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The term DNA sequencing encompasses biochemical methods for determining the order of the nucleotide bases, adenine, guanine, cytosine, and thymine, in a DNA oligonucleotide. Biochemistry is the study of the chemical processes in living Organisms It deals with the Structure and function of cellular components such as Nucleotides are Organic compounds that consist of three joined structures a nitrogenous base a Sugar, and a Phosphate group Adenine is a Purine with a variety of roles in Biochemistry including Cellular respiration, in the form of both the energy-rich Adenosine Guanine is one of the five main Nucleobases found in the Nucleic acids DNA and RNA, the others being Adenine, Cytosine, Cytosine is one of the five main bases found in DNA and RNA. It is a Pyrimidine derivative with a Heterocyclic Aromatic ring Thymine is one of the four bases in the Nucleic acid of DNA that make up the letters ATGC Deoxyribonucleic acid ( DNA) is a Nucleic acid that contains the genetic instructions used in the development and functioning of all known An oligonucleotide (or oligo) is a short segment of RNA or DNA, typically with twenty or fewer bases. The sequence of DNA constitutes the heritable genetic information in nuclei, plasmids, mitochondria, and chloroplasts that forms the basis for the developmental programs of all living organisms. In Genetics, Heritability is the proportion of phenotypic variation in a population that is attributable to genetic variation among individuals In Cell biology, the nucleus (pl nuclei; from Latin la ''nucleus'' or la ''nuculeus'' "little nut" or kernel is a membrane-enclosed A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA In Cell biology, a mitochondrion (plural mitochondria) is a membrane-enclosed Organelle found in most eukaryotic cells. Chloroplasts are Organelles found in Plant cells and eukaryotic Algae that conduct Photosynthesis. Determining the DNA sequence is therefore useful in basic research studying fundamental biological processes, as well as in applied fields such as diagnostic or forensic research. The advent of DNA sequencing has significantly accelerated biological research and discovery. The rapid speed of sequencing attained with modern DNA sequencing technology has been instrumental in the large-scale sequencing of the human genome, in the Human Genome Project. The human genome is the Genome of Homo sapiens, which is stored on 23 chromosome pairs The Human Genome Project (HGP was an international Scientific research project with a primary goal to determine the sequence of chemical base pairs which make up DNA Related projects, often by scientific collaboration across continents, have generated the complete DNA sequences of many animal, plant, and microbial genomes.

DNA Sequence Trace
DNA Sequence Trace

Contents

Early methods

For thirty years, a large proportion of DNA sequencing has been carried out with the chain-termination method developed by Frederick Sanger and coworkers in 1975. Frederick Sanger, OM, CH, CBE, FRS (born 13 August 1918) is an English biochemist and twice [1][2] Prior to the development of rapid DNA sequencing methods in the early 1970s by Sanger in England and Walter Gilbert and Allan Maxam at Harvard,[3][4] a number of laborious methods were used. Walter Gilbert (born March 21, 1932) is an American physicist, biochemist, and Molecular biology pioneer Allan Maxam is one of the pioneers of Molecular genetics. He was one of the contributors to develop a DNA sequencing method at Harvard University, while For instance, in 1973[5] Gilbert and Maxam reported the sequence of 24 basepairs using a method known as wandering-spot analysis.

RNA sequencing, which for technical reasons is easier to perform than DNA sequencing, was one of the earliest forms of nucleotide sequencing. The major landmark of RNA sequencing, dating from the pre-recombinant DNA era, is the sequence of the first complete gene and then the complete genome of Bacteriophage MS2, identified and published by Walter Fiers and his coworkers at the University of Ghent (Ghent, Belgium), published between 1972[6] and 1976. The bacteriophage MS2. MS2 phage is an icosahedral bacteriophage with a diameter of 27-34nm and an isoelectric point (pI of 3 Walter Fiers (b Ieper, Belgium, 1931 is a Belgian molecular biologist Ghent University (in Dutch Universiteit Gent, abbreviated UGent) is one of the three large Flemish universities. Ghent (ˈɡɛnt Gent ʝɛnt in Dutch, Gand in French, and formerly Gaunt in English) is a City and a The Kingdom of Belgium is a Country in northwest Europe. It is a founding member of the European Union and hosts its headquarters as well as those [7]

