Termination of DNA synthesis by novel 3'-modified-deoxyribonucleoside 5'-triphosphates.

TitleTermination of DNA synthesis by novel 3'-modified-deoxyribonucleoside 5'-triphosphates.
Publication TypeJournal Article
Year of Publication1994
AuthorsMetzker, ML, Raghavachari, R, Richards, S, Jacutin, SE, Civitello, A, Burgess, K, Gibbs, RA
JournalNucleic Acids Res
Volume22
Issue20
Pagination4259-67
Date Published1994 Oct 11
ISSN0305-1048
KeywordsBase Sequence, Chromatography, High Pressure Liquid, Deoxyribonucleotides, DNA, DNA-Directed DNA Polymerase, Molecular Sequence Data, Molecular Structure, Templates, Genetic
Abstract

Eight 3'-modified-dNTPs were synthesized and tested in two different DNA template assays for incorporation activity. From this enzymatic screen, two 3'-O-methyl-dNTPs were shown to terminate DNA syntheses mediated by a number of polymerases and may be used as alternative terminators in Sanger sequencing. 3'-O-(2-Nitrobenzyl)-dATP is a UV sensitive nucleotide and was shown to be incorporated by several thermostable DNA polymerases. Base specific termination and efficient photolytic removal of the 3'-protecting group was demonstrated. Following deprotection, DNA synthesis was reinitiated by the incorporation of natural nucleotides into DNA. The identification of this labile terminator and the demonstration of a one cycle stop-start DNA synthesis are initial steps in the development of a novel sequencing strategy.

DOI10.1093/nar/22.20.4259
Alternate JournalNucleic Acids Res.
PubMed ID7937154
PubMed Central IDPMC331938
Grant List5 P30 HG00210 / HG / NHGRI NIH HHS / United States