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Applications of PCRMedical applications

PCR has been applied to a large number of medical procedures:

The first application of PCR was forgenetic testing, where a sample of DNA is

analyzed for the presence ofgenetic diseasemutations. Prospective parents can be

tested for beinggenetic carriers, or their children might be tested for actually being

affected by adisease. DNA samples forPrenatal testingcan be obtained

byamniocentesis,chorionic villus sampling, or even by the analysis of rare fetal cells

circulating in the mother's bloodstream. PCR analysis is also essential

toPreimplantation genetic diagnosis, where individual cells of a developing embryo are

tested for mutations.

PCR can also be used as part of a sensitive test fortissue typing,vital toorgan

transplantation. As of 2008, there is even a proposal to replace the traditional antibody-

based tests forblood typewith PCR-based tests.

Many forms of cancer involve alterations tooncogenes. By using PCR-based tests to

study these mutations, therapy regimens can sometimes be individually customized to a

patient.

Infectious disease applications

Characterization and detection of infectious disease organisms have been revolutionized by

PCR:

The Human Immunodeficiency Virus (orHIV), of antibodies to the virus circulating in

the bloodstream. However, antibodies don't appear until many weeks after infection,

maternal antibodies mask the infection of a newborn, and therapeutic agents to fight the

infection don't affect the antibodies. PCRtestshave been developed that can detect as

little as one viral genome among the DNA of over 50,000 host cells. Infections can be

detected earlier, donated blood can be screened directly for the virus, newborns can be

immediately tested for infection, and the effects of antiviral treatments can bequantified.
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Some disease organisms, such as that forTuberculosis, are difficult to sample from

patients and slow to begrownin the laboratory. PCR-based tests have allowed

detection of small numbers of disease organisms (both live or dead), in

convenientsamples. Detailed genetic analysis can also be used to detect antibiotic

resistance, allowing immediate and effective therapy. The effects of therapy can also be

immediately evaluated.

The spread of adiseaseorganism through populations ofdomesticorwildanimals can

be monitored by PCR testing. In many cases, the appearance of new virulent sub-

typescan be detected and monitored. The sub-types of an organism that were

responsible forearlier epidemicscan also be determined by PCR analysis.

Forensic applications]

The development of PCR-basedgenetic(orDNA) fingerprinting protocols has seen widespread

application inforensics:

In its most discriminating form,Genetic fingerprintingcan uniquely discriminate any

one person from the entire population of theworld. Minute samples of DNA can be

isolated from acrime scene, andcomparedto that from suspects, or from aDNA

databaseof earlier evidence or convicts. Simpler versions of these tests are often used

to rapidly rule out suspects during a criminal investigation. Evidence from decades-old

crimes can be tested, confirming orexoneratingthe people originally convicted.

Less discriminating forms ofDNA fingerprintingcan help inParental testing, where an

individual is matched with their close relatives. DNA from unidentified human remains

can be tested, and compared with that from possible parents, siblings, or children.

Similar testing can be used to confirm the biological parents of an adopted (or

kidnapped) child. The actual biological father of anewborncan also beconfirmed(or

ruled out).

Research applications

PCR has been applied to many areas of research in molecular genetics:

PCR allows rapid production of short pieces of DNA, even when nothing more than the

sequence of the two primers is known. This ability of PCR augments many methods,
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such as generatinghybridizationprobesforSouthernornorthern blothybridization.

PCR supplies these techniques with large amounts of pure DNA, sometimes as a single

strand, enabling analysis even from very small amounts of starting material.

The task ofDNA sequencingcan also be assisted by PCR. Known segments of DNAcan easily be produced from a patient with a genetic disease mutation. Modifications to

the amplification technique can extract segments from a completely unknown genome,

or can generate just a single strand of an area of interest.

PCR has numerous applications to the more traditional process ofDNA cloning. It can

extract segments for insertion into a vector from a larger genome, which may be only

available in small quantities. Using a single set of 'vector primers', it can also analyze or

extract fragments that have already been inserted into vectors. Some alterations to the

PCR protocol can generate mutations (general or site-directed) of an inserted

fragment.

Sequence-tagged sitesis a process where PCR is used as an indicator that a

particular segment of a genome is present in a particular clone. TheHuman Genome

Projectfound this application vital to mapping the cosmid clones they were sequencing,

and to coordinating the results from different laboratories.

An exciting application of PCR is thephylogenicanalysis of DNA fromancient sources,

such as that found in the recovered bones ofNeanderthals, or from frozen tissues

ofMammoths. In some cases the highly degraded DNA from these sources might be

reassembled during the early stages of amplification.

