Seven-color, homogeneous detection of six PCR products.

We describe an extension of the fluorogenic PCR 5'-nuclease assay, or "Taq-Man" assay. Sequence-specific probes consisted of a novel nonfluorescent quencher, nitrothiazole blue (NTB), at the 3' terminus 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 phthalocyanine tetrasulfonate and was utilized as a "passive reference" to calibrate concentration variations. Our test system was a set of three single-nucleotide polymorphisms (SNPs). Each SNP system consisted of two primers and two sequence-specific probes, each labeled with a different reporter dye and NTB. Following PCR, the reactions were diluted with water and measured in a microcuvette on a luminescence spectrometer in synchronous scanning mode. In this method, both the excitation and emission wavelengths were scanned, with a fixed wavelength difference (delta gamma) between excitation and emission wavelengths. The spectral overlap in the set was evaluated by calculation of the condition number of the 7 x 7 matrix (dye fluorescence vs. wavelength). The small value of the condition number (1.5) proved that the cross-talk between the dyes was minimal. SNP analyses of known, synthetic target sequences and genomic DNA were plotted both as normalized, subtracted spectra and as data points in three separate dot plots.

Optimization of the performance of the polymerase chain reaction in silicon-based microstructures.

We have demonstrated the ability to perform real-time homogeneous, sequence specific detection of PCR products in silicon microstructures. Optimal design/ processing result in equivalent performance (yield and specificity) for high surface-to-volume silicon structures as compared to larger volume reactions in polypropylene tubes. Amplifications in volumes as small as 0.5 microl and thermal cycling times reduced as much as 5-fold from that of conventional systems have been demonstrated for the microstructures.