Termination of DNA synthesis by N6-alkylated, not 3'-O-alkylated, photocleavable 2'-deoxyadenosine triphosphates.

TitleTermination of DNA synthesis by N6-alkylated, not 3'-O-alkylated, photocleavable 2'-deoxyadenosine triphosphates.
Publication TypeJournal Article
Year of Publication2007
AuthorsWu, W, Stupi, BP, Litosh, VA, Mansouri, D, Farley, D, Morris, S, Metzker, S, Metzker, ML
JournalNucleic Acids Res
Volume35
Issue19
Pagination6339-49
Date Published2007
ISSN1362-4962
KeywordsAlkylation, Base Pair Mismatch, Deoxyadenine Nucleotides, Deoxyadenosines, DNA, DNA-Directed DNA Polymerase, Photochemistry, Sequence Analysis, DNA, Ultraviolet Rays
Abstract

The Human Genome Project has facilitated the sequencing of many species, yet the current Sanger method is too expensive, labor intensive and time consuming to accomplish medical resequencing of human genomes en masse. Of the 'next-generation' technologies, cyclic reversible termination (CRT) is a promising method with the goal of producing accurate sequence information at a fraction of the cost and effort. The foundation of this approach is the reversible terminator (RT), its chemical and biological properties of which directly impact the performance of the sequencing technology. Here, we have discovered a novel paradigm in RT chemistry, the attachment of a photocleavable, 2-nitrobenzyl group to the N(6)-position of 2'-deoxyadenosine triphosphate (dATP), which, upon incorporation, terminates DNA synthesis. The 3'-OH group of the N(6)-(2-nitrobenzyl)-dATP remains unblocked, providing favorable incorporation and termination properties for several commercially available DNA polymerases while maintaining good discrimination against mismatch incorporations. Upon removal of the 2-nitrobenzyl group with UV light, the natural nucleotide is restored without molecular scarring. A five-base experiment, illustrating the exquisite, stepwise addition through a homopolymer repeat, demonstrates the applicability of the N(6)-(2-nitrobenzyl)-dATP as an ideal RT for CRT sequencing.

DOI10.1093/nar/gkm689
Alternate JournalNucleic Acids Res.
PubMed ID17881370
PubMed Central IDPMC2095803
Grant ListR01 HG003573 / HG / NHGRI NIH HHS / United States
R41 HG003072 / HG / NHGRI NIH HHS / United States
R43 HG003443 / HG / NHGRI NIH HHS / United States