A common application of PCR is the study of patterns ofgene expression. Tissues (or

even individual cells) can be analyzed at different stages to see which genes have

become active, or which have been switched off. This application can also useQ-

PCRto quantitate the actual levels of expression

The ability of PCR to simultaneously amplify several loci from individual sperm[4]has

greatly enhanced the more traditional task ofgenetic mappingby

studyingchromosomal crossoversaftermeiosis. Rare crossover events between very

close loci have been directly observed by analyzing thousands of individual sperms.

Similarly, unusual deletions, insertions, translocations, or inversions can be analyzed, all
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without having to wait (or pay for) the long and laborious processes of fertilization,

embryogenesis, etc.

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DNA sequencing

DNA sequencing is the process of determining the precise order ofnucleotideswithin

aDNAmolecule. It includes any method or technology that is used to determine the order of the

four basesadenine,guanine,cytosine, andthyminein a strand of DNA. The advent of rapid

DNA sequencing methods has greatly accelerated biological and medical research and

discovery.

Knowledge of DNA sequences has become indispensable for basic biological research, and in

numerous applied fields such as diagnostic, biotechnology,forensic biology, and

biologicalsystematics. The rapid speed of sequencing attained with modern DNA sequencing

technology has been instrumental in the sequencing of complete DNA sequences,

orgenomesof numerous types and species of life, including thehuman genomeand other

complete DNA sequences of many animal, plant, andmicrobialspecies.

The first DNA sequences were obtained in the early 1970s by academic researchers using

laborious methods based ontwo-dimensional chromatography. Following the developmentoffluorescence-based sequencing methods withautomated analysis,[1]DNA sequencing has

become easier and orders of magnitude faster.

Use of sequencing

DNA sequencing may be used to determine the sequence of individual genes, larger genetic

regions (i.e. clusters of genes oroperons), full chromosomes or entire genomes. Depending on

the methods used, sequencing may provide the order of nucleotides in DNA orRNAisolated

from cells of animals, plants, bacteria,archaea, or virtually any other source of genetic

information. The resulting sequences may be used by researchers inmolecular

biologyorgeneticsto further scientific progress or may be used by medical personnel to make

treatment decisions or aid ingenetic counseling.
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History

Though the structure of DNA was established as adouble helixin 1953,]several decades would

pass before fragments of DNA could be reliably analyzed for their sequence in the laboratory.

RNA sequencing was one of the earliest forms of nucleotide sequencing. The major landmark ofRNA sequencing is the sequence of the first complete gene and the complete genome

ofBacteriophage MS2, identified and published byWalter Fiersand his coworkers at

theUniversity of Ghent(Ghent,Belgium), in 1972 and 1976.

Several notable advancements in DNA sequencing were made during the 1970s. Frederick

Sangerdeveloped rapid DNA sequencing methods at theMRC Centre,Cambridge, UK and

published a method for "DNA sequencing with chain-terminating inhibitors" in 1977.Walter

GilbertandAllan MaxamatHarvardalso developed sequencing methods, including one for

"DNA sequencing by chemical degradation". In 1973, Gilbert and Maxam reported the sequence

of 24 basepairs using a method known as wandering-spot analysis.[ Advancements in

sequencing were aided by the concurrent development ofrecombinant DNAtechnology,

allowing DNA samples to be isolated from sources other than viruses.

The first full DNA genome to be sequenced was that ofbacteriophage X174in 1977Medical

Research Councilscientists deciphered the complete DNA sequence of theEpstein-Barr virusin

1984, finding it to be 170 thousand base-pairs long.

Leroy E. Hood's laboratory at theCalifornia Institute of Technologyand Smith announced the

first semi-automated DNA sequencing machine in 1986. This was followed byAppliedBiosystems' marketing of the first fully automated sequencing machine, the ABI 370, in 1987. By

1990, the U.S.National Institutes of Health(NIH) had begun large-scale sequencing trials

onMycoplasma capricolum,Escherichia coli,Caenorhabditis elegans, andSaccharomyces

cerevisiaeat a cost of US$0.75 per base. Meanwhile, sequencing of humancDNAsequences

calledexpressed sequence tagsbegan inCraig Venter's lab, an attempt to capture the coding

fraction of thehuman genome.

