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<p><font sizespan style="4">[[Sequencing technology]]<br />[[Sequencing companies]]<br />[[Sequencing assembly program]]<br /></font><br /><br />&nbsp;</p><hr /><p><font size="6">What is sequencing?<br /><font size="3">Sequencing means to determine the order of signals in a polymer.</font>&nbsp;<br /><br /></font><font -size=:24px"3">In genetics and biochemistry, <strong>sequencing</strong> means to determine the primary structure (sequence) of an unbranched biopolymer. DNA Sequencing results in a symbolic linear depiction known as a <strong>sequence</strong> which succinctly summarizes much of the atomic-level structure of the sequenced molecule. </fontspan></p> 

 <h2p><span classstyle="mwfont-headlinesize:20px">[[DNA sequencing]]</span></h2><p><font size="3"strong>DNA sequencing What is the process of determining the nucleotide order of a given <font color="#810081">DNASequencing?</fontstrong> 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.</fontspan></p><p><font size="3">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.</font></p>

 <h3p><span class="mw-headline">Sanger sequencing</span></h3><div class="thumb tright"><div style="width: 162px" class="thumbinner"><img class="thumbimage" border="0" alt="Part of a radioactively labelled sequencing gel" width="160" height="332" src="http://upload.wikimedia.org/wikipedia/commons/c/cb/Sequencing.jpg" /><div class="thumbcaption"><div style="float: right" class="magnify"><img alt="" width="15" height="11" src="http://en.wikipedia.org/skinsfont-1.5/common/images/magnify-clip.png" /></div>Part of a radioactively labelled sequencing gel</div></div></div><p><font size="3:14px">In chain terminator DNA sequencing (Sanger sequencing), extension is initiated at a specific site on the template DNA by using a short oligonucleotide 'primer' complementary to process of determining the template at that region. The oligonucleotide primer is extended using nucleotide order of a given DNA polymerasefragment. Thus far, an enzyme that replicates most DNA. Included with sequencing has been performed using 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 <strong>ditermination method developed by Frederick Sanger. This technique uses sequence-</strong>deoxynucleotide). Limited incorporation specific termination 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 synthesis reaction using modified nucleotide is usedsubstrates. The fragments However, new sequencing technologies such as Pyrosequencing 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.</font></p><div class="thumb tright"><div style="width: 182px" class="thumbinner"><img class="thumbimage" border="0" alt="View gaining an increasing share of the start of an example dye-terminator read (click to expand)" width="180" height="42" src="http://uploadsequencing market.wikimedia.org/wikipedia/commons/thumb/4/44/Sanger_sequencing_read_display.gif/180px-Sanger_sequencing_read_display.gif" /><div class="thumbcaption"><div style="float: right" class="magnify"><img alt="" width="15" height="11" src="http://en.wikipedia.org/skins-1.5/common/images/magnify-clip.png" /></div>View of the start of an example dye-terminator read (click to expand)</div></div></div><p><font size="3">An alternative to the labelling of the primer More genome data is to label the terminators instead, commonly called 'dye terminator being produced by pyrosequencing than Sanger DNA sequencing'these days. The major advantage of this approach is the complete Pyrosequencing has enabled rapid genome sequencing set . Bacterial genome can be performed sequenced in a single reaction, rather than the four needed run with the labeled-primer approach. This is accomplished by labelling each of the dideoxynucleotide chain-terminators several X coverage with a separate fluorescent dye, which fluoresces at a different wavelengththis technique. This method is easier and quicker than the dye primer approach, but may produce more uneven data peaks (different heights), due technique was also used to a template dependent difference in sequence the incorporation genome of the large dye chain-terminators. This problem has been significantly reduced with the introduction of new enzymes and dyes that minimize incorporation variabilityJames Watson recently.</fontspan></p><p><font size="3">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.</font></p>

<h3><span class="mw-headline">Pyrosequencing</span></h3><p><font size="3">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 emPCR. These DNA-bound beads are then placed into wells on a fiber-optic chip along with enzymes which produce light in the presence of ATP. When free nucleotides are washed over this chip, light is produced as ATP is generated when nucleotides join with their complementary base pairs. 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. [1]</font></p><p><span style="font-size: medium20px"><bstrong>Synthesis based Why sequencing by Illuminais important?</bstrong></span></p><p><font size="3">Solexa, now part of Illumina developed a sequencing technology based on reversible dye-terminators. DNA molecules are first attached to primers on a slide and amplified so that local clonal colonies are formed (bridge amplification). Four types of ddNTPs are added, and non-incorporated nucleotides are washed away. Unlike pyrosequencing, the DNA can only be extended one nucleotide at a time. A camera takes images of the fluorescently labeled nucleotides then the dye along with the terminal 3' blocker is chemically removed from the DNA, allowing a next cycle. The final product of the SBS is many [[DNA reads]].</font>&nbsp;</p>

 <p><b span style="font-size: medium;20px">ligation based <strong>How sequencing&nbsp;is operated?</strong></bspan></p> 

