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Encyclopedia > DNA sequencing

The term DNA sequencing encompasses biochemical methods for determining the order of the nucleotide bases, adenine, guanine, cytosine, and thymine, in a DNA oligonucleotide. 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. 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 attainable with modern DNA sequencing technology has been instrumental in the large-scale 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. Biochemistry is the study of the chemical processes and transformations in living organisms. ... A nucleotide is a chemical compound that consists of a heterocyclic base, a sugar, and one or more phosphate groups. ... This article or section does not adequately cite its references or sources. ... Guanine is one of the five main nucleobases found in the nucleic acids DNA and RNA; the others being adenine, cytosine, thymine, and uracil. ... Cytosine is one of the 5 main nucleobases used in storing and transporting genetic information within a cell in the nucleic acids DNA and RNA. It is a pyrimidine derivative, with a heterocyclic aromatic ring and two substituents attached (an amine group at position 4 and a keto group at... For the similarly-spelled vitamin compound, see Thiamine Thymine, also known as 5-methyluracil, is a pyrimidine nucleobase. ... The structure of part of a DNA double helix Deoxyribonucleic acid, or DNA, is a nucleic acid molecule that contains the genetic instructions used in the development and functioning of all known living organisms. ... Oligonucleotides are short sequences of nucleotides (RNA or DNA), typically with twenty or fewer bases. ... HeLa cells stained for DNA with the Blue Hoechst dye. ... Figure 1 : Schematic drawing of a bacterium with plasmids enclosed. ... In cell biology, a mitochondrion is an organelle found in the cells of most eukaryotes. ... Chloroplasts are organelles found in plant cells and eukaryotic algae which conduct photosynthesis. ... Forensics or forensic science is the application of science to questions which are of interest to the legal system. ... A graphical representation of the normal human karyotype. ... // The Human Genome Project (HGP) is a project to de-code (i. ...

DNA Sequence Trace
DNA Sequence Trace

Contents

Image File history File links No higher resolution available. ... Image File history File links No higher resolution available. ...

Early methods

For thirty years, a large proportion of DNA sequencing has been carried out with the chain-termination method [1], developed by Frederick Sanger and coworkers in 1975. Prior to the development of rapid DNA sequencing methods in the early 1970s by Sanger in England and Gilbert et al. at Harvard,[1], a number of laborious methods were used. For instance, in 1973[2] Gilbert and Maxam reported the sequence of 24 basepairs using a method known as wandering-spot analysis. Dr Frederick Sanger, OM, CH, CBE, FRS (born 13 August 1918) is an English biochemist and a two times Nobel laureate in chemistry. ...


It is noteworthy that RNA sequencing, which for technical reasons is easier to perform than DNA sequencing, could be considered 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 phage MS2 genome, identified and published by Walter Fiers and coworkers. [3]


Maxam-Gilbert sequencing 00

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[2]. Although Maxam and Gilbert published their chemical sequencing method two years after the ground-breaking paper of Sanger and Coulson on plus-minus sequencing,[4][5] 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 (see below), chemicals used in the Maxam-Gilbert method cannot easily be customized for use in a standard molecular biology kit. Allan Maxam is one of the pioneers of molecular genetics. ... Walter Gilbert Walter Gilbert (born March 21, 1932) is an American physicist, biochemist,and molecular biology pioneer. ...