In 1995, Venter,Hamilton Smith, and colleagues atThe Institute for Genomic Research(TIGR)

published the first complete genome of a free-living organism, the bacteriumHaemophilus

influenzae. The circular chromosome contains 1,830,137 bases and its publication in the journal

Science]marked the first published use of whole-genome shotgun sequencing, eliminating the

need for initial mapping efforts. By 2001, shotgun sequencing methods had been used to

produce a draft sequence of the human genome.
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ipedia.org/wiki/DNA_sequencing#cite_note-12http://en.wikipedia.org/wiki/DNA_sequencing#cite_note-12http://en.wikipedia.org/wiki/DNA_sequencing#cite_note-12http://en.wikipedia.org/wiki/DNA_sequencing#cite_note-12http://en.wikipedia.org/wiki/Haemophilus_influenzaehttp://en.wikipedia.org/wiki/Haemophilus_influenzaehttp://en.wikipedia.org/wiki/The_Institute_for_Genomic_Researchhttp://en.wikipedia.org/wiki/Hamilton_O._Smithhttp://en.wikipedia.org/wiki/Human_genomehttp://en.wikipedia.org/wiki/Craig_Venterhttp://en.wikipedia.org/wiki/Expressed_sequence_taghttp://en.wikipedia.org/wiki/CDNAhttp://en.wikipedia.org/wiki/Saccharomyces_cerevisiaehttp://en.wikipedia.org/wiki/Saccharomyces_cerevisiaehttp://en.wikipedia.org/wiki/Caenorhabditis_eleganshttp://en.wikipedia.org/wiki/Escherichia_colihttp://en.wikipedia.org/wiki/Mycoplasma_capricolumhttp://en.wikipedia.org/wiki/National_Institutes_of_Healthhttp://en.wikipedia.org/wiki/Applied_Biosystemshttp://en.wikipedia.org/wiki/Applied_Biosystemshttp://en.wikipedia.org/wiki/California_Institute_of_Technologyhttp://en.wikipedia.org/wiki/Leroy_E._Hoodhttp://en.wikipedia.org/wiki/Epstein-Barr_virushttp://en.wikipedia.org/wiki/Medical_Research_Council_(UK)http://en.wikipedia.org/wiki/Medical_Research_Council_(UK)http://en.wikipedia.org/wiki/Bacteriophage_%CF%86X174http://en.wikipedia.org/wiki/Recombinant_DNAhttp://en.wikipedia.org/wiki/DNA_sequencing#cite_note-9http://en.wikipedia.org/wiki/Harvard_Universityhttp://en.wikipedia.org/wiki/Allan_Maxamhttp://en.wikipedia.org/wiki/Walter_Gilberthttp://en.wikipedia.org/wiki/Walter_Gilberthttp://en.wikipedia.org/wiki/Cambridgehttp://en.wikipedia.org/wiki/Medical_Research_Council_(United_Kingdom)http://en.wikipedia.org/wiki/Frederick_Sangerhttp://en.wikipedia.org/wiki/Frederick_Sangerhttp://en.wikipedia.org/wiki/Belgiumhttp://en.wikipedia.org/wiki/Ghenthttp://en.wikipedia.org/wiki/University_of_Ghenthttp://en.wikipedia.org/wiki/Walter_Fiershttp://en.wikipedia.org/wiki/Bacteriophage_MS2http://en.wikipedia.org/wiki/DNA_sequencing#cite_note-pmid13168976-3http://en.wikipedia.org/wiki/DNA_double_helix


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Several new methods for DNA sequencing were developed in the mid to late 1990s. These

techniques comprise the first of the "next-generation" sequencing methods. In 1996, Pl

Nyrnand his studentMostafa Ronaghiat the Royal Institute of Technology

inStockholmpublished their method ofpyrosequencing

A year later, Pascal Mayer and Laurent Farinelli submitted patents to the World Intellectual

Property Organization describing DNA colony sequencing. Lynx Therapeutics published and

marketed "Massively parallel signature sequencing", or MPSS, in 2000. This method

incorporated a parallelized, adapter/ligation-mediated, bead-based sequencing technology and

served as the first commercially available "next-generation" sequencing method, though

noDNA sequencerswere sold to independent laboratories. In 2004,454 Life

Sciencesmarketed a parallelized version of pyrosequencing. The first version of their machine

reduced sequencing costs 6-fold compared to automated Sanger sequencing, and was the

second of the new generation of sequencing technologies, after MPSS.

The large quantities of data produced by DNA sequencing have also required development of

new methods and programs for sequence analysis. Phil Green and Brent Ewing of the

University of Washington described theirphred quality scorefor sequencer data analysis in

1998.

Basic methods

Maxam-Gilbert sequencing

Allan MaxamandWalter Gilbertpublished a DNA sequencing method in 1977 based onchemical modification of DNA and subsequent cleavage at specific bases.[7]Also known as

chemical sequencing, this method allowed purified samples of double-stranded DNA to be used

without further cloning. This method's use of radioactive labeling and its technical complexity

discouraged extensive use after refinements in the Sanger methods had been made.