 <p><span style="font-size: medium;14px">In this method, octomer-oligonucleotide probe is used, and instead The whole genome can&#39;t be sequenced all at once because available methods of DNA polymerase, sequencing can only handle short stretches of DNA ligase is used, as its name impliesat a time. Each of probes are dyed according to its first two nucleotides. FirstSo instead, primer is annealed to scientists must break the template strandgenome into small pieces, probe followssequence the pieces, and DNA ligase is applied on then reassemble them in the junctionproper order. An automatic sequencing machine spits out what genome scientists call &quot;raw&quot; sequence. unbound probe is washed awayIn raw sequence, and signal from bound probe is detectedthe reads or short DNA sequences are all jumbled together. End The process of probe transforming the fragmented rough draft into a long, continuous final product without breaks or errors is cleaved away from 3 nucleotide behind from first two nucleotidescalled finishing. Cycle is repeated with another primer, which has different starting baseFinishing often takes longer than the sequencing itself. The data are collected and compared to compensate empty nucleotides. This method is usually used for short strands.&nbsp;</span></p> 

 <h2p><span classstyle="mwfont-headlinesize:14px">RNA To facilitate their full genome sequencing</span></h2><p><font size="3"><font color="#810081">RNA</font> is less stable in the cellinitiatives, and also more prone to nuclease attack experimentally&ldquo;Illumina&rdquo; licensed nanopore sequencing technology from Oxford Nanopore Technologies. Illumina sequencing system based on reversible terminators for sequence determination. As RNA It is generated by transcription from an approach that generates several billion bases of accurate nucleotide sequence per experiment at low cost. Single molecules of DNAare attached to a flat surface, the information is already present amplified in the cell's DNAsitu and used as templates for synthetic sequencing with fluorescent reversible terminator deoxyribonucleotides. However, it Another possible way to accomplish cost-effective high-throughput sequencing is sometimes desirable to sequence RNA moleculesby utilizing fluorophore technology. In particular, Pacific Biosciences is currently using this approach in Eukaryotes RNA molecules are not necessarily co-linear with their SMRT (Single Molecule Real Time) DNA template, as introns are excisedsequencing technology. To sequence RNA, the usual method SMRT sequencing is first to convert a harnesses the sample to complimertary natural process of DNA replication and enables real-time observation of DNA fragments, synthesis by reverse transcriptaseusing phosphorlinked nucleotides. This can then be sequenced as described above.&nbsp;</fontspan></p> <h3>&nbsp;</h3> <p><span style="font -size="3:20px"><br strong>References</strong></fontspan></p> 

<h2><span class="mw-headline">Protein sequencing</span></h2><p><font size="3">Methods for performing protein sequencing include:</font></pol><ul> <li><font sizespan style="3">Edman degradation </font></li> <li><font -size="3:14px">Peptide mass fingerprinting <N/font>A. (2008). Accurate Whole Human Genome Sequencing using Reversible Terminator Chemistry. </liem> <li><font size="3">Mass spectrometry Nature.</fontem></li> <li><font size="3">Protease digests </font></liem>456</ulem><p><font size="3">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 end7218) by one of the above methods may be sufficient to enable the identification of a clone carrying the gene: 53&ndash;59.</fontspan></pli> <p>&nbsp;</p><h2li><span classstyle="mwfont-headline">Polysaccharide sequencing</span></h2><p><font size="3:14px">Though polysaccharides are also biopolymersXiaoge, it is not so common to talk of 'sequencing' a polysaccharideG., Kevin, for several reasonsL. Although many polysaccharides are linear, many have branchesKaren, O. Many different units (individual monosaccharides) can be used, and bonded in different waysJenny Z. However, the main theoretical reason is that whereas the other polymers listed here are primarily generated in a 'template-dependent' manner by one processive enzymeSandeep S. D., each individual join in a polysaccharide may be formed by a different enzyme. In many cases the assembly is not uniquely specified&amp; depending on which enzyme acts, one of several different units may be incorporatedSue J. This can lead to a family of similar molecules being formedR. This is particularly true for plant polysaccharides(2015). Methods SMRT Sequencing for the structure determination Parallel Analysis of oligosaccharides Multiple Targets and polysaccharides include NMR spectroscopy and methylation analysis<sup id="_ref-0" class="reference">[1]</sup>Accurate SNP Phasing.</font></p><p>&nbsp;</p><h2><span class="mw-headline">See also</span></h2><ul> <li><font size="3">Genetic code </font></li> <li><font size="3"em>Sequence motif G3</fontem></li> <li><font size="3">[http://sequenceome.org Sequenceomeg3.org] </font></li> <li><font size="3">[http://glycome115.net Glycome.net]</font></li></ul><p><a id="References" name="References"></a></p><h2><span class="mw-headline">References023317</span></h2><div class="references-small"><ol class="references"> <li id="_note-0"><strong><a title="" href="http://en.wikipedia.org/wiki/Sequencing#_ref-0">^</a></strong> <a class="external text" title="http://www.stenutz.eu/sop" rel="nofollow" href="http://www.stenutz.eu/sop">A practical guide to structural analysis of carbohydrates</a></li>

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