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 [1]. Allan Maxam is one of the pioneers of Molecular genetics. He was one of the contributors to develop a DNA sequencing method at Harvard University, while Walter Gilbert (born March 21, 1932) is an American physicist, biochemist, and Molecular biology pioneer Although Maxam and Gilbert published their chemical sequencing method two years after the ground-breaking paper of Sanger and Coulson on plus-minus sequencing,[8][9] 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. However, with the development and improvement of the chain-termination method (see below), Maxam-Gilbert sequencing has fallen out of favour due to its technical complexity, extensive use of hazardous chemicals, and difficulties with scale-up. In addition, unlike the chain-termination method, chemicals used in the Maxam-Gilbert method cannot easily be customized for use in a standard molecular biology kit.

In brief, the method requires radioactive labelling at one end 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). Thus a series of labelled fragments is generated, from the radiolabelled end to the first 'cut' site in each molecule. The fragments are then size-separated by gel electrophoresis, with the four reactions arranged side by side. To visualize the fragments generated in each reaction, the gel is exposed to X-ray film for autoradiography, yielding an image of a series of dark 'bands' corresponding to the radiolabelled DNA fragments, from which the sequence may be inferred. For medical radiography see Radiology Radiography is the use of X-rays to view unseen or hard-to-image objects An autoradiograph is an image on an X-ray film or nuclear emulsion produced by the pattern of decay emissions (e Isotopic labeling is a technique for tracking the passage of a sample of substance through a system

Also sometimes known as 'chemical sequencing', this method originated in the study of DNA-protein interactions (footprinting), nucleic acid structure and epigenetic modifications to DNA, and within these it still has important applications.

Chain-termination methods

Part of a radioactively labelled sequencing gel
Part of a radioactively labelled sequencing gel

While the chemical sequencing method of Maxam and Gilbert, and the plus-minus method of Sanger and Coulson were orders of magnitude faster than previous methods, the chain-terminator method developed by Sanger was even more efficient, and rapidly became the method of choice. The Maxam-Gilbert technique requires the use of highly toxic chemicals, and large amounts of radiolabeled DNA, whereas the chain-terminator method uses fewer toxic chemicals and lower amounts of radioactivity. Isotopic labeling is a technique for tracking the passage of a sample of substance through a system The key principle of the Sanger method was the use of dideoxynucleotides triphosphates (ddNTPs) as DNA chain terminators. Dideoxynucleotides, or ddNTPs are Nucleotides lacking a 3'-hydroxyl (-OH group on their Deoxyribose sugar

The classical chain-termination or Sanger method requires a single-stranded DNA template, a DNA primer, a DNA polymerase, radioactively or fluorescently labeled nucleotides, and modified nucleotides that terminate DNA strand elongation. A primer is a strand of Nucleic acid that serves as a starting point for DNA replication. A DNA Polymerase is an Enzyme that assists in DNA replication. Fluorescence is a Luminescence that is mostly found as an The DNA sample is divided into four separate sequencing reactions, containing the four standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase. Nucleotides are Organic compounds that consist of three joined structures a nitrogenous base a Sugar, and a Phosphate group A DNA Polymerase is an Enzyme that assists in DNA replication. To each reaction is added only one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP). Dideoxynucleotides, or ddNTPs are Nucleotides lacking a 3'-hydroxyl (-OH group on their Deoxyribose sugar These dideoxynucleotides are the chain-terminating nucleotides, lacking a 3'-OH group required for the formation of a phosphodiester bond between two nucleotides during DNA strand elongation. Hydroxyl in Chemistry stands for a molecule consisting of an Oxygen atom and a Hydrogen atom connected by a Covalent bond. A phosphodiester bond is a group of strong covalent bonds between the phosphorus atom in a Phosphate group and two other Molecules over two Incorporation of a dideoxynucleotide into the nascent (elongating) DNA strand therefore terminates DNA strand extension, resulting in various DNA fragments of varying length. The dideoxynucleotides are added at lower concentration than the standard deoxynucleotides to allow strand elongation sufficient for sequence analysis.

