High Throughput Sequencing: Difference between revisions
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High Throughput 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]]. ( | High Throughput 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>.<br> | ||
===== Step 1 ===== | ===== Step 1 ===== | ||
Attach adapters to either end of the fragmented [[DsDNA|dsDNA]]. Two adapters are added separately so both ends of the [[DNA|dsDNA]] have an adapter present. | Attach adapters to either end of the fragmented [[DsDNA|dsDNA]]. Two adapters are added separately so both ends of the [[DNA|dsDNA]] have an adapter present.<br> | ||
<br> | ===== Step 2<br> ===== | ||
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.<br> | |||
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. | |||
<br> | |||
===== Step 3 ===== | ===== Step 3 ===== | ||
Bridge Amplification- Free end of the fragment hybridises with a separate [[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 [[DNA|DNA]]. | Bridge Amplification- Free end of the fragment hybridises with a separate [[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 [[DNA|DNA]].<br> | ||
<br> | |||
===== Step 4 ===== | ===== Step 4 ===== | ||
Denature [[DNA|DNA]] back into single stranded fragments producing clusters of identical [[DNA|DNA]] around the original sequence. | Denature [[DNA|DNA]] back into single stranded fragments producing clusters of identical [[DNA|DNA]] around the original sequence.<br> | ||
'''Step 5''' | |||
<br> | 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' 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.<br> | ||
=== References === | === References === | ||
<references /> | |||
Revision as of 18:26, 15 November 2010
High Throughput Sequencing is a technique used to sequence DNA or cDNA via fragmenting the genetic information randomly. RNA can also be sequenced but involves additional steps to those shown below. Firstly mRNA is hydrolysed by magnesium catalysed reaction to give fragments and then the sequences are randomly primed by reverse transcription [1].
Step 1
Attach adapters to either end of the fragmented dsDNA. Two adapters are added separately so both ends of the dsDNA have an adapter present.
Step 2
Denature the dsDNA to ssDNA and wash over flow cell channels. The adapters will enable binding to the dense lawn of primers on the flow cell.
Step 3
Bridge Amplification- Free end of the fragment hybridises with a separate primer on the flow cell forming a 'bridge'. Unlabelled nucleotides and enzymes are then added to the flow cell to initiate amplification of the DNA.
Step 4
Denature DNA back into single stranded fragments producing clusters of identical DNA around the original sequence.
Step 5
First Chemistry Cycle- Add four reversible terminators, primers and DNA polymerase to the flow cell. Each cluster will show up as spot after laser excitation due to the added bases fluorescence. The blocked 3' 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
- ↑ Mardis ER. (2008), "Next-generation DNA sequencing methods", Annu Rev Genomics Hum Genet.,9:387-402