Polymerase Usage Sequencing
Pu-seq provides direct replication-origin location and efficiency data, as well as indirect estimates of replication timing (Daigaku et al., 2015).
Alkali treatment of duplex ribonucleotide-containing DNA results in phosphate-backbone cleavage 3ê to the ribose, resulting in a 5ê-hydroxyl end. If the denatured DNA is used as a template for random-hexamer primer extension, 5ê to 3ê synthesis results in a flush end adjacent to the initial ribose. By generating a library from single-stranded DNA and placing distinct index primers at each end, sequencing reads can be mapped to individual strands, to determine the original ribonucleotide position with single-base accuracy.
- Single-base accuracy
- Only applied to yeast
Illumina Library prep and Array Kit Selector
None available yet
Keszthelyi A., Daigaku Y., Ptasinska K., Miyabe I. and Carr A. M. Mapping ribonucleotides in genomic DNA and exploring replication dynamics by polymerase usage sequencing (Pu-seq). Nat Protoc. 2015;10:1786-1801