Abstract
We have synthesized 3'-substituted-2'-deoxyribonucleotide-5'-triphosphates corresponding to A, T, G and C. The 3' position was esterified by a separate anthranylic derivative (3'-tag) giving specific fluorescent properties to each nucleotide (nt). These nt acted as substrates with several DNA polymerases leading to chain termination. Upon alkali or enzymatic treatment of the terminated DNA chain, free 3'-hydroxyl groups were recovered and found able to undergo chain extension when incubated with a mixture of dNTPs and a DNA polymerase. Because each tag has different fluorescent properties in itself, i.e., as a free acid, it theoretically is possible, after removal and characterization of the tag, to infer which nt has been inserted. Reiteration of the process can then be used to determine a nt sequence with a non-gel-based method amenable to automation.
Keywords
BiologyPolymeraseDeoxyribonucleotideDNADNA polymeraseFluorescenceBiochemistryNucleotideMolecular biologyPrimer (cosmetics)Polymerase chain reactionOligonucleotideCombinatorial chemistryGeneChemistry
MeSH Terms
Adenosine TriphosphateBase SequenceCytidine TriphosphateDNA-Directed DNA PolymeraseDeoxyribonucleotidesFluorescent DyesGuanosine TriphosphateMolecular Sequence DataRNA-Directed DNA PolymeraseSequence AnalysisDNAThymine Nucleotidesortho-Aminobenzoates
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Publication Info
- Year
- 1994
- Type
- article
- Volume
- 148
- Issue
- 1
- Pages
- 1-6
- Citations
- 90
- Access
- Closed
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Cite This
Bruno Canard,
Robert S. Sarfati
(1994).
DNA polymerase fluorescent substrates with reversible 3′-tags.
Gene
, 148
(1)
, 1-6.
https://doi.org/10.1016/0378-1119(94)90226-7
Identifiers
- DOI
- 10.1016/0378-1119(94)90226-7
- PMID
- 7523248