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. Gel electrophoresis is the separation of deoxyribonucleic acid, ribonucleic acid, or protein through an electric charge. ... A radiograph of a right elbow-joint Radiography is the use of certain types of electromagnetic radiation—usually ionizing—to view objects. ... An autoradiograph is an image produced on a photographic film by the radiation from a radioactive substance. ... Radioisotopic 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. The key principle of the Sanger method was the use of dideoxynucleotides triphosphates (ddNTPs) as DNA chain terminators. DNA sequencing gel. ... DNA sequencing gel. ... Radioisotopic labeling is a technique for tracking the passage of a sample of substance through a system. ... Dideoxynucleotides, or ddNTPs, are nucleotides lacking 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. The DNA sample is divided into four separate sequencing reactions, containing the four standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase. To each reaction is added only one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP). 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. 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. A primer is a nucleic acid strand, or a related molecule that serves as a starting point for DNA replication. ... 3D structure of the DNA-binding helix-hairpin-helix motifs in human DNA polymerase beta A DNA polymerase is an enzyme that assists in DNA replication. ... Fluorescence induced by exposure to ultraviolet light in vials containing various sized Cadmium selenide (CdSe) quantum dots. ... A nucleotide is a monomer or the structural unit of nucleotide chains forming nucleic acids as RNA and DNA. A nucleotide consists of a heterocyclic nucleobase, a pentose sugar, and a phosphate or polyphosphate group. ... 3D structure of the DNA-binding helix-hairpin-helix motifs in human DNA polymerase beta A DNA polymerase is an enzyme that assists in DNA replication. ... Dideoxynucleotides, or ddNTPs, are nucleotides lacking 3-hydroxyl (-OH) group on their deoxyribose sugar. ... // Hydroxyl group The term hydroxyl group is used to describe the functional group -OH when it is a substituent in an organic compound. ... Diagram of phosphodiester bonds between nucleotides A phosphodiester bond is a group of strong covalent bonds between the phosphorus atom in a phosphate group and two other molecules over two ester bonds. ...


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. 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. 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. The dissociation of a double-stranded DNA molecule is often referred to as melting because it occurs quickly once a certain temperature has been reached. ... Gel electrophoresis is the separation of deoxyribonucleic acid, ribonucleic acid, or protein through an electric charge. ... // A polyacrylamide gel is a separation matrix used in electrophoresis of biomolecules, such as proteins or DNA fragments. ... An autoradiograph is an image produced on a photographic film by the radiation from a radioactive substance. ... A radiograph of a right elbow-joint Radiography is the use of certain types of electromagnetic radiation—usually ionizing—to view objects. ...

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. Alternatively, a primer labeled at the 5’ end with a fluorescent dye is used for the tagging. 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[6][7] of fluorescently labeled ddNTPs and primers set the stage for automated, high-throughput DNA sequencing. Image File history File links Size of this preview: 450 × 600 pixelsFull resolution (540 × 720 pixel, file size: 37 KB, MIME type: image/jpeg) Created by Abizar Lakdawalla. ... Image File history File links Size of this preview: 450 × 600 pixelsFull resolution (540 × 720 pixel, file size: 37 KB, MIME type: image/jpeg) Created by Abizar Lakdawalla. ... Radioisotopic labeling is a technique for tracking the passage of a sample of substance through a system. ... Fluorescence induced by exposure to ultraviolet light in vials containing various sized Cadmium selenide (CdSe) quantum dots. ...

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. 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. Image File history File links No higher resolution available. ... Image File history File links No higher resolution available. ...


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. 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). 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. Image File history File links No higher resolution available. ... Image File history File links No higher resolution available. ... The wavelength is the distance between repeating units of a wave pattern. ... Pictured is a sophisticated gas chromatography system. ... Capillary electrophoresis (CE) can be used to separate ionic species by their charge and frictional forces. ...


Automation and sample preparation

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. However, automated DNA sequencers carry out only DNA size separation by capillary electrophoresis, 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. Image File history File links Download high resolution version (994x230, 19 KB)Example of (the start of) a Sanger sequencing read. ... Image File history File links Download high resolution version (994x230, 19 KB)Example of (the start of) a Sanger sequencing read. ... A DNA sequencer is a machine to automatize the DNA sequencing process. ... Capillary electrophoresis (CE) can be used to separate ionic species by their charge and frictional forces. ... Pictured is a sophisticated gas chromatography system. ... Thermal cycler Thermal cycler or thermocycler or PCR machine is a laboratory apparatus used for PCR. The device has a thermal block with holes where tubes with the PCR reaction mixtures can be inserted. ... Acids and bases: Acid-base reaction theories pH Self-ionization of water Buffer solutions Systematic naming Electrochemistry Acid-base extraction Acids: Strong acids Weak acids Superacids Lewis acids Mineral acids Organic acids Bases: Strong bases Weak bases Superbases Lewis bases Organic bases edit Buffer solutions are solutions which resist change...

