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.

Keywords

BiologyDNA polymeraseDNASanger sequencingNucleotideSequencing by ligationDNA synthesisPolymeraseTerminator (solar)BiochemistryDNA clampBase pairDNA sequencingMolecular biologyPolymerase chain reactionBase sequenceGeneGenomic libraryReverse transcriptase

MeSH Terms

Base SequenceChromatographyHigh Pressure LiquidDNADNA-Directed DNA PolymeraseDeoxyribonucleotidesMolecular Sequence DataMolecular StructureTemplatesGenetic

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Publication Info

Year
1994
Type
article
Volume
22
Issue
20
Pages
4259-4267
Citations
91
Access
Closed

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Cite This

Michael L. Metzker, Ramesh Raghavachari, Stephen M. Richards et al. (1994). Termination of DNA synthesis by novel 3'-modifieddeoxyribonucleoside 5'-triphosphates. Nucleic Acids Research , 22 (20) , 4259-4267. https://doi.org/10.1093/nar/22.20.4259

Identifiers

DOI
10.1093/nar/22.20.4259
PMID
7937154
PMCID
PMC331938

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Data completeness: 86%