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

Authors
  • Wu, Weidong
  • Stupi, Brian P
  • Litosh, Vladislav A
  • Mansouri, Dena
  • Farley, Demetra
  • Morris, Sidney
  • Metzker, Sherry
  • Metzker, Michael L
Type
Published Article
Journal
Nucleic Acids Research
Publisher
Oxford University Press
Publication Date
Jan 01, 2007
Volume
35
Issue
19
Pages
6339–6349
Identifiers
PMID: 17881370
Source
Medline
License
Unknown

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.

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