Maxam-Gilbert sequencing requires radioactive labeling at one 5' end of the DNA and

purification of the DNA fragment to be sequenced. Chemical treatment then 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). The concentration of the modifying chemicals is controlled to introduce on average onemodification per DNA molecule. Thus a series of labeled fragments is generated, from the

radiolabeled end to the first "cut" site in each molecule. 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.
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Chain-termination methods

Thechain-termination methoddeveloped by Frederick Sanger and coworkers in 1977 soon

became the method of choice, owing to its relative ease and reliability. When invented, the

chain-terminator method used fewer toxic chemicals and lower amounts of radioactivity than the

Maxam and Gilbert method. Because of its comparative ease, the Sanger method was soon

automated and was the method used in the first generation ofDNA sequencers.

Sanger sequencing is the method which prevailed from the 80's until the mid-2000's. Over that

period, great advances were made in the technique, such as fluorescent labelling, capillary

electrophoresis, and general automation. These developments allowed much more efficient

sequencing, leading to lower costs. The Sanger method, in mass production form, is the

technology which produced thefirst human genomein 2001, ushering in the age ofgenomics.

However, later in the decade, radically different approaches reached the market, bringing the

cost per genome down from $100 million in 2001 to $10,000 in 2011.

Advanced methods and de novo sequencing
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Genomic DNA is fragmented into random pieces and cloned as a bacterial library. DNA from

individual bacterial clones is sequenced and the sequence is assembled by using overlapping

DNA regions.(click to expand)

Large-scale sequencing often aims at sequencing very long DNA pieces, such as

wholechromosomes, although large-scale sequencing can also be used to generate very large

numbers of short sequences, such as found inphage display. For longer targets such as

chromosomes, common approaches consist of cutting (withrestriction enzymes) or shearing

(with mechanical forces) large DNA fragments into shorter DNA fragments. The fragmented

DNA may then beclonedinto aDNA vectorand amplified in a bacterial host such

asEscherichia coli. Short DNA fragments purified from individual bacterial colonies are

individually sequenced andassembled electronicallyinto one long, contiguous sequence.

The term "de novo sequencing" specifically refers to methods used to determine the sequence

of DNA with no previously known sequence. De novo translates from Latin as "from the

beginning". Gaps in the assembled sequence may be filled byprimer walking. The different

strategies have different tradeoffs in speed and accuracy;shotgun methodsare often used for

sequencing large genomes, but its assembly is complex and difficult, particularly withsequence

repeatsoften causing gaps in genome assembly.

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. Apolymerase 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 developed by Marguilis et al. (commercialized by454 Life

Sciences), Shendure and Porreca et al. (also known as "Polony sequencing") andSOLiD

sequencing, (developed byAgencourt, laterApplied Biosystems, nowLife Technologies).

Shotgun sequencing

Shotgun sequencing is a sequencing method designed for analysis of DNA sequences longer

than 1000 base pairs, up to and including entire chromosomes. This method requires the target

DNA to be broken into random fragments. After sequencing individual fragments, the sequences

can be reassembled on the basis of their overlapping regions.

Bridge PCR

Another method forin vitroclonal amplification is bridge PCR, in which fragments are amplified

upon primers attached to a solid surface and form "DNA colonies" or "DNA clusters". This
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method is used in theIlluminaGenome Analyzersequencers. Single-molecule methods, such

as that developed byStephen Quake's laboratory (later commercialized byHelicos) are an

exception: they use bright fluorophores and laser excitation to detect base addition events from

individual DNA molecules fixed to a surface, eliminating the need for molecular amplification.

Next-generation methods

The high demand for low-cost sequencing has driven the development of high-throughput

sequencing (or next-generation sequencing) technologies thatparallelizethe sequencing

process, producing thousands or millions of sequences concurrently. High-throughput

sequencing technologies are intended to lower the cost of DNA sequencing beyond what is

possible with standard dye-terminator methods.]In ultra-high-throughput sequencing as many

as 500,000 sequencing-by-synthesis operations may be run in parallel.

Multiple, fragmented sequence reads must be assembled together on the basis of theiroverlapping areas.

