base modifications can be involved in packaging, with regions that have low or no gene expression usually containing high levels of methylation of cytosine bases. For example, cytosine methylation, produces 5-methylcytosine, which is important for X-chromosome inactivation.Sony VAIO VPCW12S1E/P Battery

The average level of methylation varies between organisms – the wormCaenorhabditis elegans lacks cytosine methylation, while vertebrates have higher levels, with up to 1% of their DNA containing 5-methylcytosine. Despite the importance of 5-methylcytosine, it can deaminate to leave a thymine base, so methylated cytosines are particularly prone to mutations.Sony VAIO VPCW12S1E/T Battery

Other base modifications include adenine methylation in bacteria, the presence of 5-hydroxymethylcytosine in the brain, and the glycosylation of uracil to produce the "J-base" in kinetoplastids.

Damage

DNA can be damaged by many sorts of mutagens, which change the DNA sequence. Sony VAIO VPCW12S1E/W Battery

Mutagens include oxidizing agents, alkylating agentsand also high-energy electromagnetic radiation such as ultraviolet light and X-rays. The type of DNA damage produced depends on the type of mutagen. For example, UV light can damage DNA by producing thymine dimers, which are cross-links between pyrimidine bases.Sony VAIO VPCW213AG/L Battery

On the other hand, oxidants such as free radicals or hydrogen peroxide produce multiple forms of damage, including base modifications, particularly of guanosine, and double-strand breaks. A typical human cell contains about 150,000 bases that have suffered oxidative damage.Sony VAIO VPCW213AG/P Battery

Of these oxidative lesions, the most dangerous are double-strand breaks, as these are difficult to repair and can produce point mutations, insertions and deletions from the DNA sequence, as well as chromosomal translocations.

Many mutagens fit into the space between two adjacent base pairs, this is called intercalation. Most intercalators are aromaticand planar molecules; examples include ethidium bromide, acridines, daunomycin, and doxorubicin.Sony VAIO VPCW213AG/T Battery

In order for an intercalator to fit between base pairs, the bases must separate, distorting the DNA strands by unwinding of the double helix. This inhibits both transcription and DNA replication, causing toxicity and mutations. As a result, DNA intercalators may be carcinogens, and in the case of thalidomide, a teratogen. Sony VAIO VPCW213AG/W Battery

Others such as benzo[a]pyrene diol epoxide and aflatoxin form DNA adducts which induce errors in replication. Nevertheless, due to their ability to inhibit DNA transcription and replication, other similar toxins are also used in chemotherapy to inhibit rapidly growing cancer cells.Sony VAIO VPCW215AG/L Battery

Biological functions

DNA usually occurs as linear chromosomes in eukaryotes, and circular chromosomes in prokaryotes. The set of chromosomes in a cell makes up its genome; the human genome has approximately 3 billion base pairs of DNA arranged into 46 chromosomes.Sony VAIO VPCW217JC Battery

The information carried by DNA is held in the sequence of pieces of DNA called genes. Transmission of genetic information in genes is achieved via complementary base pairing. For example, in transcription, when a cell uses the information in a gene, the DNA sequence is copied into a complementary RNA sequence through the attraction between the DNA and the correct RNA nucleotides.Sony VAIO VPCW217JC/L Battery

Usually, this RNA copy is then used to make a matching protein sequence in a process called translation, which depends on the same interaction between RNA nucleotides. In alternative fashion, a cell may simply copy its genetic information in a process called DNA replication. Sony VAIO VPCW217JC/P Battery

The details of these functions are covered in other articles; here we focus on the interactions between DNA and other molecules that mediate the function of the genome.

Genes and genomes

Genomic DNA is tightly and orderly packed in the process called DNA condensation to fit the small available volumes of the cell.Sony VAIO VPCW217JC/T Battery

In eukaryotes, DNA is located in thecell nucleus, as well as small amounts in mitochondria and chloroplasts. In prokaryotes, the DNA is held within an irregularly shaped body in the cytoplasm called the nucleoid. The genetic information in a genome is held within genes, and the complete set of this information in an organism is called its genotype. Sony VAIO VPCW217JC/W Battery

A gene is a unit of heredity and is a region of DNA that influences a particular characteristic in an organism. Genes contain an open reading frame that can be transcribed, as well as regulatory sequences such as promoters and enhancers, which control the transcription of the open reading frame.Sony VAIO VPCW218JC Battery

In many species, only a small fraction of the total sequence of the genome encodes protein. For example, only about 1.5% of the human genome consists of protein-coding exons, with over 50% of human DNA consisting of non-coding repetitive sequences. Sony VAIO VPCW218JC/L Battery

The reasons for the presence of so much noncoding DNA in eukaryotic genomes and the extraordinary differences in genome size, or C-value, among species represent a long-standing puzzle known as the "C-value enigma". However, DNA sequences that do not code protein may still encode functional non-coding RNA molecules, which are involved in the regulation of gene expression.Sony VAIO VPCW218JC/P Battery

Some noncoding DNA sequences play structural roles in chromosomes. Telomeres and centromeres typically contain few genes, but are important for the function and stability of chromosomes. An abundant form of noncoding DNA in humans are pseudogenes, which are copies of genes that have been disabled by mutation.Sony VAIO VPCW218JC/T Battery

These sequences are usually just molecular fossils, although they can occasionally serve as raw genetic material for the creation of new genes through the process of gene duplication anddivergence.

Transcription and translation

A gene is a sequence of DNA that contains genetic information and can influence the phenotype of an organism. Sony VAIO VPCW218JC/W Battery

Within a gene, the sequence of bases along a DNA strand defines a messenger RNA sequence, which then defines one or more protein sequences. The relationship between the nucleotide sequences of genes and the amino-acid sequences of proteins is determined by the rules oftranslation, known collectively as the genetic code.Sony VAIO VPCW219AJ/L Battery

The genetic code consists of three-letter 'words' called codons formed from a sequence of three nucleotides (e.g. ACT, CAG, TTT).