Large-scale sequencing strategies

Current methods can directly sequence only relatively short (300-1000 nucleotides long) DNA fragments in a single reaction. [3]. 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.[citation needed] A nucleotide is an organic molecule consisting of a heterocyclic nucleobase (a purine or a pyrimidine), a pentose sugar (deoxyribose in DNA or ribose in RNA), and a phosphate or polyphosphate group. ...

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. Therefore, some 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, usually a bacterial plasmid, and amplified in Escherichia coli. 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). 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). 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. 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 euchromotic sequence (99% complete at the time), excluding these intractable repetitive regions.[8] Image File history File links Size of this preview: 450 × 600 pixelsFull resolution (540 × 720 pixel, file size: 65 KB, MIME type: image/jpeg) Created by Abizar Lakdawalla. ... Image File history File links Size of this preview: 450 × 600 pixelsFull resolution (540 × 720 pixel, file size: 65 KB, MIME type: image/jpeg) Created by Abizar Lakdawalla. ... In biology the genome of an organism is the whole hereditary information of an organism that is encoded in the DNA (or, for some viruses, RNA). ... Chromosome 1 is, by convention, the designation for the largest human chromosome. ... A nucleotide is a chemical compound that consists of a heterocyclic base, a sugar, and one or more phosphate groups. ... A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded DNA. The enzyme makes two incisions, one through each of the sugar-phosphate backbones (i. ... Molecular cloning refers to the procedure of isolating a defined DNA sequence and obtaining multiple copies of it in vivo. ... A Vector DNA is a small piece of DNA containing regulatory and coding sequences of interest. ... Figure 1: Schematic drawing of a bacterium with plasmids enclosed. ... E. coli redirects here. ... In bioinformatics, sequence assembly refers to aligning and merging fragments of a DNA sequence to reconstruct the original sequence, typically fragments of the genome resulting from shotgun sequencing, or fragments of a gene transcript (ESTs). ... Shotgun sequencing is a method used in genetics for sequencing long DNA strands. ... This article or section does not cite its references or sources. ... Transposons are sequences of DNA that can move around to different positions within the genome of a single cell, a process called transposition. ... A microsatellite is a short, noncoding DNA sequence (a Tandemly Repetitive DNA sequence) that is repeated many times within the genome of an organism. ... // The Human Genome Project (HGP) is a project to de-code (i. ...

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;[9] 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. Image File history File links No higher resolution available. ... Image File history File links No higher resolution available. ... In mathematics, computing, linguistics, and related disciplines, an algorithm is a finite list of well-defined instructions for accomplishing some task that, given an initial state, will terminate in a defined end-state. ...


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.[10][11] 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, recently acquired by Roche) and Shendure and Porreca et al. (also known as "polony sequencing", commercialized by Agencourt and recently acquired by Applied Biosystems).[12][13] 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). 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.[14] PCR tubes in a stand after a colony PCR The polymerase chain reaction (PCR) is a biochemistry and molecular biology technique[1] for exponentially amplifying DNA, via enzymatic replication, without using a living organism (such as E. coli or yeast). ... 454 Life Sciences is a biotechnology company based in Branford, Connecticut specializing in high-throughput DNA sequencing using a novel massively parallel sequencing-by-synthesis approach. ... Applied Biosystems, Inc. ... Illumina (NASDAQ: ILMN) has developed a targeted set of instruments and chips for organizations conducting larger-scale research in genotyping and gene expression profiling. ...

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. 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.[12][15] 3D structure of the DNA-binding helix-hairpin-helix motifs in human DNA polymerase beta A DNA polymerase is an enzyme that assists in DNA replication. ... The introduction to this article provides insufficient context for those unfamiliar with the subject matter. ...


"Sequencing by ligation" is another enzymatic method of sequencing, using a DNA ligase enzyme rather than polymerase to identify the target sequence.[13][16] Used in the polony method (and in the SOLiD technology offered by Applied Biosystems), this method uses a pool of random oligonucleotides 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. 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. ... It has been suggested that sticky end/blunt end be merged into this article or section. ...


