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<p><u><span style="font-size:24px"><strong>DNA Sequencing</strong></span></u>[[Sanger sequencing]]</p>

<p>What is sequencing?In genetics and biochemistry, <span style="font-size:20px"strong>sequencing</strong>What is means to determine the primary structure (or primary sequence) of an unbranched biopolymer. Sequencing?results in a symbolic linear depiction known as a </strong>sequence</spanstrong>which succinctly summarizes much of the atomic-level structure of the sequenced molecule.</p>

<p><span style="font-size:14px">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.</span>&nbsp;</p>

<p>&nbsp;<span style="font-size:20px"/p> <h2>DNA sequencing</h2> <strongp>Why 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 important?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.</strongp> </spanp>The sequence of DNA encodes the necessary information for living things to survive and reproduce. Determining the sequence is therefore useful in &#39;pure&#39; 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.</p>

<ph3>Sanger sequencing</h3> <div class="thumb tright"><div class="thumbinner" style="WIDTH: 162px"><img alt="Part of a radioactively labelled sequencing gel" class="thumbimage" src="http://upload.wikimedia.org/wikipedia/commons/c/cb/Sequencing.jpg" style="height:332px; width:160px" /><div class="thumbcaption"><span div class="magnify" style="fontFLOAT: right"><img alt="" src="http://en.wikipedia.org/skins-1.5/common/images/magnify-sizeclip.png" style="height:11px; width:14px15px"/></div>Part of a radioactively labelled sequencing gel</div></div></div> <p>Genome In chain terminator sequencing (Sanger sequencing ), extension is initiated at a specific site on the template DNA by using a short oligonucleotide &#39;primer&#39; complementary to the template at that region. The oligonucleotide primer is figuring out 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 <strong>di-</strong>deoxynucleotide). Limited incorporation of the chain terminating nucleotide by the order DNA polymerase results in a series of related DNA nucleotidesfragments 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 basesmore commonly now, in a narrow glass tube (capillary) filled with a viscous polymer.</p> <div class="thumb tright"><div class="thumbinner" style="WIDTH: 182px"><img alt="View of the start of an example dye-terminator read (click to expand)" class="thumbimage" src="http://upload.wikimedia.org/wikipedia/commons/thumb/4/44/Sanger_sequencing_read_display.gif/180px-Sanger_sequencing_read_display.gif" style="height:42px; width:180px" /><div class="thumbcaption"><div class="magnify" style="FLOAT: right"><img alt="" src="http://en.wikipedia.org/skins-1.5/common/images/magnify-clip. When you png" style="height:11px; width:15px" /></div>View of the start of an example dye-terminator read (click to expand)</div></div></div> <p>An alternative to the labelling of the primer is to label the terminators instead, commonly called &#39;dye terminator sequencing&#39;. The major advantage of this approach is the complete sequencing set can be performed in a sentencesingle reaction, rather than the four needed with the meaning labeled-primer approach. This is not just in the sequence accomplished by labelling each of the lettersdideoxynucleotide chain-terminators with a separate fluorescent dye, which fluoresces at a different wavelength. It This method is also in easier and quicker than the words those letters make and dye primer approach, but may produce more uneven data peaks (different heights), due to a template dependent difference in the grammar incorporation of the languagelarge dye chain-terminators. Similarly, This problem has been significantly reduced with the human genome introduction of new enzymes and dyes that minimize incorporation variability.</p> <p>This method is more than just its sequence. Sequencing now used for the genome vast majority of sequencing reactions as it is an important step towards understanding itboth simpler and cheaper. Scientists also hope The major reason for this is that being able to study the entire genome sequence will help them understand how the genome works&mdash;how genes work together primers do not have to direct the growthbe separately labelled (which can be a significant expense for a single-use custom primer), development and maintenance although this is less of an entire organisma concern with frequently used &#39;universal&#39; primers. Finally</p> <p>&nbsp;</p> <h3>Pyrosequencing</h3> <p>Pyrosequencing, which was originally developed by Mostafa Ronaghi, genes account has been commercialized by Biotage (for low throughput sequencing) and 454 Life Sciences (for less than 25 percent of 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 genomepresence of ATP. When free nucleotides are washed over this chip, and so knowing the entire genome sequence will help scientists study the parts 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 genome outside CCD camera in the genesinstrument. This includes The signal strength is proportional to the regulatory regions that control how genes are turned on an offnumber of nucleotides, for example, as well as long homopolymer stretches of junk DNA, incorporated in a single nucleotide flow.</span>[1]</p>

