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Nucleic Acids Research Advance Access published online on September 18, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm689
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© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Chemistry

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

Weidong Wu1, Brian P. Stupi1, Vladislav A. Litosh1, Dena Mansouri2, Demetra Farley3, Sidney Morris1,3, Sherry Metzker1 and Michael L. Metzker1,2,3,*

1LaserGen, Inc., Houston, TX 77054, 2Department of Molecular & Human Genetics and 3Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX 77030, USA

*To whom correspondence should be addressed. Tel: +1 713 798 7565; Fax: +1 713 798 5741; Email: mmetzker{at}bcm.edu

Received July 25, 2007. Revised August 17, 2007. Accepted August 21, 2007.

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 N6-position of 2'-deoxyadenosine triphosphate (dATP), which, upon incorporation, terminates DNA synthesis. The 3'-OH group of the N6-(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 N6-(2-nitrobenzyl)-dATP as an ideal RT for CRT sequencing.


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