In transcription, the codons of a gene are copied into messenger RNA by RNA polymerase. This RNA copy is then decoded by a ribosome that reads the RNA sequence by base-pairing the messenger RNA to transfer RNA, which carries amino acids.Sony VAIO VPCW219AJ/P Battery

Since there are 4 bases in 3-letter combinations, there are 64 possible codons (43combinations). These encode the twenty standard amino acids, giving most amino acids more than one possible codon. There are also three 'stop' or 'nonsense' codons signifying the end of the coding region; these are the TAA, TGA and TAG codons.Sony VAIO VPCW219AJ/W Battery

Replication

Cell division is essential for an organism to grow, but, when a cell divides, it must replicate the DNA in its genome so that the two daughter cells have the same genetic information as their parent. The double-stranded structure of DNA provides a simple mechanism for DNA replication. Sony VAIO VPCW21AAJ Battery

Here, the two strands are separated and then each strand's complementary DNA sequence is recreated by an enzyme called DNA polymerase. This enzyme makes the complementary strand by finding the correct base through complementary base pairing, and bonding it onto the original strand. Sony VAIO VPCW21AKJ Battery

As DNA polymerases can only extend a DNA strand in a 5? to 3? direction, different mechanisms are used to copy the antiparallel strands of the double helix. In this way, the base on the old strand dictates which base appears on the new strand, and the cell ends up with a perfect copy of its DNA.Sony VAIO VPCW21AVJ Battery

Interactions with proteins

All the functions of DNA depend on interactions with proteins. These protein interactions can be non-specific, or the protein can bind specifically to a single DNA sequence. Enzymes can also bind to DNA and of these, the polymerases that copy the DNA base sequence in transcription and DNA replication are particularly important.Sony VAIO VPCY115FGS Battery

DNA-binding proteins

Structural proteins that bind DNA are well-understood examples of non-specific DNA-protein interactions. Within chromosomes, DNA is held in complexes with structural proteins. These proteins organize the DNA into a compact structure called chromatin.Sony VAIO VPCY115FX/BI Battery

In eukaryotes this structure involves DNA binding to a complex of small basic proteins called histones, while in prokaryotes multiple types of proteins are involved.The histones form a disk-shaped complex called a nucleosome, which contains two complete turns of double-stranded DNA wrapped around its surface. Sony VAIO VPCY115FXBI Battery

These non-specific interactions are formed through basic residues in the histones making ionic bonds to the acidic sugar-phosphate backbone of the DNA, and are therefore largely independent of the base sequence. Chemical modifications of these basic amino acid residues include methylation,phosphorylation and acetylation.Sony VAIO VPCY118EC Battery

These chemical changes alter the strength of the interaction between the DNA and the histones, making the DNA more or less accessible to transcription factors and changing the rate of transcription. Other non-specific DNA-binding proteins in chromatin include the high-mobility group proteins, which bind to bent or distorted DNA.Sony VAIO VPCY118GX/BI Battery

These proteins are important in bending arrays of nucleosomes and arranging them into the larger structures that make up chromosomes.

A distinct group of DNA-binding proteins are the DNA-binding proteins that specifically bind single-stranded DNA. Sony VAIO VPCY119FJ/S Battery

In humans, replication protein A is the best-understood member of this family and is used in processes where the double helix is separated, including DNA replication, recombination and DNA repair. These binding proteins seem to stabilize single-stranded DNA and protect it from formingstem-loops or being degraded by nucleases.Sony VAIO VPCY11AFJ Battery

In contrast, other proteins have evolved to bind to particular DNA sequences. The most intensively studied of these are the varioustranscription factors, which are proteins that regulate transcription. Each transcription factor binds to one particular set of DNA sequences and activates or inhibits the transcription of genes that have these sequences close to their promoters. Sony VAIO VPCY11AGJ Battery

The transcription factors do this in two ways. Firstly, they can bind the RNA polymerase responsible for transcription, either directly or through other mediator proteins; this locates the polymerase at the promoter and allows it to begin transcription. Alternatively, transcription factors can bind enzymes that modify the histones at the promoter; this will change the accessibility of the DNA template to the polymerase. Sony VAIO VPCY11AHJ Battery

As these DNA targets can occur throughout an organism's genome, changes in the activity of one type of transcription factor can affect thousands of genes. Consequently, these proteins are often the targets of the signal transduction processes that control responses to environmental changes or cellular differentiation and development. Sony VAIO VPCY11AVJ Battery

The specificity of these transcription factors' interactions with DNA come from the proteins making multiple contacts to the edges of the DNA bases, allowing them to "read" the DNA sequence. Most of these base-interactions are made in the major groove, where the bases are most accessible.Sony VAIO VPCY11M1E/S Battery

Nucleases and ligases

Nucleases are enzymes that cut DNA strands by catalyzing the hydrolysis of the phosphodiester bonds. Nucleases that hydrolyse nucleotides from the ends of DNA strands are called exonucleases, whileendonucleases cut within strands. Sony VAIO VPCY11S1E Battery

The most frequently used nucleases in molecular biology are therestriction endonucleases, which cut DNA at specific sequences. For instance, the EcoRV enzyme shown to the left recognizes the 6-base sequence 5?-GAT|ATC-3? and makes a cut at the vertical line. In nature, these enzymes protect bacteria against phageinfection by digesting the phage DNA when it enters the bacterial cell, acting as part of the restriction modification system.Sony VAIO VPCY11V9E/S Battery

In technology, these sequence-specific nucleases are used in molecular cloning and DNA fingerprinting.

