DNA sequencing

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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. 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. Because DNA is key to all living organisms, knowledge of the DNA sequence may be useful in almost any biological subject area. For example, in medicine it can be used to identify, diagnose and potentially develop treatments for genetic diseases. Similarly, genetic research into plant or animal pathogens may lead to treatments of various diseases caused by these pathogens.

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. This technique uses sequence-specific termination of an in vitro DNA synthesis reaction using modified nucleotides as substrate for DNA polymerases. 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.

Contents 1 Early Methods 2 Maxam-Gilbert sequencing 3 Chain-termination method 3.1 Dye-terminator sequencing 3.2 Automation and sample preparation 4 Large-scale sequencing strategies 5 Next-generation technology 6 Limitations of current technology 7 Major landmarks in DNA sequencing 8 See also 9 Citations 10 External links

[edit] Early Methods

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.

[edit] 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[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 ^{[3] [4]}, 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 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.

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.

[edit] Chain-termination method

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.

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.

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.

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^{[5] [6]} of fluorescently labeled ddNTPs and primers set the stage for automated, high-throughput DNA sequencing.

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.

[edit] Dye-terminator sequencing

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. 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.

[edit] Automation and sample preparation

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.

[edit] Large-scale sequencing strategies

Current methods can directly sequence only relatively short (300-1000 nucleotides long) DNA fragments in a single reaction. [3]. This limitation is largely due to limitations on the ability to tell apart DNA molecules that differ in length by one base, eg, if you wish to sequence a piece of DNA that is 10,000 bp long, with the sequence ATCGTC......(9,990 bp not shown)...ATTC, you would start by making a set of all possible fragments: A, AT, ATC, ATCG, ATCGT, and so forth. Since the % difference caused by adding a single base to a short piece is large, it is easy to physicaly seperate A from AT from ATC from ATCG. However, at the end of the molecule, it is necessary to seperate fragments that are, say 9,990 from 9,991 from 9,992 bases in length. Current technology is not able to do this in a cost effective manner. Limitations on ddNTP incorporation were largely solved by Tabor at Harvard Medical, Carl Fuller at USB biochemicals, and their coworkers.

It is often necessary to obtain the sequence of much larger regions. For example, even simple bacterial genomes contain millions of nucleotides, and the human chromosome 1 alone contains about 246 million bases. The basic strategy is quite simple: break the long sequence into many, many smaller pieces and reassemble the long sequence by OVERLAPPING the short fragments. Conceptually, this is like reassembling a book from random fragments that are N words long; clearly, it is a lot easier to reassemble a book by finding a set of overlappong 50 word fragments then it is to reassemble a book from a set of 20 word long fragments.
To push the analogy further, if you have a book where the same sentence is repeated many times, and the sentence is 40 words long, many of your 50 word fragments will have an end within the repeated sentence; the only way to tell the repeats apart is to find the rare 50 word fragments that contain an entire repeated sentence and BOTH ends.
If your book has some sentences that are 60 words long and repeatd, then it is not possible, simply from fragments, to reassemble the book - there will be no possible fragment that will tell you what goes at the end of the 60 word repeat. These sort of issues occur frequently in real life sequencing. The human genome is roughly 3 billion (3,000,000,000) bp long ^[7]; if the average fragment length is 500 bases, it would take a minimum of six hundred thousand fragments (3 billion/500) to sequence the human genome (not allowing for overlap). Clearly, simply keeping track of this many test tubes is a challenge. Thus, sequencing the human genome was, to a large extent, a project that involved learning how to do things on a large scale.
Several strategies have been devised for larger-scale DNA sequencing. Primer walking, often with cloning and sub-cloning steps (dependent on the size of the region to be sequenced), used to be the standard method. With the increase in computing power, shotgun sequencing is now common, or used as part of a hybrid method. 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 repeating sequences.

