Abstract
The 5'----3' exonuclease activity of the thermostable enzyme Thermus aquaticus DNA polymerase may be employed in a polymerase chain reaction product detection system to generate a specific detectable signal concomitantly with amplification. An oligonucleotide probe, nonextendable at the 3' end, labeled at the 5' end, and designed to hybridize within the target sequence, is introduced into the polymerase chain reaction assay. Annealing of probe to one of the polymerase chain reaction product strands during the course of amplification generates a substrate suitable for exonuclease activity. During amplification, the 5'----3' exonuclease activity of T. aquaticus DNA polymerase degrades the probe into smaller fragments that can be differentiated from undegraded probe. The assay is sensitive and specific and is a significant improvement over more cumbersome detection methods.
Keywords
Thermus aquaticusExonucleaseHot start PCRKlenow fragmentPolymerasePolymerase chain reactionMolecular biologyTaq polymeraseDNA polymeraseInverse polymerase chain reactionOligonucleotideBiologyPrimer dimerPolymerase chain reaction optimizationMolecular probeHybridization probeExonuclease IIIDNAMultiple displacement amplificationDNA polymerase IBiochemistryNested polymerase chain reactionMultiplex polymerase chain reactionReverse transcriptaseGeneDNA extractionEscherichia coli
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Publication Info
- Year
- 1991
- Type
- article
- Volume
- 88
- Issue
- 16
- Pages
- 7276-7280
- Citations
- 2697
- Access
- Closed
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Cite This
Pamela M. Holland,
Richard D. Abramson,
R. R. Watson
et al.
(1991).
Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase..
Proceedings of the National Academy of Sciences
, 88
(16)
, 7276-7280.
https://doi.org/10.1073/pnas.88.16.7276
Identifiers
- DOI
- 10.1073/pnas.88.16.7276