A polymerase engineered for bisulfite sequencing.
Bisulfite sequencing is a key methodology in epigenetics. However, the standard workflow of bisulfite sequencing involves heat and strongly basic conditions to convert the intermediary product 5,6-dihydrouridine-6-sulfonate (dhU6S) (generated by reaction of bisulfite with deoxycytidine (dC)) to uracil (dU). These harsh conditions generally lead to sample loss and DNA damage while milder conditions may result in incomplete conversion of intermediates to uracil. Both can lead to poor recovery of bisulfite-treated DNA by the polymerase chain reaction (PCR) as either damaged DNA and/or intermediates of bisulfite treatment are poor substrate for standard DNA polymerases. Here we describe an engineered DNA polymerase (5D4) with an enhanced ability to replicate and PCR amplify bisulfite-treated DNA due to an ability to bypass both DNA lesions and bisulfite intermediates, allowing significantly milder conversion conditions and increased sensitivity in the PCR amplification of bisulfite-treated DNA. Incorporation of the 5D4 DNA polymerase into the bisulfite sequencing workflow thus promises significant sensitivity and efficiency gains.
Genetic Signatures, Level 9, Lowy Packer Building 405, Liverpool Street, Darlinghurst 2010, Sydney, Australia.
Doug
D
Millar
MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK.
Yonka
Y
Christova
MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK ph1@mrc-lmb.cam.ac.uk.
Philipp
P
Holliger
D045744:Cell Line, Tumor; D004247:DNA / Q000096:biosynthesis; D019175:DNA Methylation; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013447:Sulfites; D019914:Taq Polymerase / Q000378:metabolism; D013698:Templates, Genetic
D011993:Recombinant Fusion Proteins; D013447:Sulfites; D004247:DNA; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase; C042345:hydrogen sulfite