The newly synthesized and labeled DNA fragments are heat denatured, and separated by size (with a resolution of just one nucleotide) by gel electrophoresis on a denaturing polyacrylamide-urea gel. A Polyacrylamide Gel is a separation matrix used in electrophoresis of Biomolecules, such as Proteins or DNA fragments Each of the four DNA synthesis reactions is run in one of four individual lanes (lanes A, T, G, C); the DNA bands are then visualized by autoradiography or UV light, and the DNA sequence can be directly read off the X-ray film or gel image. An autoradiograph is an image on an X-ray film or nuclear emulsion produced by the pattern of decay emissions (e For medical radiography see Radiology Radiography is the use of X-rays to view unseen or hard-to-image objects 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. 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 terminal nucleotide base can be identified according to which dideoxynucleotide was added in the reaction giving that band. The relative positions of the different bands among the four lanes are then used to read (from bottom to top) the DNA sequence as indicated.

DNA fragments can be labeled by using a radioactive or fluorescent tag on the primer (1), in the new DNA strand with a labeled dNTP, or with a labeled ddNTP. (click to expand)
DNA fragments can be labeled by using a radioactive or fluorescent tag on the primer (1), in the new DNA strand with a labeled dNTP, or with a labeled ddNTP. (click to expand)

There are some technical variations of chain-termination sequencing. In one method, the DNA fragments are tagged with nucleotides containing radioactive phosphorus for radiolabelling. Isotopic labeling is a technique for tracking the passage of a sample of substance through a system Alternatively, a primer labeled at the 5’ end with a fluorescent dye is used for the tagging. Fluorescence is a Luminescence that is mostly found as an Four separate reactions are still required, but DNA fragments with dye labels can be read using an optical system, facilitating faster and more economical analysis and automation. This approach is known as 'dye-primer sequencing'. The later development by L Hood and coworkers[10][11] of fluorescently labeled ddNTPs and primers set the stage for automated, high-throughput DNA sequencing.

Sequence ladder by radioactive sequencing compared to fluorescent peaks (click to expand)
Sequence ladder by radioactive sequencing compared to fluorescent peaks (click to expand)

The different chain-termination methods have greatly simplified the amount of work and planning needed for DNA sequencing. For example, the chain-termination-based "Sequenase" kit from USB Biochemicals contains most of the reagents needed for sequencing, prealiquoted and ready to use. USB Corporation offers life science products for use in basic research drug discovery and molecular diagnostics Some sequencing problems can occur with the Sanger method, such as non-specific binding of the primer to the DNA, affecting accurate read-out of the DNA sequence. In addition, secondary structures within the DNA template, or contaminating RNA randomly priming at the DNA template can also affect the fidelity of the obtained sequence. Other contaminants affecting the reaction may consist of extraneous DNA or inhibitors of the DNA polymerase.

Dye-terminator sequencing

Capillary electrophoresis (click to expand)
Capillary electrophoresis (click to expand)