It is only possible to obtain high-quality sequence data when the desired segment of DNA is relatively pure, i.e., free from other contaminants, including other DNA. This can be achieved through PCR for shorter regions (several kilobases), if a very short sequence at both ends is known. Alternatively, the DNA can be 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. Most large-scale sequencing efforts involve the preparation of a large DNA library of such clones. 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.

[edit] Next-generation technology

Because of strong demand for high-throughput sequencing technologies by publicly and privately funded research institutions and companies, many bio-pharmaceutical companies have now research programs in place to develop next-generation DNA-sequencing technologies. These new technologies may soon replace the current dye-terminator technology based on DNA separation by capillary electrophoresis.

The current market leader in DNA sequencing, the ABI division of Applera, has a new rather baroque, technology, Solid ^[8], which combines elements of emulsion polymerization from Agencourt with an oligo ligation strategy ^[9] and methods developed by Brenner et al at Lynx (now part of Solexa)^[10]

As of Feb 2007, some of the other players include Perlegen, an Affymetrix subsidiary, which uses sequencing by hybridization, originally proposed ^[11]by Drmanac et al.; Solexa, now owned by Illumina, which uses a Bridge amplification technology ^[12] originally developed by Adams and Kron; Helicos, a private company in Cambridge, MA which emphasizes single molecule sequencing; 454 corporation, a subsidiary of Curagen with funding from Roche, using the innovative pyrosequencing method developed by Ronaghi et al ^[13]; a polony based technique (from Mitra and Church at Harvard) developed by Agencourt and licensed to ABI, and doubtless others.
Other proposals include labeling the DNA polymerase ^[14], reading the sequence as a strand transits through some sort of nanopore ^[15], vision based techniques capable of single molecule resolution, such as AFM or electron microscopy ^[16], and doubtless many others. There is a long list of methods that, for one reason or another, failed to become commercial successes, such as the blot based genome sequencing method of Church and Gilbert, commercialized by Integrated Genetics ^[17], methods using tin isotope tagged nucleotides and mass spect detectors; and the "bottom wiper" of Pohl ^[18] commericalized by Hoefer.

[edit] Limitations of current technology

Claims that a complete sequence of the human genome is available seem wrong ^[19], as the current technologies are not capable of providing complete DNA sequence information for the human genome. As an example, the Lawrence Berkeley Laboratory has a paper entitled "The complete sequence of human chromosome 5" ^[20]. Careful inspection of this paper shows that despite the use of the word "complete", there are non sequenced regions, and that the authors can only estimate how much more work remains to be done.

[edit] Major landmarks in DNA sequencing

• 1953 Discovery of the structure of the DNA helix.

“With my fingers too cold to write legibly I huddled next to the fireplace, daydreaming about how several DNA chains could fold together in a pretty and hopefully scientific way. Soon, however, I abandoned thinking at the molecular level and turned to the much easier job of reading biochemical papers on the interrelations of DNA, RNA and protein synthesis.” from Chapter 21 of The Double Helix by James Dewey Watson.

• 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

• 1981 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.

• 1990 NIH begins large-scale sequencing trials on Mycoplasma capricolum, Escherichia coli, Caenorhabditis elegans, and Saccharomyces cerevisiae (@75c/base.
  • Lipman, Myers publish the BLAST algorithm for aligning sequences.
  • Barry Karger (Analytical Chemistry, January), Lloyd Smith (Nucleic Acids Research, August), and Norman Dovichi (Journal of Chromatography, September) publish on capillary electrophoresis.

• 1991 Craig Venter develops strategy to find expressed genes with ESTs (Expressed Sequence Tags).
  • Uberbacher develops GRAIL- gene-finding 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;
  • Cohen et al. chromosome 21.
  • Lander - complete mouse genetic map;
  • Weissenbach - complete human genetic map.

• 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 (1.8 Mb).
  • Richard Mathies et al. publish on sequencing dyes (PNAS, May).
  • Michael Reeve and Carl Fuller, thermostable polymerase for sequencing (Nature, August).

