Seven-Color, Homogeneous Detection of Six PCR (original) (raw)

1999

Abstract

We describe an extension of the fluoro genic PCR 5 ′ -nuclease assay, or “Taq Man ™ ” assay. Sequence-specific probe s consisted of a novel nonfluorescent quench er, nitrothiazole blue (NTB), at the 3 ′ term i nus and six different reporter dyes at the 5 ′ terminus. The six reporters were 6-FAM , dR110, dR6G, dTMR, dROX and JAZ dyes . The seventh color was from aluminum ph thalocyanine tetrasulfonate and was utilized as a “passive reference” to calibrate concen tration variations. Our test system was a se t of three single-nucleotide polymorphism s (SNPs). Each SNP system consisted of two primers and two sequence-specific probes , each labeled with a different reporter dy e and NTB. Following PCR, the reactions wer e diluted with water and measured in a m i crocuvette on a luminescence spectromete r in synchronous scanning mode. In thi s method, both the excitation and emission wavelengths were scanned, with a fixed wavelength difference (Δ λ ) between excita tion and emission wavel...

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