Termination of DNA synthesis by N6-alkylated, not 3′-O-alkylated, photocleavable 2′-deoxyadenosine triphosphates
Weidong Wu,Brian P. Stupi,Vladislav A. Litosh,Dena L. Mansouri,Demetra Farley,Sidney E. Morris,Sherry Metzker,Michael L. Metzker +7 more
TLDR
A novel paradigm in RT chemistry is discovered, the attachment of a photocleavable, 2-nitrobenzyl group to the N6-position of 2′-deoxyadenosine triphosphate (dATP), which, upon incorporation, terminates DNA synthesis.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.read more
Citations
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Sequencing technologies-the next generation
TL;DR: A technical review of template preparation, sequencing and imaging, genome alignment and assembly approaches, and recent advances in current and near-term commercially available NGS instruments is presented.
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Four-color dna sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators
TL;DR: In this paper, a process for sequencing single-stranded DNA employing modified nucleotides was proposed, which is based on the approach described in this paper. But this method requires a large number of modifications.
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The challenges of sequencing by synthesis
Carl W. Fuller,Lyle Richard Middendorf,Steven A. Benner,George M. Church,Timothy D. Harris,Xiaohua Huang,Stevan B Jovanovich,John Richard Nelson,Jeffery A. Schloss,David C. Schwartz,Dmitri V. Vezenov +10 more
TL;DR: This work frames the challenges of DNA sequencing-by-synthesis in a manner accessible to a broad community of scientists and engineers, and hopes to solicit input from the broader research community on means of accelerating the advancement of genome sequencing technology.
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Four-color DNA sequencing with 3′-O-modified nucleotide reversible terminators and chemically cleavable fluorescent dideoxynucleotides
Jia Guo,Ning Xu,Zengmin Li,Shenglong Zhang,Jian Wu,Dae H. Kim,Mong S. Marma,Qinglin Meng,Huanyan Cao,Xiaoxu Li,Shundi Shi,Lin Yu,Sergey Kalachikov,James J. Russo,Nicholas J. Turro,Jingyue Ju +15 more
TL;DR: A DNA sequencing method that is a hybrid between the Sanger dideoxynucleotide terminating reaction and SBS, using four nucleotides modified as reversible terminators by capping the 3′-OH with a small reversible moiety so that they are still recognized by DNA polymerase as substrates to perform SBS.
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