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'-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.
Whitcombe
D
D
Zeneca Diagnostics, Gadbrook Park, Northwich, Cheshire, UK. david.whitcombe@ukbla71.zeneca.com
Brownie
J
J
Gillard
H L
HL
McKechnie
D
D
Theaker
J
J
Newton
C R
CR
Little
S
S
D000483:Alleles; D024682:BRCA2 Protein; D001943:Breast Neoplasms / Q000097:blood / Q000235:genetics; D003550:Cystic Fibrosis / Q000097:blood / Q000235:genetics; D004247:DNA / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics; D005165:Factor V / Q000235:genetics; D005260:Female; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005838:Genotype; D006801:Humans; D009363:Neoplasm Proteins / Q000097:blood / Q000235:genetics; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D017422:Sequence Analysis, DNA / Q000379:methods; D014157:Transcription Factors / Q000097:blood / Q000235:genetics
D024682:BRCA2 Protein; D004273:DNA, Neoplasm; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009363:Neoplasm Proteins; D015345:Oligonucleotide Probes; D014157:Transcription Factors; C095381:factor V Leiden; C024098:6-carboxyfluorescein; D005165:Factor V; D004247:DNA