Applied Biosystems 7900HT Fast Real-Time PCR System and SDS ...

Design Tips for Allelic Discrimination AssaysDesign Tips for Allelic Discrimination AssaysDiscrimination byMultiple ProbesTaqMan ProbeDesignGuidelinesBy using different reporter dyes, cleavage of multiple probes can be detected in asingle PCR. One application of this multi-probe capability is to use allele-specificprobes to distinguish genetic polymorphisms (Bloch, 1991, Lee et al., 1993). Probesthat differ by as little as a single nucleotide will exhibit allele-specific cleavage. Thisis true even for probes with a reporter on the 5' end and the non-fluorescent quencheron the 3´ end (Bloch, 1991).IMPORTANT! When designing probes, it is important to consider probes from bothstrands.Follow the guidelines in the table below for designing TaqMan MGB probes:Table B-2Priority1Guidelines for Designing TaqMan MGB Probes for Allelic DiscriminationGuidelineAvoid probes with a guanine residue at the 5´ end of the probe.A guanine residue adjacent to the reporter dye will quench the reporterfluorescence, even after cleavage.2 Select probes with a Primer Express software–estimated T m of 65–67 °C.34Make the TaqMan MGB probes as short as possible, but no fewer than13 nucleotides in length.Avoid runs of an identical nucleotide.This is especially true for guanine, where runs of four or more should beavoided.Position the polymorphic site in the central third of the probe.Note: The polymorphic site can be shifted toward the 3´ end to meet theabove guidelines, however, the site must be located more than twonucleotides upstream from the 3´ terminus.The following figure illustrates the placement of a polymorphism in an exampleprobe (N = Nucleotide).5First, try to position the polymorphicsite in the central third of the probe.Do not placeit here.5´ 3´N N N N N N N N N N N N N N N N N N N N NPolymorphismIf necessary, place thepolymorphism here.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide B-5DRAFTSeptember 1, 2004 11:39 am, App_P&PDesign.fm