Enzymes called DNA ligases can rejoin cut or broken DNA strands. Ligases are particularly important in lagging strand DNA replication, as they join together the short segments of DNA produced at the replication fork into a complete copy of the DNA template. They are also used in DNA repair and genetic recombination.Sony VAIO VPCY218EC/BI Battery

Topoisomerases and helicases

Topoisomerases are enzymes with both nuclease and ligase activity. These proteins change the amount of supercoiling in DNA. Some of these enzymes work by cutting the DNA helix and allowing one section to rotate, thereby reducing its level of supercoiling; the enzyme then seals the DNA break.Sony VAIO VPCY218EC/G Battery

Other types of these enzymes are capable of cutting one DNA helix and then passing a second strand of DNA through this break, before rejoining the helix. Topoisomerases are required for many processes involving DNA, such as DNA replication and transcription.Sony VAIO VPCY218EC/L Battery

Helicases are proteins that are a type of molecular motor. They use the chemical energy in nucleoside triphosphates, predominantly ATP, to break hydrogen bonds between bases and unwind the DNA double helix into single strands. These enzymes are essential for most processes where enzymes need to access the DNA bases.Sony VAIO VPCY218EC/P Battery

Polymerases

Polymerases are enzymes that synthesize polynucleotide chains from nucleoside triphosphates. The sequence of their products are copies of existing polynucleotide chains – which are called templates. These enzymes function by adding nucleotides onto the 3? hydroxyl group of the previous nucleotide in a DNA strand. Sony VAIO VPCY21S1E/L Battery

As a consequence, all polymerases work in a 5? to 3? direction. In the active site of these enzymes, the incoming nucleoside triphosphate base-pairs to the template: this allows polymerases to accurately synthesize the complementary strand of their template. Polymerases are classified according to the type of template that they use.Sony VAIO VPCY21S1E/P Battery

In DNA replication, a DNA-dependent DNA polymerase makes a copy of a DNA sequence. Accuracy is vital in this process, so many of these polymerases have aproofreading activity. Here, the polymerase recognizes the occasional mistakes in the synthesis reaction by the lack of base pairing between the mismatched nucleotides. Sony VAIO VPCY21S1E/SI Battery

If a mismatch is detected, a 3? to 5? exonuclease activity is activated and the incorrect base removed. In most organisms, DNA polymerases function in a large complex called the replisome that contains multiple accessory subunits, such as the DNA clamp or helicases.Sony VAIO VPCZ110 Battery

RNA-dependent DNA polymerases are a specialized class of polymerases that copy the sequence of an RNA strand into DNA. They include reverse transcriptase, which is aviral enzyme involved in the infection of cells by retroviruses, and telomerase, which is required for the replication of telomeres.Sony VAIO VPCZ110GB/BI Battery

Telomerase is an unusual polymerase because it contains its own RNA template as part of its structure.

Transcription is carried out by a DNA-dependent RNA polymerase that copies the sequence of a DNA strand into RNA. To begin transcribing a gene, the RNA polymerase binds to a sequence of DNA called a promoter and separates the DNA strands. Sony VAIO VPCZ112GD/S Battery

It then copies the gene sequence into a messenger RNA transcript until it reaches a region of DNA called the terminator, where it halts and detaches from the DNA. As with human DNA-dependent DNA polymerases, RNA polymerase II, the enzyme that transcribes most of the genes in the human genome, operates as part of a large protein complex with multiple regulatory and accessory subunits.Sony VAIO VPCZ112GX/S Battery

Genetic recombination

A DNA helix usually does not interact with other segments of DNA, and in human cells the different chromosomes even occupy separate areas in the nucleus called "chromosome territories". Sony VAIO VPCZ114GX/S Battery

This physical separation of different chromosomes is important for the ability of DNA to function as a stable repository for information, as one of the few times chromosomes interact is during chromosomal crossover when they recombine. Chromosomal crossover is when two DNA helices break, swap a section and then rejoin.Sony VAIO VPCZ115 Battery

Recombination allows chromosomes to exchange genetic information and produces new combinations of genes, which increases the efficiency of natural selection and can be important in the rapid evolution of new proteins. Genetic recombination can also be involved in DNA repair, particularly in the cell's response to double-strand breaks.Sony VAIO VPCZ115FC/B Battery

The most common form of chromosomal crossover is homologous recombination, where the two chromosomes involved share very similar sequences. Non-homologous recombination can be damaging to cells, as it can produce chromosomal translocations and genetic abnormalities. The recombination reaction is catalyzed by enzymes known as recombinases, such as RAD51.Sony VAIO VPCZ115FC/S Battery

The first step in recombination is a double-stranded break either caused by an endonuclease or damage to the DNA. A series of steps catalyzed in part by the recombinase then leads to joining of the two helices by at least one Holliday junction, in which a segment of a single strand in each helix is annealed to the complementary strand in the other helix. Sony VAIO VPCZ116 Battery

The Holliday junction is a tetrahedral junction structure that can be moved along the pair of chromosomes, swapping one strand for another. The recombination reaction is then halted by cleavage of the junction and re-ligation of the released DNA.

Evolution

DNA contains the genetic information that allows all modern living things to function, grow and reproduce.Sony VAIO VPCZ116GX/S Battery

However, it is unclear how long in the 4-billion-year history of life DNA has performed this function, as it has been proposed that the earliest forms of life may have used RNA as their genetic material.RNA may have acted as the central part of earlycell metabolism as it can both transmit genetic information and carry out catalysis as part of ribozymes.Sony VAIO VPCZ117 Battery

This ancient RNA world where nucleic acid would have been used for both catalysis and genetics may have influenced the evolution of the current genetic code based on four nucleotide bases. This would occur, since the number of different bases in such an organism is a trade-off between a small number of bases increasing replication accuracy and a large number of bases increasing the catalytic efficiency of ribozymes.Sony VAIO VPCZ117FC/B Battery

However, there is no direct evidence of ancient genetic systems, as recovery of DNA from most fossils is impossible. This is because DNA will survive in the environment for less than one million years and slowly degrades into short fragments in solution. Claims for older DNA have been made, most notably a report of the isolation of a viable bacterium from a salt crystal 250 million years old, but these claims are controversial. Sony VAIO VPCZ118 Battery

On August 8, 2011, a report, based on NASA studies with meteorites found on Earth, was published suggesting building blocks of DNA (adenine, guanine and relatedorganic molecules) may have been formed extraterrestrially in outer space.Sony VAIO VPCZ118GC/B Battery

Genetic engineering

Methods have been developed to purify DNA from organisms, such as phenol-chloroform extraction, and to manipulate it in the laboratory, such as restriction digestsand the polymerase chain reaction. Modern biology and biochemistry make intensive use of these techniques in recombinant DNA technology.Sony VAIO VPCZ118GX/S Battery

