Hybridization probe pairs and single-labeled probes: an alternative approach for genotyping and quantification.
Real-time polymerase chain reaction (PCR) has become a standard tool in both quantitative gene expression and genetic variation analysis. Data collection is performed throughout the PCR process, thus combining amplification and detection into a single step. This can be achieved by combining a variety of different fluorescent chemistries that correlate the concentration of an amplified PCR product to changes in fluorescence intensity. Hybridization probe pairs and single-labeled probes are sequence-specific, dye-labeled oligonucleotides, used in real-time PCR approaches, in particular for genotyping of single nucleotide polymorphisms (SNPs). In that case, a detector probe is designed to cover the polymorphism. Allelic variants are identified and differentiated via post-PCR melting curve analysis. A single melting curve can distinguish different T (m)s, and differently labeled probes may be used, theoretically allowing multiplexed genotyping of several SNPs.
Froehlich
Thomas
T
Roche Diagnostics GmbH, Roche Applied Science, Penzberg, Germany.
Geulen
Oliver
O
D004247:DNA / Q000032:analysis*; D005456:Fluorescent Dyes; D005838:Genotype; D020180:Heteroduplex Analysis; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*
D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D004247:DNA