Massively parallel signature sequencing (MPSS)

The first of the next-generation sequencing technologies,massively parallel signature

sequencing(or MPSS), was developed in the 1990s at Lynx Therapeutics, a company founded

in 1992 bySydney BrennerandSam Eletr. 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. Because the technology was so complex, MPSS was only

performed 'in-house' by Lynx Therapeutics and no DNA sequencing machines were sold to

independent laboratories. Lynx Therapeutics merged with Solexa (later acquired byIllumina) in

2004, leading to the development of sequencing-by-synthesis, a more simple approach

acquired fromManteia Predictive Medicine, which rendered MPSS obsolete. However, the

essential properties of the MPSS output were typical of later "next-generation" data types,

including hundreds of thousands of short DNA sequences. In the case of MPSS, these were

typically used for sequencingcDNAfor measurements ofgene expressionlevels.

Polony sequencing

ThePolony sequencingmethod, developed in the laboratory ofGeorge M. Churchat Harvard,

was among the first next-generation sequencing systems and was used to sequence a full

genome in 2005. It combined an in vitro paired-tag library with emulsion PCR, an automated

microscope, and ligation-based sequencing chemistry to sequence an E. coligenome at an

accuracy of >99.9999% and a cost approximately 1/9 that of Sanger sequencing.[45]The
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technology was licensed to Agencourt Biosciences, subsequently spun out into Agencourt

Personal Genomics, and eventually incorporated into the Applied Biosystems SOLiD platform,

which is now owned byLife Technologies.

454 pyrosequencing

A parallelized version ofpyrosequencingwas developed by454 Life Sciences, which has since

been acquired byRoche 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. The sequencing machine contains

manypicoliter-volume wells each containing a single bead and sequencing enzymes.

Pyrosequencing usesluciferaseto generate light for detection of the individual nucleotides

added to the nascent DNA, and the combined data are used to generate sequence read-

outs. 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 ofIllumina, developed a sequencing method based on reversible dye-

terminators technology, and engineered polymerases, that it developed internally. The

terminated chemistry was developed internally at Solexa and the concept of the Solexa system

was invented by Balasubramanian and Klennerman from Cambridge University's chemistry

department. In 2004, Solexa acquired the companyManteia Predictive Medicinein order to gain

a massivelly parallel sequencing technology based on "DNA Clusters", which involves the clonal

amplification of DNA on a surface. The cluster technology was co-acquired with Lynx

Therapeutics of California. Solexa Ltd. later merged with Lynx to form Solexa Inc.

In this method, DNA molecules and primers are first attached on a slide and amplified

withpolymeraseso that local clonal DNA colonies, later coined "DNA clusters", are formed. To

determine the sequence, four types of reversible terminator bases (RT-bases) are added and

non-incorporated nucleotides are washed away. A camera takes images of thefluorescently

labelednucleotides, then the dye, along with the terminal 3' blocker, is chemically removed from

the DNA, allowing for the next cycle to begin. Unlike pyrosequencing, the DNA chains are

extended one nucleotide at a time and image acquisition can be performed at a delayed

moment, allowing for very large arrays of DNA colonies to be captured by sequential images

taken from a single camera.

Decoupling the enzymatic reaction and the image capture allows for optimal throughput and

theoretically unlimited sequencing capacity. With an optimal configuration, the ultimately

reachable instrument throughput is thus dictated solely by the analog-to-digital conversion rate
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of the camera, multiplied by the number of cameras and divided by the number of pixels per

DNA colony required for visualizing them optimally (approximately 10 pixels/colony). In 2012,

with cameras operating at more than 10 MHz A/D conversion rates and available optics, fluidics

and enzymatics, throughput can be multiples of 1 million nucleotides/second, corresponding

roughly to 1 human genome equivalent at 1xcoverageper hour per instrument, and 1 human

genome re-sequenced (at approx. 30x) per day per instrument (equipped with a single camera).

SOLiD sequencing

Library preparation for the SOLiD platform

Applied Biosystems' (now aLife Technologiesbrand) SOLiD technology employssequencing

by ligation. Here, a pool of all possible oligonucleotides of a fixed length are labeled according

to the sequenced position. Oligonucleotides are annealed and ligated; the preferential ligation

byDNA ligasefor matching sequences results in a signal informative of the nucleotide at that

position. Before sequencing, the DNA is amplified by emulsion PCR. The resulting beads, each

containing single copies of the same DNA molecule, are deposited on a glass slide. The result

is sequences of quantities and lengths comparable to Illumina sequencing.

Ion semiconductor sequencing[

Ion Torrent Systems Inc. (now owned byLife Technologies) developed a system based on

using standard sequencing chemistry, but with a novel, semiconductor based detection system.

