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Old 04-04-2012, 11:26 AM   #1
kmpphd
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Location: Birmingham, AL

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Default Cluster formation/ddNTP blocking

I have a ddNTP question based on the Bentley et al NATURE 2008 Article, “Accurate whole human genome sequencing using reversible terminator chemistry."

In the Supplementary Information, page 4, “Processing of clusters for single read experiments,” it is stated “ All exposed 3’-OH termini of DNA…were blocked by dideoxy chain termination using a terminal transferase and ddNTPs.”

Again on page 4, “Processing of clusters for paired read experiments,” it is stated “Clusters were 3’-dephosphorylated using T4 polynucleotide kinase…”

Following the final round of bridge amplification to form the clusters, my assumption is that ddNTPs with 3’-monophosphates were used for blocking (ddCTP specifically given the sequences of the primers used?). The flow cell-fixed primers that had been treated with USER would already have
3'-monophosphate groups, so essentially all exposed 3’-hydroxyls are capped with 3’-phosphates. Treatment with T4 polynucleotide kinase then converts all 3’-phosphates to 3’-hydroxyls, allowing extension to take place.

Is my assumption correct?
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Old 04-04-2012, 12:39 PM   #2
kmpphd
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Default

Follow up to my ddNTP question. I just skimmed through the Bentley et al patent application entitled "Method for Sequencing a Polynucleotide Template," Patent Application Number 20110009276. In it is referenced WO 2004/070005, "Double Ended Sequencing."

The latter patent states in part “The reversibly blocked primers should be blocked with different chemical groups. By choosing the appropriate method of deblocking, one primer may be deblocked without affecting the blocking groups of the other primers. In a preferred embodiment, the blocking group is P04. That is, the second primer is blocked by P04 and deblocking is accomplished by T4 polynucleotide kinase (utilizing its 3'-phosphatase activity) or calf-intestinal alkaline phosphatase (CIAP) or any suitable phosphatase.”

Still not a confirmation of what is going on within the cBot but it is consistent with the Bentley paper.
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