Sequencing
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:''For the sense of "sequencing" used in [[electronic music]], see the [[music sequencer]] article.''
In [[genetics]] and [[biochemistry]], '''sequencing''' means to determine the [[primary structure]] (or primary sequence) of an unbranched [[biopolymer]]. Sequencing results in a symbolic linear depiction known as a '''sequence''' which succinctly summarizes much of the atomic-level structure of the sequenced molecule.
==DNA sequencing==
{{main|DNA sequencing}}
DNA sequencing is the process of determining the [[nucleotide]] order of a given [[DNA]] fragment. Thus far, most DNA sequencing has been performed using the [[chain termination method]] developed by [[Frederick Sanger]]. This technique uses sequence-specific termination of a DNA synthesis reaction using modified nucleotide substrates. However, new sequencing technologies such as [[Pyrosequencing]] are gaining an increasing share of the sequencing market. More genome data is being produced by pyrosequencing than Sanger DNA sequencing these days. Pyrosequencing has enabled rapid genome sequencing. Bacterial genome can be sequenced in a single run with several X coverage with this technique. This technique was also used to sequence the genome of [[James Watson]] recently.
The sequence of DNA encodes the necessary information for living things to survive and reproduce. Determining the sequence is therefore useful in 'pure' research into why and how organisms live, as well as in applied subjects. Because of the key nature of DNA to living things, knowledge of DNA sequence may come in useful in practically any biological research. For example, in medicine it can be used to identify, diagnose and potentially develop treatments for genetic diseases. Similarly, research into [[pathogens]] may lead to treatments for contagious diseases. [[Biotechnology]] is a burgeoning discipline, with the potential for many useful products and services.
===Sanger sequencing===
[[Image:Sequencing.jpg|thumb|right|Part of a radioactively labelled sequencing gel]]In chain terminator sequencing (Sanger sequencing), extension is initiated at a specific site on the template DNA by using a short oligonucleotide 'primer' complementary to the template at that region. The oligonucleotide primer is extended using a [[DNA polymerase]], an enzyme that replicates DNA. Included with the primer and DNA polymerase are the four deoxynucleotide bases (DNA building blocks), along with a low concentration of a chain terminating nucleotide (most commonly a '''di-'''deoxynucleotide). Limited incorporation of the chain terminating nucleotide by the DNA polymerase results in a series of related DNA fragments that are terminated only at positions where that particular nucleotide is used. The fragments are then size-separated by electrophoresis in a slab polyacrylamide gel, or more commonly now, in a narrow glass tube (capillary) filled with a viscous polymer.
[[Image:Sanger sequencing read display.gif|thumb|right|View of the start of an example dye-terminator read (click to expand)]]
An alternative to the labelling of the primer is to label the terminators instead, commonly called 'dye terminator sequencing'. The major advantage of this approach is the complete sequencing set can be performed in a single reaction, rather than the four needed with the labeled-primer approach. This is accomplished by labelling each of the dideoxynucleotide chain-terminators with a separate fluorescent dye, which fluoresces at a different [[wavelength]]. This method is easier and quicker than the dye primer approach, but may produce more uneven data peaks (different heights), due to a template dependent difference in the incorporation of the large dye chain-terminators. This problem has been significantly reduced with the introduction of new enzymes and dyes that minimize incorporation variability.
This method is now used for the vast majority of sequencing reactions as it is both simpler and cheaper. The major reason for this is that the primers do not have to be separately labelled (which can be a significant expense for a single-use custom primer), although this is less of a concern with frequently used 'universal' primers.
===Pyrosequencing===
[[Pyrosequencing]], which was originally developed by Mostafa Ronaghi, has been commercialized by Biotage (for low throughput sequencing) and 454 Life Sciences (for high-throughput sequencing). The latter platform sequences roughly 100 megabases in a 7-hour run with a single machine. In the array-based method (commercialized by 454 Life Sciences), single-stranded DNA is annealed to beads and amplified via em[[PCR]]. These DNA-bound beads are then placed into wells on a fiber-optic chip along with [[enzymes]] which produce light in the presence of [[adenosine triphosphate|ATP]]. When free nucleotides are washed over this chip, light is produced as ATP is generated when nucleotides join with their complementary [[base pair]]s. Addition of one (or more) nucleotide(s) results in a reaction that generates a light signal that is recorded by the CCD camera in the instrument. The signal strength is proportional to the number of nucleotides, for example, homopolymer stretches, incorporated in a single nucleotide flow. [http://www.454.com]
==RNA sequencing==
[[RNA]] is less stable in the cell, and also more prone to nuclease attack experimentally. As RNA is generated by [[Transcription (genetics)|transcription]] from DNA, the information is already present in the cell's DNA. However, it is sometimes desirable to sequence RNA molecules. In particular, in [[Eukaryote]]s RNA molecules are not necessarily [[co-linear]] with their DNA template, as [[intron]]s are excised. To sequence RNA, the usual method is first to [[reverse transcriptase|reverse transcribe]] the sample to generate DNA fragments. This can then be sequenced as described above.
==Protein sequencing==
{{main|protein sequencing}}
Methods for performing [[protein]] sequencing
include:
*[[Edman degradation]]
*[[Peptide mass fingerprinting]]
*[[Mass spectrometry]]
*[[Protease digests]]
If the gene encoding the protein can be identified it is currently much easier to sequence the DNA and infer the protein sequence. Determining part of a protein's amino-acid sequence (often one end) by one of the above methods may be sufficient to enable the identification of a [[clone (genetics)|clone]] carrying the gene.
==Polysaccharide sequencing==
Though [[polysaccharide]]s are also biopolymers, it is not so common to talk of 'sequencing' a polysaccharide, for several reasons. Although many polysaccharides are linear, many have branches. Many different units (individual [[monosaccharide]]s) can be used, and [[chemical bond|bonded]] in different ways. However, the main theoretical reason is that whereas the other polymers listed here are primarily generated in a 'template-dependent' manner by one processive enzyme, each individual join in a polysaccharide may be formed by a different [[enzyme]]. In many cases the assembly is not uniquely specified; depending on which enzyme acts, one of several different units may be incorporated. This can lead to a family of similar molecules being formed. This is particularly true for plant polysaccharides. Methods for the [[structure determination]] of [[oligosaccharide]]s and [[polysaccharide]]s include [[NMR]] spectroscopy and [[methylation analysis]]<ref>[http://www.stenutz.eu/sop A practical guide to structural analysis of carbohydrates]</ref>.
==See also==
* [[Genetic code]]
* [[Sequence motif]]
==References==
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[[Category:Biochemistry methods]]
[[Category:Molecular biology]]
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