<p><span style="font-size:20px"><strongh2>How RNA sequencing is operated?</strongh2> </spanp>RNA is less stable in the cell, and also more prone to nuclease attack experimentally. As RNA is generated by transcription from DNA, the information is already present in the cell&#39;s DNA. However, it is sometimes desirable to sequence RNA molecules. In particular, in Eukaryotes RNA molecules are not necessarily co-linear with their DNA template, as introns are excised. To sequence RNA, the usual method is first to reverse transcribe the sample to generate DNA fragments. This can then be sequenced as described above.</p>

<h2>Protein sequencing</h2> <p>Methods for performing protein sequencing include:</p> <ul> <li>Edman degradation</li> <li>Peptide mass fingerprinting</li> <li>Mass spectrometry<span style="font-size:14px"/li> <li>Protease digests</li></ul> <p>The whole genome If the gene encoding the protein can&#39;t be sequenced all at once because available methods of DNA sequencing can only handle short stretches of DNA at a time. So instead, scientists must break the genome into small pieces, identified it is currently much easier to sequence the pieces, DNA and then reassemble them in infer the proper orderprotein sequence. An automatic sequencing machine spits out what genome scientists call Determining part of a protein&quot;raw&quot#39; s amino-acid sequence. In raw sequence, (often one end) by one of the above methods may be sufficient to enable the reads or short DNA sequences are all jumbled together. The process identification of transforming the fragmented rough draft into a long, continuous final product without breaks or errors is called finishing. Finishing often takes longer than clone carrying the sequencing itselfgene.</span></p>

<ph2>Polysaccharide sequencing<span style="font-size:20px"/h2> <strongp>Which kinds Though polysaccharides are also biopolymers, it is not so common to talk of &#39;sequencing techniques &#39; a polysaccharide, for several reasons. Although many polysaccharides are linear, many have branches. Many different units (individual monosaccharides) can be used, and bonded in different ways. However, the main theoretical reason is that whereas the other polymers listed here are available?primarily generated in a &#39;template-dependent&#39; 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 oligosaccharides and polysaccharides include NMR spectroscopy and methylation analysis</strongsup>[1]</spansup>.</p>

<p><span style="font-size:14px">To facilitate their full genome sequencing initiatives, &ldquo;Illumina&rdquo; licensed nanopore sequencing technology from Oxford Nanopore Technologies. Illumina sequencing system based on reversible terminators for sequence determination. It is an approach that generates several billion bases of accurate nucleotide sequence per experiment at low cost. Single molecules of DNA are attached to a flat surface, amplified in situ and used as templates for synthetic sequencing with fluorescent reversible terminator deoxyribonucleotides. Another possible way to accomplish cost-effective high-throughput sequencing is by utilizing fluorophore technology. Pacific Biosciences is currently using this approach in their SMRT (Single Molecule Real Time) DNA sequencing technology. SMRT sequencing is a harnesses the natural process of DNA replication and enables real-time observation of DNA synthesis by using phosphorlinked nucleotides.</spanh2>See also</ph2>

<h3ul>&nbsp; <li>Genetic code</li> <li>Sequence motif</li> <li>[http://sequenceome.org Sequenceome.org]</li> <li>[http://glycome.net Glycome.net]</li></h3ul>

<p><span stylea id="font-size:20pxReferences" name="><strong>References</strong"></spana></p>

<ph2>&nbsp;References</ph2>

<li><span stylestrong><a href="font-sizehttp:14px">N/A. (2008). Accurate Whole Human Genome Sequencing using Reversible Terminator Chemistry/en. <em>Naturewikipedia.<org/em> <em>456<wiki/emSequencing#_ref-0" title="">(7218): 53&ndash;59.^</spana></listrong> <li><span stylea class="external text" href="font-sizehttp:14px">Xiaoge, G//www., Kevin, Lstenutz., Karen, O., Jenny Zeu/sop" rel="nofollow" title="http://www., Sandeep Sstenutz. D., &amp; Sue J.R. (2015). SMRT Sequencing for Parallel Analysis of Multiple Targets and Accurate SNP Phasing. <em>G3<eu/emsop">. g3.115.023317A practical guide to structural analysis of carbohydrates</spana></li>

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