An alternative to primer labelling is labelling of the chain terminators, a method commonly called 'dye-terminator sequencing'. The major advantage of this method is that the sequencing can be performed in a single reaction, rather than four reactions as in the labelled-primer method. In dye-terminator sequencing, each of the four dideoxynucleotide chain terminators is labelled with a different fluorescent dye, each fluorescing at a different wavelength. In Physics wavelength is the distance between repeating units of a propagating Wave of a given Frequency. This method is attractive because of its greater expediency and speed and is now the mainstay in automated sequencing with computer-controlled sequence analyzers (see below). Its potential 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 after capillary electrophoresis (see figure to the right). Chromatography (from Greek χρώμα chroma, color and γραφειν"graphein" to write is the collective term for a family of Laboratory Capillary electrophoresis ( CE) also known as capillary zone electrophoresis (CZE can be used to separate ionic species by their charge and frictional forces This problem has largely been overcome with the introduction of new DNA polymerase enzyme systems and dyes that minimize incorporation variability, as well as methods for eliminating "dye blobs", caused by certain chemical characteristics of the dyes that can result in artifacts in DNA sequence traces. The dye-terminator sequencing method, along with automated high-throughput DNA sequence analyzers, is now being used for the vast majority of sequencing projects, as it is both easier to perform and lower in cost than most previous sequencing methods.

Challenges

Modern sequencing typically produces a sequence that has poor quality in the first 15-40 bases, a high quality region of 700-900 bases, and then quickly deteriorating quality. Base calling software typically outputs an estimate of quality along with the sequence to aid in quality trimming.

Before the DNA can be sequenced, linker sequences are attached to its ends, and it is inserted into a cloning vector. A cloning vector is a small DNA vehicle into which a foreign DNA fragment can be inserted The resulting sequence can therefore often contain parts of the vector or the linker sequences, which must be filtered out prior to analysis. In contrast, emerging sequencing technologies based on pyrosequencing often avoid using cloning vectors. Pyrosequencing is a method of DNA sequencing (determining the order of Nucleotides in DNA based on the "sequencing by synthesis" principle

During PCR amplification, unrelated sequences can hybridize, and the resulting clone can be a chimaeric sequence, containing fragments from both sequences. Another problem is polymerase stuttering, where the polymerase repeatedly outputs the same fragments, giving an artificially long low-complexity part of the sequence. Polymerase stuttering is the process by which a Polymerase Transcribes a nucleotide several times without progressing further on the MRNA chain A polymerase (EC 2776/7/19/48/49 is an Enzyme whose central function is associated with Polymers of Nucleic acids such as RNA and DNA

Automation and sample preparation

View of the start of an example dye-terminator read (click to expand)
View of the start of an example dye-terminator read (click to expand)

Modern automated DNA sequencing instruments (DNA sequencers) can sequence up to 384 fluorescently labelled samples in a single batch (run) and perform as many as 24 runs a day. A DNA sequencer is a Scientific instrument used to automate the DNA sequencing process However, automated DNA sequencers carry out only DNA-size-based separation (by capillary electrophoresis), detection and recording of dye fluorescence, and data output as fluorescent peak trace chromatograms. Capillary electrophoresis ( CE) also known as capillary zone electrophoresis (CZE can be used to separate ionic species by their charge and frictional forces Chromatography (from Greek χρώμα chroma, color and γραφειν"graphein" to write is the collective term for a family of Laboratory Sequencing reactions by thermocycling, cleanup and re-suspension in a buffer solution before loading onto the sequencer are performed separately. The Thermal Cycler (also known as a Thermocycler, PCR Machine or DNA Amplifier) is a Laboratory apparatus used to amplify segments of DNA For an individual weak acid or weak base component see Buffering agent. In the past, an operator had to trim the low quality ends (see image in the right) of every sequence manually in order to remove the sequencing errors. However, today, software like Fast Chromatogram Viewer can automatically trim the ends at batch.