Other sequencing technologies

Additional 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. 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.[17] 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).[18] sequencing by hybridization ... It has been suggested that Gene chip technology be merged into this article or section. ... Mass spectrometry (also known as mass spectroscopy (deprecated)[1] or informally, mass-spec and MS) is an analytical technique used to measure the mass-to-charge ratio of ions. ...


There are new proposals for DNA sequencing, which are in development, but remain to be proven. These include labeling the DNA polymerase,[19] reading the sequence as a DNA strand transits through nanopores,[20] 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 labelling with heavier elements (e.g., halogens) for visual detection and recording.[21] 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."[22] Topographic scan of a glass surface The atomic force microscope (AFM) is a very high-resolution type of scanning probe microscope, with demonstrated resolution of fractions of a nanometer, more than 1000 times better than the optical diffraction limit. ... This article does not cite any references or sources. ... The original Ansari X Prize was presented on November 6, 2004. ... The Archon X Prize in genomics, the second X Prize to be awarded by the X Prize Foundation, based in Santa Monica, California, was announced October 4th, 2006. ...


Major landmarks in DNA sequencing

  • 1953 Discovery of the structure of the DNA double helix.
  • 1972 Development of recombinant DNA technology, which permits isolation of defined fragments of DNA; prior to this, the only accessible samples for sequencing were from bacteriophage or virus DNA.
  • 1977 Allan Maxam and Walter Gilbert publish DNA Sequencing by chemical degradation [4]. Fred Sanger, independently, publishes DNA sequencing by enzymatic synthesis.
  • 1980 Fred Sanger and Wally Gilbert receive the Nobel Prize
  • 1982 Genbank started as a public repository of DNA sequences.
    • Andre Marion and Sam Eletr from Hewlett Packard start Applied Biosystems, that comes to dominate automated sequencing, in May.
  • 1982 Akiyoshi Wada proposes automated sequencing and gets support to build robots with help from Hitachi.
  • 1984 MRC scientists decipher the complete DNA sequence of the Epstein-Barr virus, 170 kb.
  • 1985 Mullis and colleagues develop PCR, a technique to replicate small amounts of DNA
  • 1986 Leroy E. Hood's Laboratory at the California Institute of Technology and Smith announce the first semi-automated DNA sequencing machine.
  • 1987 Applied Biosystems markets first automated sequencing machine, the Prism 373.
    • Walter Gilbert leaves the U.S. National Research Council genome panel to start Genome Corp., with the goal of sequencing and commercializing the data.
  • 1991 Craig Venter develops strategy to find expressed genes with ESTs (Expressed Sequence Tags).
    • Uberbacher develops GRAIL, a gene-prediction program.
  • 1992 Craig Venter leaves NIH to set up The Institute for Genomic Research (TIGR).
    • William Haseltine heads Human Genome Sciences, to commercialize TIGR products.
    • Wellcome Trust begins participation in the Human Genome Project.
    • Simon et al. develop BACs (Bacterial Artificial Chromosomes) for cloning. **First chromosome physical maps published:
    • Page et al. - Y chromosome;[citation needed]
    • Cohen et al. chromosome 21.[citation needed]
    • Lander - complete mouse genetic map;[citation needed]
    • Weissenbach - complete human genetic map.[citation needed]
  • 1993 Wellcome Trust and MRC open Sanger Centre, near Cambridge, UK.
    • The GenBank database migrates from Los Alamos (DOE) to NCBI (NIH).
  • 1995 Venter, Fraser and Smith publish first sequence of free-living organism, Haemophilus influenzae (genome size of 1.8 Mb).
    • Richard Mathies et al. publish on sequencing dyes (PNAS, May).[citation needed]
    • Michael Reeve and Carl Fuller, thermostable polymerase for sequencing (Nature, August).[citation needed]
  • 1996 International HGP partners agree to release sequence data into public databases within 24 hours.
    • International consortium releases genome sequence of yeast S. cerevisiae (genome size of 12.1 Mb).
    • Yoshihide Hayashizaki's at RIKEN completes the first set of full-length mouse cDNAs.
    • ABI introduces a capillary electrophoresis system, the ABI310 sequence analyzer.
  • 1997 Blattner, Plunkett et al. publish the sequence of E. coli (genome size of 5 Mb)[citation needed]
  • 1998 Phil Green and Brent Ewing of Washington University publish “phred” for interpreting sequencer data (in use since ‘95).[citation needed]
    • Venter starts new company “Celera”; “will sequence HG in 3 yrs for $300m.”
    • Applied Biosystems introduces the 3700 capillary sequencing machine.
    • Wellcome Trust doubles support for the HGP to $330 million for 1/3 of the sequencing.
    • NIH & DOE goal: "working draft" of the human genome by 2001.
    • Sulston, Waterston et al finish sequence of C. elegans (genome size of 97Mb).[citation needed]
  • 1999 NIH moves up completion date for rough draft, to spring 2000.
    • NIH launches the mouse genome sequencing project.
    • First sequence of human chromosome 22 published.[citation needed]
  • 2000 Celera and collaborators sequence fruit fly Drosophila melanogaster (genome size of 180Mb) - validation of Venter's shotgun method. HGP and Celera debate issues related to data release.
    • HGP consortium publishes sequence of chromosome 21.[23]
    • HGP & Celera jointly announce working drafts of HG sequence, promise joint publication.
    • Estimates for the number of genes in the human genome range from 35,000 to 120,000. International consortium completes first plant sequence, Arabidopsis thaliana (genome size of 125 Mb).
  • 2001 HGP consortium publishes Human Genome Sequence draft in Nature (15 Feb).[citation needed]
    • Celera publishes the Human Genome sequence in Science (16 Feb).[citation needed]
  • 2005 420,000 VariantSEQr human resequencing primer sequences published on new NCBI Probe database.