This method of sequencing is based on the detection ofhydrogen ionsthat are released during

thepolymerisationofDNA, as opposed to the optical methods used in other sequencing
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systems. A microwell containing a template DNA strand to be sequenced is flooded with a

single type ofnucleotide. If the introduced nucleotide iscomplementaryto 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. Ifhomopolymerrepeats 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.

Sequencing of the TAGGCT template with IonTorrent, PacBioRS and GridION

DNA nanoball sequencing

DNA nanoball sequencingis a type ofhigh throughput sequencingtechnology used to

determine the entiregenomic sequenceof an organism. The companyComplete

Genomicsuses this technology to sequence samples submitted by independent researchers.

The method usesrolling circle replicationto amplify small fragments of genomic DNA into DNA

nanoballs. Unchained sequencing by ligation is then used to determine the nucleotide

sequence. This method of DNA sequencing allows large numbers of DNA nanoballs to be

sequenced per run and at lowreagentcosts 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 areference genomedifficult. This technology has been

used for multiple genome sequencing projects and is scheduled to be used for more.

Heliscope single molecule sequencing

Heliscope sequencing is a method of single-molecule sequencing developed byHelicos

Biosciences. It uses DNA fragments with added poly-A tail adapters which are attached to the
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flow cell surface. 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 Heliscope sequencer. The reads are short, up

to 55 bases per run, but recent improvements allow for more accurate reads of stretches of one

type of nucleotides

This sequencing method and equipment were used to sequence the genome of theM13

bacteriophage.

Single molecule real time (SMRT) sequencing[

SMRT sequencing is based on the sequencing by synthesis approach. The DNA is synthesized

in zero-mode wave-guides (ZMWs) small well-like containers with the capturing tools located

at the bottom of the well. 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. The fluorescent label is detached from the nucleotide at its incorporation

into the DNA strand, leaving an unmodified DNA strand. According toPacific Biosciences, the

SMRT technology developer, this methodology allows detection of nucleotide modifications

(such as cytosine methylation). This happens through the observation of polymerase kinetics.

This approach allows reads of up to 15,000 nucleotides, with mean read lengths of 2.5 to 2.9

kilobases.

Methods in development

DNA sequencing methods currently under development include labeling the DNA polymerase,

reading the sequence as a DNA strand transits throughnanopores, and microscopy-based

techniques, such asatomic force microscopyortransmission electron microscopythat 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. 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.

Nanopore DNA sequencing

This method is based on the readout of electrical signals occurring at nucleotides passing by

alpha-hemolysinpores covalently bound withcyclodextrin. The DNA passing through the

nanopore changes its ion current. 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
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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.

Sequencing by hybridization

Sequencing by hybridizationis a non-enzymatic method that uses aDNA microarray. A singlepool 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.

Sequencing with mass spectrometry

Mass spectrometrymay be used to determine DNA sequences. Matrix-assisted laser desorption

ionization time-of-flight mass spectrometry, orMALDI-TOF MS, has specifically been

investigated as an alternative method to gel electrophoresis for visualizing DNA fragments. With

this method, DNA fragments generated by chain-termination sequencing reactions arecompared by mass rather than by size. The mass of each nucleotide is different from the others

and this difference is detectable by mass spectrometry. Single-nucleotide mutations in a

fragment can be more easily detected with MS than by gel electrophoresis alone. MALDI-TOF

MS can more easily detect differences between RNA fragments, so researchers may indirectly

sequence DNA with MS-based methods by converting it to RNA first.

The higher resolution of DNA fragments permitted by MS-based methods is of special interest to

researchers in forensic science, as they may wish to findsingle-nucleotide polymorphismsin

human DNA samples to identify individuals. These samples may be highly degraded so forensicresearchers often prefermitochondrial DNAfor its higher stability and applications for lineage

studies. MS-based sequencing methods have been used to compare the sequences of human

mitochondrial DNA from samples in aFederal Bureau of Investigationdatabase and from bones

found in mass graves of World War I soldiers.

Early chain-termination and TOF MS methods demonstrated read lengths of up to 100 base

pairs. Researchers have been unable to exceed this average read size; like chain-termination

sequencing alone, MS-based DNA sequencing may not be suitable for large de

novo sequencing projects. Even so, a recent study did use the short sequence reads and mass

spectroscopy to compare single-nucleotide polymorphisms in pathogenicStreptococcusstrains.

Microfluidic Sanger sequencing

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 .[70]In some instances researchers
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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.

Microscopy-based techniques

This approach directly visualizes the sequence of DNA molecules using electron microscopy.

The first identification of DNA base pairs within intact DNA molecules by enzymatically

incorporating modified bases, which contain atoms of increased atomic number, direct

visualization and identification of individually labeled bases within a synthetic 3,272 base-pair

DNA molecule and a 7,249 base-pair viral genome has been demonstrated.

