ABI Prism® 7900HT Sequence Detection System ... - OpenWetWare

Design Probes and
Primers
The following sections contain general guidelines for designing primers and probes.
For specific design tips, refer to the appropriate section: for Allelic Discrimination see
page C-5 and for Quantitative PCR see page C-6.
Design Probe(s) for the Assay
Adhere to the following guidelines when designing TaqMan probes:
♦ Keep the G-C content in the range of 30–80%.
♦ Avoid runs of an identical nucleotide (especially guanine, where runs of four or
more Gs should be avoided).
♦ No G on 5´ end.
♦ Keep the melting temperature (Tm ) in the range of 68-70 °C for quantitative PCR
and 65-67 °C for allelic discrimination (using the Primer Express software).
♦ Select the strand that gives the probe with more Cs than Gs.
♦ For allelic discrimination (see page C-5):
– Adjust probe length so that both probes have the same Tm .
– Position the polymorphism site approximately in the center of each probe.
♦ For multiplex PCR applications (involving multiple probes), design the probes with
different fluorescent reporter dyes as explained below:
Reporter Dye
Application
a
First Probe Second Probe
Allelic Discrimination FAM VIC
a. The use of the FAM and VIC reporter dyes for multiplex applications provides the greatest degree of
spectral separation.
Design Primers for the Assay
Adhere to the following guidelines when designing primers for 5´-nuclease assays:
♦ Keep the G-C content in the range of 30–80%.
♦ Avoid runs of an identical nucleotide (especially guanine, where runs of four or
more bases should be avoided).
♦ Keep the Tm in the range of 58-60 °C (using the Primer Express software).
♦ Limit the G and/or C bases on the 3´ end. The five nucleotides at the 3´ end
shouldhavenomorethantwoGand/orCbases.
♦ Place the forward and reverse primers as close as possible to the probe without
overlapping the it.
♦ Use an annealing temperature of 60 °C for quantitative PCR, and 62 °C for allelic
discrimination (except for TaqMan® PDARs for Allelic Discrimination).
Designing TaqMan Assays C-3