Large-scale sequencing strategies

Current methods can directly sequence only relatively short (300-1000 nucleotides long) DNA fragments in a single reaction. Nucleotides are Organic compounds that consist of three joined structures a nitrogenous base a Sugar, and a Phosphate group [2]. 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. Limitations on ddNTP incorporation were largely solved by Tabor at Harvard Medical, Carl Fuller at USB biochemicals, and their coworkers. [12]

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 regions.(click to expand)
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 regions. (click to expand)

Large-scale sequencing aims at sequencing very long DNA fragments. Even relatively small bacterial genomes contain millions of nucleotides, and the human chromosome 1 alone contains about 246 million bases. In classical genetics the genome of a Diploid Organism including Eukarya refers to a full set of chromosomes or genes in a Gamete, thereby Chromosome 1 is the designation for the largest Human Chromosome. Nucleotides are Organic compounds that consist of three joined structures a nitrogenous base a Sugar, and a Phosphate group Therefore, some approaches consist of cutting (with restriction enzymes) or shearing (with mechanical forces) large DNA fragments into shorter DNA fragments. A restriction enzyme (or restriction Endonuclease) is an Enzyme that cuts double-stranded DNA at specific recognition Nucleotide The fragmented DNA is cloned into a DNA vector, usually a bacterial artificial chromosome (BAC), and amplified in Escherichia coli. Molecular cloning refers to the procedure of isolating a defined DNA sequence and obtaining multiple copies of it In vivo. In Molecular biology, a vector is any vehicle used to transfer foreign genetic material into another cell A bacterial artificial chromosome (BAC is a DNA construct, based on a fertility Plasmid (or F-plasmid) used for transforming and Cloning The amplified DNA can then be purified from the bacterial cells (a disadvantage of bacterial clones for sequencing is that some DNA sequences may be inherently un-clonable in some or all available bacterial strains, due to deleterious effect of the cloned sequence on the host bacterium or other effects). These short DNA fragments purified from individual bacterial colonies are then individually and completely sequenced and assembled electronically into one long, contiguous sequence by identifying 100%-identical overlapping sequences between them (shotgun sequencing). In Bioinformatics, sequence assembly refers to aligning and merging many fragments of a much longer DNA sequence in order to reconstruct the original In Genetics, shotgun sequencing, also known as shotgun cloning, is a method used for Sequencing long DNA strands This method does not require any pre-existing information about the sequence of the DNA and is often referred to as de novo sequencing. Gaps in the assembled sequence may be filled by Primer walking, often with sub-cloning steps (or transposon-based sequencing depending on the size of the remaining region to be sequenced). Primer walking is a Sequencing method of choice for sequencing DNA fragments between 1 Transposons are sequences of DNA that can move around to different positions within the Genome of a single cell, a process called transposition These strategies all involve taking many small reads of the DNA by one of the above methods and subsequently assembling them into a contiguous sequence. The different strategies have different tradeoffs in speed and accuracy; the shotgun method is the most practical for sequencing large genomes, but its assembly process is complex and potentially error-prone - particularly in the presence of sequence repeats. Microsatellites, or Simple Sequence Repeats (SSRs are polymorphic loci present in nuclear and organellar DNA that consist of repeating Because of this, the assembly of the human genome is not literally complete — the repetitive sequences of the centromeres, telomeres, and some other parts of chromosomes result in gaps in the genome assembly. Despite having only 93% of the full genome assembled, the Human Genome Project was declared complete because their definition of human genome sequencing was limited to euchromatic sequence (99% complete at the time), excluding these intractable repetitive regions. The Human Genome Project (HGP was an international Scientific research project with a primary goal to determine the sequence of chemical base pairs which make up DNA [13]

Resequencing steps. Sample prep: Extraction of nucleic acid. Template prep: Amplification and preparation of a small region of the target region. Sequencing steps. (click to expand)
Resequencing steps. Sample prep: Extraction of nucleic acid. Template prep: Amplification and preparation of a small region of the target region. Sequencing steps. (click to expand)

The human genome is about 3 billion (3,000,000,000) bp long;[14] if the average fragment length is 500 bases, it would take a minimum of six million (3 billion/500) to sequence the human genome (not allowing for overlap = 1-fold coverage). Keeping track of such a high number of sequences presents significant challenges, only held down by developing and coordinating several procedural and computational algorithms, such as efficient database development and management. In Mathematics, Computing, Linguistics and related subjects an algorithm is a sequence of finite instructions often used for Calculation

Resequencing or targeted sequencing is utilized for determining a change in DNA sequence from a "reference" sequence. It is often performed using PCR to amplify the region of interest (pre-existing DNA sequence is required to design the PCR primers). Resequencing uses three steps, extraction of DNA or RNA from biological tissue; amplification of the RNA or DNA (often by PCR); followed by sequencing. The resultant sequence is compared to a reference or a normal sample to detect mutations.

