High Throughput Sequencing

2012-10-23T10:40:47Z

110109778:

High Throughput Sequencing (also known as Illumina Solexa DNA sequencing) is a technique used to sequence [[DNA|DNA]] or [[CDNA|cDNA]] via fragmenting the genetic information randomly. RNA can also be sequenced but involves additional steps to those shown below. Firstly [[MRNA|mRNA]] is hydrolysed by magnesium [[Catalysts|catalysed]] reaction to give fragments and then the sequences are randomly primed by [[Reverse transcription|reverse transcription]] <ref>Mardis ER. (2008), "Next-generation DNA sequencing methods", Annu Rev Genomics Hum Genet.,9:387-402</ref>. 

'''The Process:'''

===== Step 1 =====

Double stranded DNA (dsDNA) is randomly fragmented to blund ended pieces. Attach adapters to either end of the fragmented [[DsDNA|dsDNA]] via blunt end ligation. Two adapters are added separately so both ends of the [[DNA|dsDNA]] have an adapter present. 

===== Step 2 =====

Denature the [[DNA|dsDNA]] to [[SsDNA|ssDNA]] and wash over flow cell channels. The adapters will enable binding to the dense lawn of [[Primers|primers]] on the flow cell. 

===== Step 3 =====

Bridge Amplification- The free end of the fragment bends and hybridises to a separate complimentary [[Primers|primer]] on the flow cell forming a 'bridge'. Unlabelled nucleotides and [[Enzyme|enzymes]] are then added to the flow cell to initiate amplification of the second strand of [[DNA|DNA]]. 

===== Step 4 =====

Denature [[DNA|DNA]] back into single stranded fragments and the process is repeated to produce clusters of identical [[DNA|DNA]] around the original sequence. 

'''Step 5'''

First Chemistry Cycle- Add four reversible terminators, [[Primers|primers]] and [[DNA|DNA]] [[Polymerase|polymerase]] to the flow cell. Each cluster will show up as spot after laser excitation due to the added bases fluorescence. The blocked 3' OH terminus and the flurophone from each base are then removed to allow another base to be added to the sequence. This will eventually allow the full sequence to be built up. 

=== References ===

<references />

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

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2011-11-23T21:27:58Z

110109778: Created page with " SNAREs are proteins involved in the exocytosis of vesicles. There are two types of SNARE protein, v-SNARE and t-SNARE. The v-SNARE is attatched to the membrane of the vesic..."

 SNAREs are proteins involved in the exocytosis of vesicles. There are two types of SNARE protein, v-SNARE and t-SNARE. The v-SNARE is attatched to the membrane of the vesicle to be released. The t-SNARE is integrated into the target organelles membrane.

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High Throughput Sequencing

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