RNAP sequencing

This method is based on use ofRNA polymerase(RNAP), which is attached to

apolystyrenebead. One end of DNA to be sequenced is attached to another bead, with bothbeads 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. The sequence is deduced based on the four readouts with lowered concentrations of

each of the four nucleotide types, similarly to the Sanger method.

In vitrovirus high-throughput sequencing

A method has been developed to analyze full sets ofprotein interactionsusing a combination of

454 pyrosequencing and an in vitro virusmRNA displaymethod. Specifically, this method

covalently links proteins of interest to the mRNAs encoding them, then detects the mRNApieces using reverse transcriptionPCRs. The mRNA may then be amplified and sequenced.

The combined method was titled IVV-HiTSeq and can be performed under cell-free conditions,

though its results may not be representative ofin vivo conditions.

Development initiatives

In October 2006, theX Prize Foundationestablished an initiative to promote the development

offull genome sequencingtechnologies, called theArchon 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."
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Each year theNational Human Genome Research Institute, or NHGRI, promotes grants for new

research and developments ingenomics. 2010 grants and 2011 candidates include continuing

work in microfluidic, polony and base-heavy sequencing methodologies.

Chemical DNA sequencing of DNA

The field ofbiotechnologyis one of constant change. The rapid growth anddevelopment of cutting-edge research is dependent on the innovation and creativity of scientists and their

ability to see the potential in a basic molecular technique and apply it to new processes. The advent

ofPCRopened up many doors in genetic research, including a means of DNA analysis and identificationof different genes based on theirDNAsequences. DNA sequencing is also dependent on our ability tousegel electrophoresisto separate strands of DNA that differ in size by as little as one base pair.

In the late 1970's, two DNA sequencing techniques for longer DNA molecules were invented. These werethe Sanger (or dideoxy) method and theMaxam-Gilbert (chemical cleavage) method. The Maxam-Gilbert method is based on nucleotide-specfic cleavage by chemicals and is best used to sequence

oligonucleotides (short nucleotide polymers, usually smaller than 50 base-pairs in length). The Sangermethod is more commonly used because it has been proven technically easier to apply, and, with theadvent of PCR and automation of the technique, is easily applied to long strands of DNA including some

entire genes. This technique is based on chain termination by dideoxy nucleotides during PCR elongationreactions.

In the Sanger method, the DNA strand to be analyzed is used as a template and DNA polymerase is used,in a PCR reaction, to generate complimentary strands using primers. Four different PCR reactionmixtures are prepared, each containing a certain percentage of dideoxynucleoside triphosphate (ddNTP)analogs to one of the four nucleotides (ATP, CTP, GTP or TTP). Synthesis of the new DNA strandcontinues until one of these analogs is incorporated, at which time the strand is prematurely truncated.

Each PCR reaction will end up containing a mixture of different lengths of DNA strands, all ending withthe nucleotide that was dideoxy labeled for that reaction. Gel electrophoresis is then used to separate thestrands of the four reactions, in four separate lanes, and determine the sequence of the original template

based on what lengths of strands end with what nucleotide.

In the automated Sanger reaction, primers are used that are labeled with four different colouredfluorescent tags. PCR reactions, in the presence of the different dideoxy nucleotides, are performed asdescribed above. However, next, the four reaction mixtures are then combined and applied to a single laneof a gel. The colour of each fragment is detected using a laser beam and the information is collected by acomputer which generates chromatograms showing peaks for each colour, from which the template DNAsequence can be determined.
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Typically, the automated sequencing method is only accurate for sequences up to a maximum of about700-800 base-pairs in length. However, it is possible to obtain full sequences of larger genes and, in fact,

whole genomes, using step-wise methods such as Primer Walking and Shotgun sequencing.

In Primer Walking, a workable portion of a larger gene is sequenced using the Sanger method. New

primers are generated from a reliable segment of the sequence, and used to continue sequencing theportion of the gene that was out of range of the original reactions.

Shotgun sequencing entails randomly cutting the DNA segment of interest into more appropriate

(manageable) sized fragments, sequencing each fragment, and arranging the pieces based on overlappingsequences. This technique has been made easier by the application of computer software for arr

=====================================================================================

RNA-Seq

RNA-seq, also called "Whole Transcriptome Shotgun Sequencing" ("WTSS"), refers to the use

ofhigh-throughput sequencingtechnologies to sequencecDNAin order to get information about

a sample'sRNAcontent. The technique has been adopted in studies of diseases like cancer.