New sequencing methods

High-throughput sequencing

The high demand for low cost sequencing has given rise to a number of high-throughput sequencing technologies. [15][16] These efforts have been funded by public and private institutions as well as privately researched and commercialized by biotechnology companies. High-throughput sequencing technologies are intended to lower the cost of sequencing DNA libraries beyond what is possible with the current dye-terminator method based on DNA separation by capillary electrophoresis. Many of the new high-throughput methods use methods that parallelize the sequencing process, producing thousands or millions of sequences at once.

In vitro clonal amplification

As molecular detection methods are often not sensitive enough for single molecule sequencing, most approaches use an in vitro cloning step to generate many copies of each individual molecule. Emulsion PCR is one method, isolating individual DNA molecules along with primer-coated beads in aqueous bubbles within an oil phase. A polymerase chain reaction (PCR) then coats each bead with clonal copies of the isolated library molecule and these beads are subsequently immobilized for later sequencing. Emulsion PCR is used in the methods published by Marguilis et al. (commercialized by 454 Life Sciences, acquired by Roche), Shendure and Porreca et al. Technology 454 Sequencing is a large-scale parallel Pyrosequencing system capable of sequencing roughly 400-600 megabases of DNA per 10-hour run on the Genome (also known as "polony sequencing") and SOLiD sequencing, (developed by Agencourt and acquired by Applied Biosystems). [17][18][19] Another method for in vitro clonal amplification is "bridge PCR", where fragments are amplified upon primers attached to a solid surface, developed and used by Solexa (now owned by Illumina). Illumina Inc ( incorporated in April 1998 develops manufactures and markets integrated systems for the analysis of genetic variation and biological function These methods both produce many physically isolated locations which each contain many copies of a single fragment. The single-molecule method developed by Stephen Quake's laboratory (later commercialized by Helicos) skips this amplification step, directly fixing DNA molecules to a surface. [20]

Parallelized sequencing

Once clonal DNA sequences are physically localized to separate positions on a surface, various sequencing approaches may be used to determine the DNA sequences of all locations, in parallel. "Sequencing by synthesis", like the popular dye-termination electrophoretic sequencing, uses the process of DNA synthesis by DNA polymerase to identify the bases present in the complementary DNA molecule. A DNA Polymerase is an Enzyme that assists in DNA replication. Reversible terminator methods (used by Illumina and Helicos) use reversible versions of dye-terminators, adding one nucleotide at a time, detecting fluorescence corresponding to that position, then removing the blocking group to allow the polymerization of another nucleotide. Pyrosequencing (used by 454) also uses DNA polymerization to add nucleotides, adding one type of nucleotide at a time, then detecting and quantifying the number of nucleotides added to a given location through the light emitted by the release of attached pyrophosphates. Pyrosequencing is a method of DNA sequencing (determining the order of Nucleotides in DNA based on the "sequencing by synthesis" principle [17][21]

"Sequencing by ligation" is another enzymatic method of sequencing, using a DNA ligase enzyme rather than polymerase to identify the target sequence. Sequencing by ligation is a DNA sequencing method that uses the enzyme DNA ligase to identify the Nucleotide present at a given position in a DNA sequence In Molecular biology, DNA ligase is a special type of Ligase ( that can link together two DNA strands that have single-strand breaks (a break in both complementary [22][18][19] Used in the polony method and in the SOLiD technology offered by Applied Biosystems, this method uses a pool of all possible oligonucleotides of a fixed length, labeled according to the sequenced position. Oligonucleotides are annealed and ligated; the preferential ligation by DNA ligase for matching sequences results in a signal corresponding to the complementary sequence at that position.

