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?
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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