Year 1953 (MCMLIII) was a common year starting on Thursday (link will display full calendar) of the Gregorian calendar. ... Year 1972 (MCMLXXII) was a leap year starting on Saturday (link will display full calendar) of the Gregorian calendar. ... Also: 1977 (album) by Ash. ... Year 1980 (MCMLXXX) was a leap year starting on Tuesday (link displays the 1980 Gregorian calendar). ... Year 1982 (MCMLXXXII) was a common year starting on Friday (link displays the 1982 Gregorian calendar). ... Year 1982 (MCMLXXXII) was a common year starting on Friday (link displays the 1982 Gregorian calendar). ... Year 1984 (MCMLXXXIV) was a leap year starting on Sunday (link displays the 1984 Gregorian calendar). ... Year 1985 (MCMLXXXV) was a common year starting on Tuesday (link displays 1985 Gregorian calendar). ... Year 1986 (MCMLXXXVI) was a common year starting on Wednesday (link displays 1986 Gregorian calendar). ... Year 1987 (MCMLXXXVII) was a common year starting on Thursday (link displays 1987 Gregorian calendar). ... Year 1990 (MCMXC) was a common year starting on Monday (link displays the 1990 Gregorian calendar). ... E. coli redirects here. ... Binomial name Maupas, 1900 Caenorhabditis elegans (IPA: ) is a free-living nematode (roundworm), about 1 mm in length, which lives in temperate soil environments. ... Binomial name Saccharomyces cerevisiae Meyen ex E.C. Hansen Saccharomyces cerevisiae is a species of budding yeast. ... In bioinformatics, Basic Local Alignment Search Tool, or BLAST, is an algorithm for comparing primary biological sequence information, such as the amino-acid sequences of different proteins or the nucleotides of DNA sequences. ... In mathematics, computing, linguistics, and related disciplines, an algorithm is a finite list of well-defined instructions for accomplishing some task that, given an initial state, will terminate in a defined end-state. ... Capillary electrophoresis (CE) can be used to separate ionic species by their charge and frictional forces. ... Year 1991 (MCMXCI) was a common year starting on Tuesday (link will display the 1991 Gregorian calendar). ... Year 1992 (MCMXCII) was a leap year starting on Wednesday (link will display full 1992 Gregorian calendar). ... NIH can refer to: National Institutes of Health Norwegian School of Sports Sciences: (Norges idrettshøgskole - NIH) Not Invented Here This is a disambiguation page — a navigational aid which lists other pages that might otherwise share the same title. ... TIGR, abbreviation for Trst (Trieste), Istra (Istria), Gorica (Gorizia) and Reka (Rijeka (Fiume)), was the first antifascist national-defensive organization in Europe, consisting of Slovenians in Slovenian region of Primorje (Primorski Slovenci). ... The Wellcome Trusts Gibbs Building on Euston Road The Wellcome Trust is a United Kingdom-based charity established in 1936 to administer the fortune of the American-born pharmaceutical magnate Sir Henry Wellcome. ... // The Human Genome Project (HGP) is a project to de-code (i. ... Year 1993 (MCMXCIII) was a common year starting on Friday (link will display full 1993 Gregorian calendar). ... The Wellcome Trust Sanger Institute (formerly the Sanger Centre) is a genome research centre in Cambridgeshire, England. ... The National Center for Biotechnology Information (NCBI) is part of the US National Library of Medicine (NLM), which is a branch of the US National Institutes of Health. ... Year 1995 (MCMXCV) was a common year starting on Sunday (link will display full 1995 Gregorian calendar). ... Binomial name Haemophilus influenzae (Lehmann & Neumann 1896) Winslow 1917 Haemophilus influenzae, formerly called Pfeiffers bacillus or Bacillus influenzae, is a non-motile Gram-negative coccobacillus first described in 1892 by Dr. Richard Pfeiffer during an influenza pandemic. ... Year 1996 (MCMXCVI) was a leap year starting on Monday (link will display full 1996 Gregorian calendar). ... Year 1997 (MCMXCVII) was a common year starting on Wednesday (link will display full 1997 Gregorian calendar). ... Year 1998 (MCMXCVIII) was a common year starting on Thursday (link will display full 1998 Gregorian calendar). ... This article is about the year. ... Year 2000 (MM) was a leap year starting on Saturday (link will display full 2000 Gregorian calendar). ... Year 2001 (MMI) was a common year starting on Monday (link displays the 2001 Gregorian calendar). ... Year 2005 (MMV) was a common year starting on Saturday (link displays full calendar) of the Gregorian calendar. ...