Recombinant DNA is a man-made DNA sequence that has been assembled from other DNA sequences. They can be transformed into organisms in the form of plasmids or in the appropriate format, by using a viral vector.The genetically modified organisms produced can be used to produce products such as recombinant proteins, used in medical research,or be grown in agriculture.Sony VAIO VPCZ119 Battery

Forensics

Forensic scientists can use DNA in blood, semen, skin, saliva or hair found at a crime scene to identify a matching DNA of an individual, such as a perpetrator. This process is formally termed DNA profiling, but may also be called "genetic fingerprinting".Sony VAIO VPCZ119FJ/S Battery

In DNA profiling, the lengths of variable sections of repetitive DNA, such as short tandem repeats and minisatellites, are compared between people. This method is usually an extremely reliable technique for identifying a matching DNA. However, identification can be complicated if the scene is contaminated with DNA from several people.Sony VAIO VPCZ119GC/X Battery

DNA profiling was developed in 1984 by British geneticist Sir Alec Jeffreys, and first used in forensic science to convict Colin Pitchfork in the 1988 Enderby murders case.

People convicted of certain types of crimes may be required to provide a sample of DNA for a database. Sony VAIO VPCZ119L Battery

This has helped investigators solve old cases where only a DNA sample was obtained from the scene. DNA profiling can also be used to identify victims of mass casualty incidents. On the other hand, many convicted people have been released from prison on the basis of DNA techniques, which were not available when a crime had originally been committed.Sony VAIO VPCZ119R/B Battery

Bioinformatics

Bioinformatics involves the manipulation, searching, and data mining of biological data, and this includes DNA sequence data. The development of techniques to store and search DNA sequences have led to widely applied advances in computer science, especially string searching algorithms, machine learning and database theory.Sony VAIO VPCZ119R/S Battery

String searching or matching algorithms, which find an occurrence of a sequence of letters inside a larger sequence of letters, were developed to search for specific sequences of nucleotides. The DNA sequenced may be aligned with other DNA sequences to identify homologous sequences and locate the specific mutations that make them distinct. Sony VAIO VPCZ11AFJ Battery

These techniques, especially multiple sequence alignment, are used in studying phylogenetic relationships and protein function. Data sets representing entire genomes' worth of DNA sequences, such as those produced by the Human Genome Project, are difficult to use without the annotations that identify the locations of genes and regulatory elements on each chromosome.Sony VAIO VPCZ11AGJ Battery

Regions of DNA sequence that have the characteristic patterns associated with protein- or RNA-coding genes can be identified by gene finding algorithms, which allow researchers to predict the presence of particular gene products and their possible functions in an organism even before they have been isolated experimentally.Sony VAIO VPCZ11AVJ Battery

Entire genomes may also be compared which can shed light on the evolutionary history of particular organism and permit the examination of complex evolutionary events.

DNA nanotechnology

DNA nanotechnology uses the unique molecular recognition properties of DNA and other nucleic acids to create self-assembling branched DNA complexes with useful properties.Sony VAIO VPCZ11CGX/X Battery

DNA is thus used as a structural material rather than as a carrier of biological information. This has led to the creation of two-dimensional periodic lattices (both tile-based as well as using the "DNA origami" method) as well as three-dimensional structures in the shapes of polyhedra.Sony VAIO VPCZ11DGX/SJ Battery

Nanomechanical devices and algorithmic self-assembly have also been demonstrated, and these DNA structures have been used to template the arrangement of other molecules such as gold nanoparticles and streptavidin proteins.

History and anthropology

Because DNA collects mutations over time, which are then inherited, it contains historical information, and, by comparing DNA sequences, geneticists can infer the evolutionary history of organisms, theirphylogeny.Sony VAIO VPCZ11FHX/XQ Battery

This field of phylogenetics is a powerful tool in evolutionary biology. If DNA sequences within a species are compared, population geneticists can learn the history of particular populations. This can be used in studies ranging from ecological genetics to anthropology; For example, DNA evidence is being used to try to identify the Ten Lost Tribes of Israel.Sony VAIO VPCZ11V9R/B Battery

DNA has also been used to look at modern family relationships, such as establishing family relationships between the descendants of Sally Hemings and Thomas Jefferson. This usage is closely related to the use of DNA in criminal investigations detailed above. Indeed, some criminal investigations have been solved when DNA from crime scenes has matched relatives of the guilty individual.Sony VAIO VPCZ11X9E/B Battery

History of DNA research

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". In 1878, Albrecht Kossel isolated the non-protein component of "nuclein", nucleic acid, and later isolated its five primary nucleobases.Sony VAIO VPCZ11Z9E/B Battery

In 1919, Phoebus Levene identified the base, sugar and phosphate nucleotide unit.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. Sony VAIO VPCZ125GX/S Battery

In 1937 William Astbury produced the first X-ray diffraction patterns that showed that DNA had a regular structure.

In 1927 Nikolai Koltsov proposed that inherited traits would be inherited via a "giant hereditary molecule" which would be made up of "two mirror strands that would replicate in a semi-conservative fashion using each strand as a template".Sony VAIO VPCZ127FC Battery

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. This system provided the first clear suggestion that DNA carries genetic information—the Avery–MacLeod–McCarty experiment—whenOswald Avery, along with coworkers Colin MacLeod and Maclyn McCarty, identified DNA as thetransforming principle in 1943.Sony VAIO VPCZ128GC Battery

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.

In 1953, James D. Watson and Francis Crick suggested what is now accepted as the first correct double-helix model of DNA structure in the journal Nature.Sony VAIO VPCZ12M9E/B Battery

Their double-helix, molecular model of DNA was then based on a single X-ray diffraction image (labeled as "Photo 51") taken by Rosalind Franklin and Raymond Gosling in May 1952, as well as the information that the DNA bases are paired — also obtained through private communications from Erwin Chargaff in the previous years.Sony VAIO VPCZ13M9E/B Battery

Chargaff's rules played a very important role in establishing double-helix configurations for B-DNA as well as A-DNA.

