A homogeneous fluorescence assay for PCR amplicons: its application to real-time, single-tube genotyping

We have developed a method whereby a single TaqMan probe can be used for many PCR reactions. We demonstrate its application as an integrated system for the direct measurement of allele-specific amplicon generation coupled to the suppression of primer-dimer accumulation in PCR. The system uses a 5�...

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Bibliographic Details
Published in:Clinical chemistry (Baltimore, Md.) Md.), 1998-05, Vol.44 (5), p.918-923
Main Authors: Whitcombe, David, Brownie, Jannine, Gillard, Helen L, McKechnie, Doug, Theaker, Jane, Newton, Clive R, Little, Stephen
Format: Article
Language:English
Subjects:
Alleles
Biological and medical sciences
Biotechnology
BRCA2 Protein
Breast Neoplasms - blood
Breast Neoplasms - genetics
Cystic Fibrosis - blood
Cystic Fibrosis - genetics
DNA - genetics
DNA, Neoplasm - genetics
Factor V - genetics
Female
Fluoresceins
Fluorescent Dyes
Fundamental and applied biological sciences. Psychology
Genetic engineering
Genetic technics
Genotype
Humans
In vitro gene amplification. Pcr technique
Methods. Procedures. Technologies
Neoplasm Proteins - blood
Neoplasm Proteins - genetics
Oligonucleotide Probes
Polymerase Chain Reaction - methods
Polymorphism, Genetic
Sequence Analysis, DNA - methods
Transcription Factors - blood
Transcription Factors - genetics
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Summary:We have developed a method whereby a single TaqMan probe can be used for many PCR reactions. We demonstrate its application as an integrated system for the direct measurement of allele-specific amplicon generation coupled to the suppression of primer-dimer accumulation in PCR. The system uses a 5'-exonuclease assay of amplicon annealed fluorogenic probes that operates in conjunction with the Amplification Refractory Mutation System, whereby relative changes in reporter fluorescent emission are monitored in real-time using an analytical thermal cycler. We have called this system Three-STAR, and it is universal in that it can either use a single probe for the detection of any one target DNA sequence or a single pair of probes for genotyping any bi-allelic polymorphism. Three-STAR is, therefore, particularly useful for the single-tube genotype analysis of a variety of human DNA polymorphisms and mutations.
ISSN:0009-9147
1530-8561
DOI:10.1093/clinchem/44.5.918