title: Termination of DNA synthesis by N6-alkylated, not 3'-O-alkylated, photocleavable 2'-deoxyadenosine triphosphates.
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.
authors: LaserGen, Inc., Houston, TX 77054, USA.
authors: Weidong
authors: W
authors: Wu
authors: Brian P
authors: BP
authors: Stupi
authors: Vladislav A
authors: VA
authors: Litosh
authors: Dena
authors: D
authors: Mansouri
authors: Demetra
authors: D
authors: Farley
authors: Sidney
authors: S
authors: Morris
authors: Sherry
authors: S
authors: Metzker
authors: Michael L
authors: ML
authors: Metzker
mesh_terms: D000478:Alkylation; D020137:Base Pair Mismatch; D004247:DNA / Q000096:biosynthesis*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000138:chemical synthesis / Q000737:chemistry* / Q000528:radiation effects; D003839:Deoxyadenosines / Q000138:chemical synthesis / Q000737:chemistry; D010777:Photochemistry; D017422:Sequence Analysis, DNA / Q000379:methods*; D014466:Ultraviolet Rays
chemical_list: D003838:Deoxyadenine Nucleotides; D003839:Deoxyadenosines; C525215:N(6)-(2-nitrobenzyl)deoxyadenosine triphosphate; D004247:DNA; D004259:DNA-Directed DNA Polymerase; C026600:2'-deoxyadenosine triphosphate