Experimental evidence supporting the Watson and Crick model were published in a series of five articles in the same issue of Nature.Sony VAIO VPCZ13V9E/X Battery

Of these, Franklin and Gosling's paper was the first publication of their own X-ray diffraction data and original analysis method that partially supported the Watson and Crick model; this issue also contained an article on DNA structure by Maurice Wilkins and two of his colleagues,Sony VAIO VPCZ13Z9E/X Battery

whose analysis and in vivo B-DNA X-ray patterns also supported the presence in vivo of the double-helical DNA configurations as proposed by Crick and Watson for their double-helix molecular model of DNA in the previous two pages of Nature. In 1962, after Franklin's death, Watson, Crick, and Wilkins jointly received the Nobel Prize in Physiology or Medicine.Sony VAIO VPCZZZHJ Battery

However, Nobel rules of the time allowed only living recipients, but a vigorous debate continues on who should receive credit for the discovery.

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".Sony VAIO VPZ117 Battery

Final confirmation of the replication mechanism that was implied by the double-helical structure followed in 1958 through the Meselson–Stahl experiment.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.Sony VAIO VPZ118 Battery

These findings represent the birth of molecular biology.

DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine,guanine, cytosine, and thymine—in a molecule of DNA.Sony VAIO VPZ119 Battery

Knowledge of DNA sequences has become indispensable for basic biological research, other research branches utilizing DNA sequencing, and in numerous applied fields such as diagnostic, biotechnology, forensic biology and biological systematics. The advent of DNA sequencing has significantly accelerated biological research and discovery. Sony VPCM11M1E/B Battery

The rapid speed of sequencing attained with modern DNA sequencing technology has been instrumental in the sequencing of the human genome, in the Human Genome Project. Related projects, often by scientific collaboration across continents, have generated the complete DNA sequences of many animal, plant, and microbial genomes.Sony VPCM11M1E/W Battery

The first DNA sequences were obtained in the early 1970s by academic researchers using laborious methods based on two-dimensional chromatography. Following the development of dye-based sequencing methods with automated analysis, DNA sequencing has become easier and orders of magnitude faster.Sony VPCM12M1E/L Battery

istory

RNA sequencing was one of the earliest forms of nucleotide sequencing. The major landmark of RNA sequencing is the sequence of the first complete gene and the complete genome of Bacteriophage MS2, identified and published by Walter Fiers and his coworkers at the University of Ghent (Ghent, Belgium), between 1972 and 1976.Sony VPCM12M1E/P Battery

Prior to the development of rapid DNA sequencing methods in the early 1970s by Frederick Sanger at the University of Cambridge, in England and Walter Gilbert andAllan Maxam at Harvard, a number of laborious methods were used. For instance, in 1973, Gilbert and Maxam reported the sequence of 24 basepairs using a method known as wandering-spot analysis.Sony VPCM12M1E/W Battery

The chain-termination method developed by Sanger and coworkers in 1977 soon became the method of choice, owing to its relative ease and reliability. It involves separating DNA bases from different DNA fragments.

Maxam–Gilbert sequencing

In 1976–1977, Allan Maxam and Walter Gilbert developed a DNA sequencing method based on chemical modification of DNA and subsequent cleavage at specific bases.Sony VPCM13M1E/L Battery

Although Maxam and Gilbert published their chemical sequencing method two years after the ground-breaking paper of Sanger and Coulson on plus-minus sequencing, Maxam–Gilbert sequencing rapidly became more popular, since purified DNA could be used directly, while the initial Sanger method required that each read start be cloned for production of single-stranded DNA.Sony VPCM13M1E/P Battery

However, with the improvement of the chain-termination method (see below), Maxam-Gilbert sequencing has fallen out of favour due to its technical complexity prohibiting its use in standard molecular biology kits, extensive use of hazardous chemicals, and difficulties with scale-up. Sony VPCM13M1E/W Battery

The method requires radioactive labeling at one 5' end of the DNA (typically by a kinase reaction using gamma-32P ATP) and purification of the DNA fragment to be sequenced. Chemical treatment generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T). Sony VAIO VGN-Z11MN/B Battery

For example, the purines (A+G) are depurinated using formic acid, the guanines (and to some extent the adenines) are methylated by dimethyl sulfate, and the pyrimidines (C+T) are methylated using hydrazine. The addition of salt (sodium chloride) to the hydrazine reaction inhibits the methylation of thymine for the C-only reaction. Sony VAIO VGN-Z11VN/X Battery

The modified DNAs are then cleaved by hot piperidine at the position of the modified base. The concentration of the modifying chemicals is controlled to introduce on average one modification per DNA molecule. Thus a series of labeled fragments is generated, from the radiolabeled end to the first "cut" site in each molecule. Sony VAIO VGN-Z11WN/B Battery

The fragments in the four reactions are electrophoresed side by side in denaturing acrylamide gels for size separation. To visualize the fragments, the gel is exposed to X-ray film for autoradiography, yielding a series of dark bands each corresponding to a radiolabeled DNA fragment, from which the sequence may be inferred.Sony VAIO VGN-Z11XN/B Battery

Also sometimes known as "chemical sequencing", this method led to the Methylation Interference Assay used to map DNA-binding sites for DNA-binding proteins.

Chain-termination methods

Because the chain-terminator method (or Sanger method after its developer Frederick Sanger) is more efficient and uses fewer toxic chemicals and lower amounts of radioactivity than the method of Maxam and Gilbert, it rapidly became the method of choice. Sony VAIO VGN-Z15 Battery

The key principle of the Sanger method was the use of dideoxynucleotide triphosphates (ddNTPs) as DNA chain terminators.

