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
Date Published1994 Oct 11
KeywordsBase Sequence, Chromatography, High Pressure Liquid, Deoxyribonucleotides, DNA, DNA-Directed DNA Polymerase, Molecular Sequence Data, Molecular Structure, Templates, Genetic

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.

Alternate JournalNucleic Acids Res
PubMed ID7937154
PubMed Central IDPMC331938
Grant List5 P30 HG00210 / HG / NHGRI NIH HHS / United States

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