See also

  • Sequencing
  • Genome project - how entire genomes are assembled from these short sequences.
  • Applied Biosystems - provided most of the chemistry and equipment for the genome projects. Next-generation technology for very high data generation rates.
  • 454 Life Sciences - company specializing in high-throughput DNA sequencing using a sequencing-by-synthesis approach.
  • Illumina (company) - Advancing genetic analysis one billion bases at a time; whole genome sequencing.
  • Joint Genome Institute - sequencing center from the US Department of Energy whose mission is to provide integrated high-throughput sequencing and computational analysis to enable genomic-scale/systems-based scientific approaches to DOE-relevant challenges in energy and the environment.
  • DNA field-effect transistor

In genetics and biochemistry, sequencing means to determine the primary structure (or primary sequence) of an unbranched biopolymer. ... Genome projects are scientific endeavours that ultimately aim to determine the complete genome sequence of an organism (be it an animal, a plant, a fungus, a bacterium, an archaean, a protist or a virus). ... Applied Biosystems, Inc. ... 454 Life Sciences is a biotechnology company based in Branford, Connecticut specializing in high-throughput DNA sequencing using a novel massively parallel sequencing-by-synthesis approach. ... Illumina (NASDAQ: ILMN) has developed a targeted set of instruments and chips for organizations conducting larger-scale research in genotyping and gene expression profiling. ... The Joint Genome Institute Production Genomics Facility is located in Walnut Creek, California. ... The United States Department of Energy (DOE) is a Cabinet-level department of the United States government responsible for energy policy and nuclear safety. ... 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. ...

