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DNA sequencing

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<p>The term <strong>DNA sequencing</strong> 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 attained 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.</p>

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<h2><span class="mw-headline">Early methods</span></h2>

<h2><span class="mw-headline">Chain-termination methods</span></h2>

<p>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.</p>

<p>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<sup class="reference" id="cite_ref-9"><font color="#800080">[10]</font></sup><sup class="reference" id="cite_ref-10"><font color="#800080">[11]</font></sup> of fluorescently labeled ddNTPs and primers set the stage for automated, high-throughput DNA sequencing.</p>

<h3><span class="mw-headline">Dye-terminator sequencing</span></h3>

<h3><span class="mw-headline">Automation and sample preparation</span></h3>

<p>Current methods can directly sequence only relatively short (300-1000 nucleotides long) DNA fragments in a single reaction. [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.<sup class="reference" id="cite_ref-Reeve.2CFuller_11-0"><font color="#800080">[12]</font></sup></p>

<p>Large-scale sequencing aims at sequencing very long DNA fragments. Even relatively small bacterial <font color="#800080">genomes</font> 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 artificial chromosome (BAC), and amplified in <em>Escherichia coli</em>. 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 <em>un-clonable</em> 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 <em>de novo</em> 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 <em>reads</em> 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 euchromatic sequence (99% complete at the time), excluding these intractable repetitive regions.<sup class="reference" id="cite_ref-12"><font color="#800080">[13]</font></sup></p>

<h2><span class="mw-headline">External links</span></h2>

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<li><a class="external text" title="http://www.jgi.doe.gov/education/how/how30minflash.html" rel="nofollow" href="http://www.jgi.doe.gov/education/how/how30minflash.html~~" rel="nofollow~~">DNA Sequencing: Dye Terminator Animation</a> </li> <li><a class="external text" title="http://www.genomics.xprize.org" rel="nofollow" href="http://www.genomics.xprize.org/~~" rel="nofollow~~">Archon Genomics X PRIZE</a> - $10 million competition for fast and inexpensive sequencing technology </li>

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