With deep coverage and base-level resolution,next-generation sequencingprovides information

on differential expression of genes, including gene alleles and differently spliced transcripts;

non-coding RNAs; post-transcriptional mutations or editing; and gene fusions.

Introduction

The introduction ofnext-generation sequencingorhigh-throughput sequencingtechnologies

opened new doors into the field ofDNA sequencing, however as understanding of these

technologies becomes more widespread and new tools are developed, new innovative ways of

applying these technologies are being created.

Givenhigh-throughput sequencingtechnologies' low requirements of nucleotide sequence

product, together with its deep coverage and base-scale resolution, its use has expanded to the

field oftranscriptomics.]

Transcriptomics is an area of research characterizing the RNAtranscribed from a particulargenomeunder investigation.

Although transcriptomes are more dynamic than genomicDNA, these molecules provide direct

access togene regulationand protein information. Sequencing transcriptomes is not a new

idea, however: various methods have been developed previously to directly determine cDNA

sequences, based mostly around traditional (and more expensive)Sanger sequencing, while
http://en.wikipedia.org/wiki/High-throughput_sequencinghttp://en.wikipedia.org/wiki/High-throughput_sequencinghttp://en.wikipedia.org/wiki/High-throughput_sequencinghttp://en.wikipedia.org/wiki/CDNAhttp://en.wikipedia.org/wiki/CDNAhttp://en.wikipedia.org/wiki/CDNAhttp://en.wikipedia.org/wiki/RNAhttp://en.wikipedia.org/wiki/RNAhttp://en.wikipedia.org/wiki/RNAhttp://en.wikipedia.org/wiki/Cancerhttp://en.wikipedia.org/wiki/Cancerhttp://en.wikipedia.org/wiki/Cancerhttp://en.wikipedia.org/wiki/Next-generation_sequencinghttp://en.wikipedia.org/wiki/Next-generation_sequencinghttp://en.wikipedia.org/wiki/Next-generation_sequencinghttp://en.wikipedia.org/wiki/Next-generation_sequencinghttp://en.wikipedia.org/wiki/Next-generation_sequencinghttp://en.wikipedia.org/wiki/Next-generation_sequencinghttp://en.wikipedia.org/wiki/High-throughput_sequencinghttp://en.wikipedia.org/wiki/High-throughput_sequencinghttp://en.wikipedia.org/wiki/High-throughput_sequencinghttp://en.wikipedia.org/wiki/DNA_sequencinghttp://en.wikipedia.org/wiki/DNA_sequencinghttp://en.wikipedia.org/wiki/DNA_sequencinghttp://en.wikipedia.org/wiki/High-throughput_sequencinghttp://en.wikipedia.org/wiki/High-throughput_sequencinghttp://en.wikipedia.org/wiki/High-throughput_sequencinghttp://en.wikipedia.org/wiki/Transcriptomicshttp://en.wikipedia.org/wiki/Transcriptomicshttp://en.wikipedia.org/wiki/RNA-Seq#cite_note-wang2009-4http://en.wikipedia.org/wiki/RNA-Seq#cite_note-wang2009-4http://en.wikipedia.org/wiki/RNA-Seq#cite_note-wang2009-4http://en.wikipedia.org/wiki/Genomehttp://en.wikipedia.org/wiki/Genomehttp://en.wikipedia.org/wiki/Genomehttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/Gene_regulationhttp://en.wikipedia.org/wiki/Gene_regulationhttp://en.wikipedia.org/wiki/Gene_regulationhttp://en.wikipedia.org/wiki/Sanger_sequencinghttp://en.wikipedia.org/wiki/Sanger_sequencinghttp://en.wikipedia.org/wiki/Sanger_sequencinghttp://en.wikipedia.org/wiki/Sanger_sequencinghttp://en.wikipedia.org/wiki/Gene_regulationhttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/Genomehttp://en.wikipedia.org/wiki/RNA-Seq#cite_note-wang2009-4http://en.wikipedia.org/wiki/Transcriptomicshttp://en.wikipedia.org/wiki/High-throughput_sequencinghttp://en.wikipedia.org/wiki/DNA_sequencinghttp://en.wikipedia.org/wiki/High-throughput_sequencinghttp://en.wikipedia.org/wiki/Next-generation_sequencinghttp://en.wikipedia.org/wiki/Next-generation_sequencinghttp://en.wikipedia.org/wiki/Cancerhttp://en.wikipedia.org/wiki/RNAhttp://en.wikipedia.org/wiki/CDNAhttp://en.wikipedia.org/wiki/High-throughput_sequencing


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others include methodologies such asSeri

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