The classical chain-termination method requires a single-stranded DNA template, a DNA primer, a DNA polymerase, normal deoxynucleotidetriphosphates (dNTPs), and modified nucleotides (dideoxyNTPs) that terminate DNA strand elongation. Sony VAIO VGN-Z15N Battery

These ddNTPs will also be radioactively or fluorescently labelled for detection in automated sequencing machines. The DNA sample is divided into four separate sequencing reactions, containing all four of the standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase. Sony VAIO VGN-Z17 Battery

To each reaction is added only one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP) which are the chain-terminating nucleotides, lacking a 3'-OH group required for the formation of a phosphodiester bond between two nucleotides, thus terminating DNA strand extension and resulting in DNA fragments of varying length.Sony VAIO VGN-Z17N Battery

The newly synthesized and labelled DNA fragments are heat denatured, and separated by size (with a resolution of just one nucleotide) bygel electrophoresis on a denaturing polyacrylamide-urea gel with each of the four reactions run in one of four individual lanes (lanes A, T, G, C);Sony VAIO VGN-Z19 Battery

the DNA bands are then visualized by autoradiography or UV light, and the DNA sequence can be directly read off the X-ray filmor gel image. In the image on the right, X-ray film was exposed to the gel, and the dark bands correspond to DNA fragments of different lengths. Sony VAIO VGN-Z19N Battery

A dark band in a lane indicates a DNA fragment that is the result of chain termination after incorporation of a dideoxynucleotide (ddATP, ddGTP, ddCTP, or ddTTP). The relative positions of the different bands among the four lanes are then used to read (from bottom to top) the DNA sequence.Sony VAIO VGN-Z21MN/B Battery

Technical variations of chain-termination sequencing include tagging with nucleotides containing radioactive phosphorus for

Chain-termination methods have greatly simplified DNA sequencing. For example, chain-termination-based kits are commercially available that contain the reagents needed for sequencing, pre-aliquoted and ready to use.Sony VAIO VGN-Z21VN/X Battery

Limitations include non-specific binding of the primer to the DNA, affecting accurate read-out of the DNA sequence, and DNA secondary structures affecting the fidelity of the sequence.

Dye-terminator sequencing

Dye-terminator sequencing utilizes labelling of the chain terminator ddNTPs, which permits sequencing in a single reaction, rather than four reactions as in the labelled-primer method.Sony VAIO VGN-Z21WN/B Battery

In dye-terminator sequencing, each of the four dideoxynucleotide chain terminators is labelled with fluorescent dyes, each of which emit light at different wavelengths.

Owing to its greater expediency and speed, dye-terminator sequencing is now the mainstay in automated sequencing. Sony VAIO VGN-Z21XN Battery

Its limitations include dye effects due to differences in the incorporation of the dye-labelled chain terminators into the DNA fragment, resulting in unequal peak heights and shapes in the electronic DNA sequence trace chromatogram aftercapillary electrophoresis (see figure to the left).Sony VAIO VGN-Z21ZN/X Battery

This problem has been addressed with the use of modified DNA polymerase enzyme systems and dyes that minimize incorporation variability, as well as methods for eliminating "dye blobs". The dye-terminator sequencing method, along with automated high-throughput DNA sequence analyzers, is now being used for the vast majority of sequencing projects.Sony VAIO VGN-Z25 Battery

Challenges

Common challenges of DNA sequencing include poor quality in the first 15–40 bases of the sequence and deteriorating quality of sequencing traces after 700–900 bases. Base calling software typically gives an estimate of quality to aid in quality trimming.Sony VAIO VGN-Z25/B Battery

In cases where DNA fragments are cloned before sequencing, the resulting sequence may contain parts of the cloning vector. In contrast, PCR-based cloning and emerging sequencing technologies based on pyrosequencing often avoid using cloning vectors.Sony VAIO VGN-Z25TN/B Battery

Recently, one-step Sanger sequencing (combined amplification and sequencing) methods such as Ampliseq and SeqSharp have been developed that allow rapid sequencing of target genes without cloning or prior amplification.

Current methods can directly sequence only relatively short (300–1000 nucleotides long) DNA fragments in a single reaction. Sony VAIO VGN-Z26TN/B Battery

The main obstacle to sequencing DNA fragments above this size limit is insufficient power of separation for resolving large DNA fragments that differ in length by only one nucleotide. In all cases the use of a primer with a free 3' end is essential.

Automation and sample preparation

Automated DNA-sequencing instruments (DNA sequencers) can sequence up to 384 DNA samples in a single batch (run) in up to 24 runs a day. Sony VAIO VGN-Z27 Battery

DNA sequencers carry out capillary electrophoresis for size separation, detection and recording of dye fluorescence, and data output as fluorescent peak trace chromatograms. Sequencing reactions by thermocycling, cleanup and re-suspension in a buffer solution before loading onto the sequencer are performed separately. Sony VAIO VGN-Z27/B Battery

A number of commercial and non-commercial software packages can trim low-quality DNA traces automatically. These programs score the quality of each peak and remove low-quality base peaks (generally located at the ends of the sequence). The accuracy of such algorithms is below visual examination by a human operator, but sufficient for automated processing of large sequence data sets.Sony VAIO VGN-Z27TN/X Battery

Amplification and clonal selection

Large-scale sequencing often aims at sequencing very long DNA pieces, such as whole chromosomes, although large scale sequencing can also be used to generate very large numbers of short sequences, such as found in phage display. Sony VAIO VGN-Z29N Battery

For longer targets, such as chromosomes, common approaches consist of cutting (with restriction enzymes) or shearing (with mechanical forces) large DNA fragments into shorter DNA fragments. The fragmented DNA is cloned into a DNA vector, and amplified in Escherichia coli. Sony VAIO VGN-Z29N/X Battery

Short DNA fragments purified from individual bacterial colonies are individually sequenced and assembled electronically into one long, contiguous sequence.

