PCR protocols for unlabeled probe genotyping

Similar to scanning and amplicon genotyping, any recipe for PCR can be used with the simple addition of LCGreen™ dyes at 1X concentration. We routinely use 50 mM Tris, pH 8.3, 500 yg/ml BSA, 200 yM each dNTP, 1-5 mM MgCl2, 0.4 U heat-stable polymerase, 88 ng of TaqStart™ antibody

(ClonTech), 0.5 ^M of the excess primer, 0.05-0.1 uM of the limiting primer, 0.5 uM of the unlabeled probe and 50 ng of human genomic DNA in 10 ^l reactions. Make sure that the unlabeled probe is on the same strand as the limiting primer. Polymerase alternatives include regular exonuclease-positive Taq (for a wide range of probe Tm values) and exonuclease-negative KlenTaql™ (for probe Tm values below the PCR extension temperature). We have also added dye to the LightCycler® FastStart DNA Master HybProbe kit with good results. Hot-start procedures are not as important as in scanning applications, especially if the probes are designed so that alleles melt below potential primer dimers. Asymmetric amplification is usually performed for 45-60 cycles, so rapid cycling techniques are convenient. A final denaturation and cooling step are not required.

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