Citations

  1. ^ http://nobelprize.org/nobel_prizes/chemistry/laureates/1980/gilbert-lecture.pdf
  2. ^ 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
  3. ^ Complete nucleotide sequence of bacteriophage MS2 RNA: primary and secondary structure of the replicase gene. Nature. 1976 Apr 8;260(5551):500-7.
  4. ^ Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448
  5. ^ http://nobelprize.org/nobel_prizes/chemistry/laureates/1980/sanger-lecture.pdf
  6. ^ 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.
  7. ^ 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 !!
  8. ^ International Human Genome Sequencing Consortium (2004). "Finishing the euchromatic sequence of the human genome.". Nature 431 (7011): 931-45. PMID 15496913.  paper available online
  9. ^ http://www.ornl.gov/sci/techresources/Human_Genome/home.shtml
  10. ^ Neil Hall (2007). "Advanced sequencing technologies and their wider impact in microbiology". The Journal of Experimental Biology 209: 1518-1525. 
  11. ^ G.M. Church (2006). "Genomes for ALL". Scientific American 294 (1): 47-54. PMID 16468433. 
  12. ^ a b M. Margulies, et al. (2005). "Genome sequencing in microfabricated high-density picolitre reactors". Nature 437: 376-380. 
  13. ^ 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. "Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome". Science 309 (5741): 1728-1732. 
  14. ^ 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. full text available online
  15. ^ 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. 
  16. ^ http://solid.appliedbiosystems.com/ - Applied Biosystems' SOLiD technology
  17. ^ 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 hybridizatino and cycle sequencing for genotyping of human immunodeficiency virus type 1 reverse transcriptase". Journal of Clinical Microbiology 38 (7): 2715. PMID 10878069. 
  18. ^ J.R. Edwards, H.Ruparel, and J. Ju. "Mass-spectrometry DNA sequencing". Mutation Research 573 (1-2): 3-12. 
  19. ^ http://visigenbio.com/technology_overview.html
  20. ^ http://mcb.harvard.edu/branton/index.htm
  21. ^ USPTO application # 20060029957 assigned to ZS genetics http://www.freepatentsonline.com/20060029957.html
  22. ^ "PRIZE Overview: Archon X PRIZE for Genomics"
  23. ^ Hattori, M., A. Fujiyama, T. D. Taylor, H. Watanabe, T. Yada, H.-S. Park, A. Toyoda, K. Ishii, Y. Totoki, D.-K. Choi, E. Soeda, M. Ohki, T. Takagi, Y. Sakaki; S. Taudienk, K. Blechschmidtk, A. Polleyk, U. Menzelk, J. Delabar, K. Kumpfk, R. Lehmannk, D. Patterson, K. Reichwaldk, A. Rumpk, M. Schillhabelk, A. Schudyk, W. Zimmermannk, A. Rosenthalk; J. KudohI, K. ShibuyaI, K. KawasakiI, S. AsakawaI, A. ShintaniI, T. SasakiI, K. NagamineI, S. MitsuyamaI, S. E. Antonarakis, S. MinoshimaI, N. ShimizuI, G. Nordsiek, K. Hornischer, P. Brandt, M. Scharfe, O. SchoÈn, A. Desario, J. Reichelt, G. Kauer, H. Bloecker; J. Ramser, A. Beck, S. Klages, S. Hennig, L. Riesselmann, E. Dagand, T. Haaf, S. Wehrmeyer, K. Borzym, K. Gardiner, D. Nizetickk, F. Francis, H. Lehrach, R. Reinhardt, and M.-L. Yaspo, (2000). The DNA sequence of human chromosome 21. Nature 405: 311-319.

External links


  Results from FactBites:
 
DNA analysis (641 words)
First, the basis for virtually all DNA sequencing was the dideoxy-chain terminating reaction, developed by Sanger and for which he received his second Nobel prize.
DNA polymerase and deoxynucleotides are added to the mixture, and the enzyme catalyzes the synthesis of the complementary DNA strand, starting at the primer and extending in the 3' direction.
When the sequencing reaction proceeds in the presence of a small amount of the dideoxynucleotide, the resulting chain-extension reaction products will consist of a set of molecules, all starting at the primer, all extending in the 3' direction, but terminating wherever the dideoxynucleotide happened to be incorporated, opposite its complementary nucleotide in the template.
Sequencer - Wikipedia, the free encyclopedia (283 words)
Sequencers started off in 1992 with a debut Album "Sahara" that was a major hit.
Sequencers got low profile from year 1996 due to the slump of music industry in Pakistan.
Now since couple of years media promotion has been shot out with launch of many private and international satellite channels that after eight years SEQUENCERS are planning for a comeback.
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