Other sequencing technologies

Other methods of DNA sequencing may have advantages in terms of efficiency or accuracy. Like traditional dye-terminator sequencing, they are limited to sequencing single isolated DNA fragments. "Sequencing by hybridization" is a non-enzymatic method that uses a DNA microarray. Sequencing by Hybridization is a class of methods for determining the order in which nucleotides occur on a strand of DNA. For terminology see glossary below A DNA microarray is a High-throughput technology used in Molecular biology and in In this method, a single pool of unknown DNA is fluorescently labeled and hybridized to an array of known sequences. If the unknown DNA hybridizes strongly to a given spot on the array, causing it to "light up", then that sequence is inferred to exist within the unknown DNA being sequenced. [23] Mass spectrometry can also be used to sequence DNA molecules; conventional chain-termination reactions produce DNA molecules of different lengths and the length of these fragments is then determined by the mass differences between them (rather than using gel separation). Mass spectrometry is an analytical technique that identifies the chemical composition of a compound or sample based on the Mass-to-charge ratio of charged particles [24]

There are new proposals for DNA sequencing, which are in development, but remain to be proven. These include labeling the DNA polymerase,[25] reading the sequence as a DNA strand transits through nanopores,[26] and microscopy-based techniques, such as AFM or electron microscopy that are used to identify the positions of individual nucleotides within long DNA fragments by nucleotide labeling with heavier elements (e. Nanopore sequencing is a method under development since 1995 for determining the order in which nucleotides occur on a strand of DNA. The atomic force microscope (AFM or scanning force microscope (SFM is a very high-resolution type of scanning probe microscope, with demonstrated resolution of fractions An electron microscope is a type of Microscope that uses Electrons to illuminate a specimen and create an enlarged image g. , halogens) for visual detection and recording. [27] In October 2006 the NIH issued a news release describing novel sequencing techniques and announcing several grant awards. "NIH" redirects here For other meanings of NIH see NIH (disambiguation. [28]

In October 2006, the X Prize Foundation established 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. The X PRIZE Foundation is a non-profit prize institute that designs and manages public competitions for the benefit of humanity The Archon X PRIZE for Genomics, the second X PRIZE to be offered by the X PRIZE Foundation, based in Santa Monica, California "[29]

