ILLUMINA (SOLEXA) DNA SEQUENCING: Difference between revisions

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Solexa DNA sequencing allows for one base at a time to be identified from a DNA sample via the use of fluorescence.
Solexa [[DNA|DNA]] sequencing is a technique used sequence DNA one [[Nucleotide|nucleotide]] base at a time with the use of fluorescence.  


The DNA sample must firstly be prepared and randomly fragmented to about 100-500bp long. Blunt end ligation is then carried out in order to attach the adaptors-these contain the purification tags for fluorescence. The DNA sample is then washed over a flow cell and the single stranded fragments attach randomly to the inside surface. Nucleotides and enzyme are then added in order to initiate bridge amplification which results in double stranded bridges. Denaturisation must then occur in order to leave single stranded templates.  
The DNA&nbsp;fragments are&nbsp;first washed over the flow cell. [[Enzymes|Enzymes]] and free nucleotides are then added in order to form double stranded cross bridges and further amplification takes place. Denaturisation then occurs in order to obtain single stranded DNA fragments that are well annealed to the surface of the cell. The modified [[DNTP|dNTP's]] are then added; these dNTP's include 3' blocking group instead of -OH which prevents further dNTP's pairing and different colour fluorescence depending on their base. Free dNTP's can then be washed off and a computer image is then taken which highlights the different colours. The [[Staudinger reaction|Staudinger reaction]] and then [[hydrolysis|hydrolysis]] occurs in order to get rid of fluorescence and remove the blocking group. The -OH present then allows for further addition of dNTP's in the second cycle&nbsp;<ref>Mardis ER (2008) Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet. 9:387-402</ref>.<br>


The first cycle then occurs in which all 4 dNTP's are washed over the flow cell and attach to the first nucleotide base of each DNA strand. These dNTP's have been modified in order to contain a 3'blocking group, which prevents further dNTP's attaching to the adjacent bases. They are also labelled with different fluorescent&nbsp;colours depending on their base.&nbsp;
=== Reference  ===


After attachment, all free dNTP's can be washed off and a computer image can be taken to identify colours and therefore the first base via complement pairing.
<references />
 
Before the second cycle occurs, the Staudinger reaction and then hydrolysis of hemiaminals occur in order to remove the 3'blocking group and the fluorescent dye. This allows for the next nucleotide base to attach as it is no longer being blocked. Multiple coloured images can be compared from each cycle to identify the sequence of the sample DNA.

Latest revision as of 09:24, 22 October 2014

Solexa DNA sequencing is a technique used sequence DNA one nucleotide base at a time with the use of fluorescence.

The DNA fragments are first washed over the flow cell. Enzymes and free nucleotides are then added in order to form double stranded cross bridges and further amplification takes place. Denaturisation then occurs in order to obtain single stranded DNA fragments that are well annealed to the surface of the cell. The modified dNTP's are then added; these dNTP's include 3' blocking group instead of -OH which prevents further dNTP's pairing and different colour fluorescence depending on their base. Free dNTP's can then be washed off and a computer image is then taken which highlights the different colours. The Staudinger reaction and then hydrolysis occurs in order to get rid of fluorescence and remove the blocking group. The -OH present then allows for further addition of dNTP's in the second cycle [1].

Reference

  1. ↑ Mardis ER (2008) Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet. 9:387-402
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