This method does not require any pre-existing information about the sequence of the DNA and is referred to as de novosequencing.Sony VAIO VGN-Z31MN/B Battery

Gaps in the assembled sequence may be filled by primer walking. The different strategies have different tradeoffs in speed and accuracy; shotgun methods are often used for sequencing large genomes, but its assembly is complex and difficult, particularly with sequence repeats often causing gaps in genome assembly.Sony VAIO VGN-Z31VN/X Battery

Most sequencing approaches use an in vitro cloning step to amplify individual DNA molecules, because their molecular detection methods are not sensitive enough for single molecule sequencing. Emulsion PCR isolates individual DNA molecules along with primer-coated beads in aqueous droplets within an oil phase. Sony VAIO VGN-Z31WN/B Battery

Polymerase chain reaction (PCR) then coats each bead with clonal copies of the DNA molecule followed by immobilization for later sequencing. Emulsion PCR is used in the methods by Marguilis et al. (commercialized by 454 Life Sciences), Shendure and Porreca et al. (also known as "Polony sequencing") and SOLiD sequencing, (developed by Agencourt, now Applied Biosystems). Sony VAIO VGN-Z31ZN/X Battery

Another method for in vitro clonal amplification is bridge PCR, where fragments are amplified upon primers attached to a solid surface, used in the Illumina Genome Analyzer. Single-molecule methods, such as that developed by Stephen Quake's laboratory (later commercialized by Helicos) is an exception: Sony VAIO VGN-Z35 Battery

it uses bright fluorophores and laser excitation to detect base addition events from individual DNA molecules fixed to a surface, eliminating the need for molecular amplification.

High-throughput sequencing

The high demand for low-cost sequencing has driven the development of high-throughput sequencing technologies that parallelize the sequencing process, producing thousands or millions of sequences at once.Sony VAIO VGN-Z35/B Battery

High-throughput sequencing technologies are intended to lower the cost of DNA sequencing beyond what is possible with standard dye-terminator methods.

Lynx Therapeutics' Massively Parallel Signature Sequencing (MPSS)

The first of the "next-generation" sequencing technologies, MPSS was developed in the 1990s at Lynx Therapeutics, a company founded in 1992 by Sydney Brenner and Sam Eletr. Sony VAIO VGN-Z35TN/B Battery

MPSS was a bead-based method that used a complex approach of adapter ligation followed by adapter decoding, reading the sequence in increments of four nucleotides; this method made it susceptible to sequence-specific bias or loss of specific sequences.Sony VAIO VGN-Z36GD Battery

Because the technology was so complex, MPSS was only performed 'in-house' by Lynx Therapeutics and no machines were sold; when the merger with Solexa later led to the development of sequencing-by-synthesis, a more simple approach with numerous advantages, MPSS became obsolete. Sony VAIO VGN-Z36GD/B Battery

However, the essential properties of the MPSS output were typical of later "next-gen" data types, including hundreds of thousands of short DNA sequences. In the case of MPSS, these were typically used for sequencing cDNA for measurements of gene expression levels. Lynx Therapeutics merged with Solexa in 2004, and this company was later purchased by Illumina.Sony VAIO VGN-Z36GD/J Battery

Polony sequencing

Polony sequencing, developed in the laboratory of George Church at Harvard, was among the first next-generation sequencing systems used to sequence a full genome in 2005. It combined an in vitro paired-tag library with emulsion PCR, Sony VAIO VGN-Z36TD/B Battery

an automated microscope, and ligation-based sequencing chemistry to sequence an E. coli genome at an accuracy of > 99.9999% and a cost approximately 1/10 that of Sanger sequencing. The technology was licensed to Agencourt Biosciences, subsequently spun out into Agencourt Personal Genomics, and ultimately incorporated into the Applied Biosystems SOLiD platform.Sony VAIO VGN-Z36TD/J Battery

454 pyrosequencing

A parallelized version of pyrosequencing was developed by 454 Life Sciences, which has since been acquired by Roche Diagnostics. The method amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony. Sony VAIO VGN-Z37D Battery

The sequencing machine contains many picolitre-volume wells each containing a single bead and sequencing enzymes. Pyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs.Sony VAIO VGN-Z37D/B Battery

This technology provides intermediate read length and price per base compared to Sanger sequencing on one end and Solexa and SOLiD on the other.

Illumina (Solexa) sequencing

Solexa, now part of Illumina, developed a sequencing technology based on reversible dye-terminators. Sony VAIO VGN-Z37GD Battery

DNA molecules are first attached to primers on a slide and amplified so that local clonal colonies are formed (bridge amplification). Four types of ddNTPs are added, and non-incorporated nucleotides are washed away. Unlike pyrosequencing, the DNA can only be extended one nucleotide at a time. Sony VAIO VGN-Z37GD/X Battery

A camera takes images of the fluorescently labeled nucleotides, then the dye along with the terminal 3' blocker is chemically removed from the DNA, allowing the next cycle.

SOLiD sequencing

Applied Biosystems' SOLiD technology employs sequencing by ligation. Here, a pool of all possible oligonucleotides of a fixed length are labeled according to the sequenced position.Sony VAIO VGN-Z39D Battery

Oligonucleotides are annealed and ligated; the preferential ligation by DNA ligase for matching sequences results in a signal informative of the nucleotide at that position. Before sequencing, the DNA is amplified by emulsion PCR. The resulting bead, each containing only copies of the same DNA molecule, are deposited on a glass slide.Sony VAIO VGN-Z39D/X Battery

The result is sequences of quantities and lengths comparable to Illumina sequencing.

Ion semiconductor sequencing

Ion Torrent Systems Inc. developed a system based on using standard sequencing chemistry, but with a novel, semiconductor based detection system. Sony VAIO VGN-Z41MD/B Battery

This method of sequencing is based on the detection of hydrogen ions that are released during the polymerisation of DNA, as opposed to the optical methods used in other sequencing systems. A microwell containing a template DNA strand to be sequenced is flooded with a single type of nucleotide.Sony VAIO VGN-Z41WD/B Battery

If the introduced nucleotide is complementary to the leading template nucleotide it is incorporated into the growing complementary strand. This causes the release of a hydrogen ion that triggers a hypersensitive ion sensor, which indicates that a reaction has occurred.Sony VAIO VGN-Z45GD/B Battery

If homopolymer repeats are present in the template sequence multiple nucleotides will be incorporated in a single cycle. This leads to a corresponding number of released hydrogens and a proportionally higher electronic signal.