Major landmarks in DNA sequencing

See also

Citations

  1. ^ Sanger F, Coulson AR. A DNA field-effect transistor (DNAFET is a Field-effect transistor which uses the field-effect due to the partial charges of DNA molecules to function as a Biosensor A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J Mol Biol. 1975 May 25;94(3):441–448
  2. ^ F. Sanger, S. Nicklen, and A. R. Coulson, DNA sequencing with chain-terminating inhibitors, Proc Natl Acad Sci U S A. 1977 December; 74(12): 5463–5467
  3. ^ Maxam AM, Gilbert W. , A new method for sequencing DNA, Proc Natl Acad Sci U S A. 1977 Feb;74(2):560-4
  4. ^ Unknown
  5. ^ Proc Natl Acad Sci U S A. 1973 December; 70(12 Pt 1-2): 3581–3584. The Nucleotide Sequence of the lac Operator, Walter Gilbert and Allan Maxam
  6. ^ Min Jou W, Haegeman G, Ysebaert M, Fiers W. , Nucleotide sequence of the gene coding for the bacteriophage MS2 coat protein, Nature. 1972 May 12;237(5350):82-8
  7. ^ Complete nucleotide sequence of bacteriophage MS2 RNA: primary and secondary structure of the replicase gene. Nature. 1976 Apr 8;260(5551):500-7.
  8. ^ Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448
  9. ^ Unknown
  10. ^ Nature. 1986 Jun 12-18;321(6071):674-9. Fluorescence detection in automated DNA sequence analysis. We have developed a method for the partial automation of DNA sequence analysis. Fluorescence detection of the DNA fragments is accomplished by means of a fluorophore covalently attached to the oligonucleotide primer used in enzymatic DNA sequence analysis. A different coloured fluorophore is used for each of the reactions specific for the bases A, C, G and T. The reaction mixtures are combined and co-electrophoresed down a single polyacrylamide gel tube, the separated fluorescent bands of DNA are detected near the bottom of the tube, and the sequence information is acquired directly by computer.
  11. ^ Nucleic Acids Res. 1985 Apr 11;13(7):2399-412. The synthesis of oligonucleotides containing an aliphatic amino group at the 5' terminus: synthesis of fluorescent DNA primers for use in DNA sequence analysis. Note that Oxford University Press, the publishers of the journal Nucleic Acids Research, make the full contents of this journal available online for free - you can download a copy of this paper for yourself !!
  12. ^ a b "A novel thermostable polymerase for DNA sequencing. " (1995-08-31). Nature 376 (6543): 796-797. doi:10.1038/376796a0. A digital object identifier ( DOI) is a permanent identifier given to an Electronic document. PMID 7651542.  
  13. ^ International Human Genome Sequencing Consortium (2004). "Finishing the euchromatic sequence of the human genome. ". Nature 431 (7011): 931-45. doi:10.1038/nature03001. A digital object identifier ( DOI) is a permanent identifier given to an Electronic document. PMID 15496913.   paper available online
  14. ^ Human Genome Project Information
  15. ^ Neil Hall (2007). "Advanced sequencing technologies and their wider impact in microbiology". The Journal of Experimental Biology 209: 1518-1525.  
  16. ^ G. M. Church (2006). "Genomes for ALL". Scientific American 294 (1): 47-54. PMID 16468433.  
  17. ^ a b M. Margulies, et al. (2005). "Genome sequencing in microfabricated high-density picolitre reactors". Nature 437: 376-380.  
  18. ^ a b J. Shendure, G. J. Porreca, N. B. Reppas, X. Lin, J. Pe McCutcheon, A. M. Rosenbaum, M. D. Wang, K. Zhang, R. D. Mitra and G. M. Church (2005). "Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome". Science 309 (5741): 1728-1732. doi:10.1126/science.1117389. A digital object identifier ( DOI) is a permanent identifier given to an Electronic document.  
  19. ^ a b http://solid.appliedbiosystems.com/ - Applied Biosystems' SOLiD technology
  20. ^ Braslavsky, I. , Hebert, H. , Kartalov, E. and Quake, S. R. (2003). "Sequence information can be obtained from single DNA molecules". Proceedings of the National Academy of Sciences of the United States of America 100: 3960–3964. doi:10.1073/pnas.0230489100. A digital object identifier ( DOI) is a permanent identifier given to an Electronic document.  full text available online
  21. ^ M. Ronaghi, S. Karamohamed, B. Pettersson, M. Uhlen, and P. Nyren (1996). "Real-time DNA sequencing using detection of pyrophosphate release". Analytical Biochemistry 242: 84=89. doi:10.1006/abio.1996.0432. A digital object identifier ( DOI) is a permanent identifier given to an Electronic document.  
  22. ^ S. C. Macevicz, US Patent 5750341, filed 1995
  23. ^ G. J. Hanna, V. A. Johnson, D. R. Kuritzkes, D. D. Richman, J. Martinez-Picado, L. Sutton, J. D. Hazelwood, R. T. D'Aquila (2000). "Comparison of sequencing by hybridization and cycle sequencing for genotyping of human immunodeficiency virus type 1 reverse transcriptase". Journal of Clinical Microbiology 38 (7): 2715. PMID 10878069.  
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  25. ^ VisiGen Biotechnologies Inc. - Technology Overview
  26. ^ The Harvard Nanopore Group
  27. ^ USPTO application # 20060029957 assigned to ZS genetics http://www.freepatentsonline.com/20060029957.html
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