• 1996 International HGP partners agree to release sequence data into public databases within 24 hours.
  • International consortium releases genome sequence of yeast S. cerevisiae (12.1 Mb).
  • Yoshihide Hayashizaki's at RIKEN completes the first set of full-length mouse cDNAs.
  • ABI introduces a capillary electrophoresis system, the 310.

• 1997 Blattner, Plunkett et al. publish the sequence of E. coli (5 Mb)

• 1998 Phil Green and Brent Ewing of Washington University publish “phred” for interpreting sequencer data (in use since ‘95).
  • 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 (97Mb).

• 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.

• 2000 Celera and collaborators sequence fruit fly Drosophila melanogaster (180Mb) - validation of Venter's shotgun method. HGP and Celera debate issues related to data release.
  • HGP consortium publishes sequence of chromosome 21.
  • HGP & Celera jointly announce working drafts of HG sequence, promise joint publication.
  • Human gene estimates range from 35,000 to 120,000. International consortium completes 1st plant sequence, Arabidopsis thaliana (125 Mb).

• 2001 HGP consortium publishes Human Genome Sequence draft in Nature (15 Feb).
  • Celera publishes the Human Genome sequence in Science (16 Feb).

• 2003 Cold Spring Harbor sponsors GeneSweep, a sweepstakes on the number of human genes.

• 2005 420,000 VariantSEQr human resequencing primer sequences published on new NCBI Probe database.

• 2007 Solexa and Applied Biosystems release next-generation sequencing technology with potential to produce 10^6 greater sequencing data than capillary electrophoresis systems. Illumina acquires Solexa.

[edit] See also

• 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.

[edit] 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. ^ Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448
4. ^ http://nobelprize.org/nobel_prizes/chemistry/laureates/1980/sanger-lecture.pdf
5. ^ 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.
6. ^ 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. A rapid and versatile method has been developed for the synthesis of oligonucleotides which contain an aliphatic amino group at their 5' terminus. This amino group reacts specifically with a variety of electrophiles, thereby allowing other chemical species to be attached to the oligonucleotide. This chemistry has been utilized to synthesize several fluorescent derivatives of an oligonucleotide primer used in DNA sequence analysis by the dideoxy (enzymatic) method. The modified primers are highly fluorescent and retain their ability to specifically prime DNA synthesis. The use of these fluorescent primers in DNA sequence analysis will enable DNA sequence analysis to be automated. 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 !!
7. ^ http://www.ornl.gov/sci/techresources/Human_Genome/home.shtml
8. ^ http://marketing.appliedbiosystems.com/mk/get/SOLID_KNOWLEDGE_LANDING?_A=80414&_D=52611&_V=0&dummy=dum&isource=fr_GAAS_MS_200610#
9. ^ Nat Biotechnol. 2003 Jun;21(6):673-8. Epub 2003
10. ^ Nat Biotechnol. 2000 Jun;18(6):630-4. Erratum in: Nat Biotechnol 2000 Oct;18(10):1021. Comment in: Nat Biotechnol. 2000 Jun;18(6):597-8. Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays.
11. ^ Genomics. 1989, pages 114-28. Sequencing of megabase plus DNA by hybridization: theory of the method. Genetic Engineering Center, Belgrade, Yugoslavia
12. ^ US Patent 5,641,658
13. ^ Nucleic Acids Research. 2004, page e166
14. ^ http://visigenbio.com/technology_overview.html
15. ^ http://mcb.harvard.edu/branton/index.htm
16. ^ USPTO application # 20060029957 assigned to ZS genetics http://www.freepatentsonline.com/20060029957.html
17. ^ http://hum-molgen.org/mail-archive/1995-Nov/msg00009.html
18. ^ Biotechniques. 1995 Sep;19(3):482-6.
19. ^ As a matter of English grammar, the word "complete" is an absolute, like unique: something is either complete or not; misuse of this word is quite common
20. ^ http://repositories.cdlib.org/lbnl/LBNL-55264/

[edit] External links

• DNA Sequencing: Dye Terminator Animation
• Sequencing-By-Synthesis Demo