DNA nanoball sequencing

DNA nanoball sequencing is a type of high throughput sequencing technology used to determine the entire genomic sequence of an organism. Sony VAIO VGN-Z45TD/B Battery

The company Complete Genomics uses this technology to sequence samples that researchers submit from several projects. The method uses rolling circle replication to amplify small fragments of genomic DNA into DNA nanoballs. Unchained sequencing by ligation is then used to determine the nucleotide sequence.Sony VAIO VGN-Z46GD/B Battery

This method of DNA sequencing allows large numbers of DNA nanoballs to be sequenced per run and at low reagent costs compared to other next generation sequencing platforms. However, only short sequences of DNA are determined from each DNA nanoball which makes mapping the short reads to a reference genome difficult.Sony VAIO VGN-Z46GD/U Battery

This technology has been used for multiple genome sequencing projects and is scheduled to be used for more.

Helioscope(TM) single molecule sequencing

Based on "true single molecule sequencing" technology, Helioscope sequencing uses DNA fragments with added polyA tail adapters, which are attached to the flow cell surface. Sony VAIO VGN-Z46MD/B Battery

The next steps involve extension-based sequencing with cyclic washes of the flow cell with fluorescently labeled nucleotides (one nucleotide type at a time, as with the Sanger method). The reads are performed by the Helioscope sequencer. The reads are short, up to 55 bases per run, but recent improvemend of the methodology allowes more accurate reads of homopolymers (stretches of one type of nucleotides) and RNA sequencing.Sony VAIO VGN-Z46SD/B Battery

Single Molecule SMRT(TM) sequencing

SMRT sequencing is based on the sequencing by synthesis approach. The DNA is synthesisd in so calles zero-mode wave-guides (ZMWs) - small well-like containers with the capturing tools located at the bottom of the well. Sony VAIO VGN-Z46TD/B Battery

The sequencing is performed with use of unmodified polymerase (attached to the ZMW bottom) and fluorescently labelled nucleotides flowing freely in the solution. The wells are constructed in a way that only the fluorescence occurring by the bottom of the well is detected. Sony VAIO VGN-Z46TD/R Battery

The fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand. According to Pacific Biosciences, the SMTR technology developer, this methodology allows detection of nucleotide modifications (such ad cytosine methylation). This happens through the observation of polymerase kinetics. This approach allows reads of 1000 nucleotides.Sony VAIO VGN-Z47GD/X Battery

Single Molecule real time (RNAP) sequencing

This method is based on RNA polymerase (RNAP), which is attached to a polystyrene bead, with distal end of sequenced DNA is attached to another bead, with both beads being placed in optical traps. RNAP motion during transcription brings the beads in closer and their relative distance changes, which can then be recorded at a single nucleotide resolution. Sony VAIO VGN-Z48GD/X Battery

The sequence is deduced based on the four readouts with lowered concentrations of each of the four nucleotide types (similarly to Sangers method).

Nanopore DNA sequencing

This method is based on the readout of electrical signal occurring at nucleotides passing by alpha-hemolysin pores covalently bound with cyclodextrin. The DNA passing through the nanopore changes its ion current. Sony VAIO VGN-Z48TD/X Battery

This change is dependent on the shape, size and length of the DNA sequence. Each type of the nucleotide blocks the ion flow through the pore for a different period of time. The method has a potential of development as it does not require modified nucleotides, however single nucleotide resolution is not yet available.Sony VAIO VGN-Z51WG/B Battery

VisiGen Biotechnologies approach

VisiGen Biotechnologies introduced a specially engineered DNA polymerase for use in their sequencing. This polymerase acts as a sensor - having incorporated a donor fluorescent dye by its active centre. This donor dye acts by FRET (fluorescent resonant energy transfer), inducing fluorescence of differently labeled nucleotides. Sony VAIO VGN-Z51XG/B Battery

This approach allows reads performed at the spead at which polymerase incorporates nucleotides into the sequence (several hundred per second). The nucleotide fluorochrome is released after the incorporation into the DNA strand. The expected read lengths in this approach should reach 1000 nucleotides, however this will have to be confirmed.Sony VAIO VGN-Z530N/B Battery

Future methods

Sequencing by hybridization is a non-enzymatic method that uses a DNA microarray. A single pool of DNA whose sequence is to be determined is fluorescently labeled and hybridized to an array containing known sequences. Strong hybridization signals from a given spot on the array identifies its sequence in the DNA being sequenced.Sony VAIO VGN-Z540EBB Battery

Mass spectrometry may be used to determine mass differences between DNA fragments produced in chain-termination reactions.

DNA sequencing methods currently under development include labeling the DNA polymerase, reading the sequence as a DNA strand transits through nanopores, Sony VAIO VGN-Z540NLB Battery

and microscopy-based techniques, such as AFM or transmission electron microscopy that are used to identify the positions of individual nucleotides within long DNA fragments (>5,000 bp) by nucleotide labeling with heavier elements (e.g., halogens) for visual detection and recording.Sony VAIO VGN-Z540NMB Battery

Third generation technologies aim to increase throughput and decrease the time to result and cost by eliminating the need for excessive reagents and harnessing the processivity of DNA polymerase.

In microfluidic Sanger sequencing the entire thermocycling amplification of DNA fragments as well as their separation by electrophoresis is done on a single glass wafer (approximately 10 cm in diameter) thus reducing the reagent usage as well as cost.Sony VAIO VGN-Z550N/B Battery

In some instances researchers have shown that they can increase the throughput of conventional sequencing through the use of microchips. Research will still need to be done in order to make this use of technology effective.

In October 2006, the X Prize Foundation established an initiative to promote the development of full genome sequencing technologies, Sony VAIO VGN-Z55F Battery

called the Archon X Prize, intending to award $10 million to "the first Team that can build a device and use it to sequence 100 human genomes within 10 days or less, with an accuracy of no more than one error in every 100,000 bases sequenced, with sequences accurately covering at least 98% of the genome, and at a recurring cost of no more than $10,000 (US) per genome."Sony VAIO VGN-Z55TG/B Battery

Each year NHGRI promotes grants for new research and developments in genomics. 2010 grants and 2011 candidates include continuing work in microfluidic, polony and base-heavy sequencing methodologies.Sony VAIO VGN-Z56GG/B Battery,Sony VAIO VGN-Z56GG/E Battery,Sony VAIO VGN-Z56GGX Battery