Applied Biosystems 7900HT - Design Tips for Allelic Discrimination Assays

DRAFT
September 1, 2004 11:39 am, App_P&PDesign.fm
Design Tips for Allelic Discrimination Assays
Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide B-5
Design Tips for Allelic Discrimination Assays
Discrimination by
Multiple Probes
By using different reporter dyes, cleavage of multiple probes can be detected in a
single PCR. One application of this multi-probe capability is to use allele-specific
probes to distinguish genetic polymorphisms (Bloch, 1991, Lee et al., 1993). Probes
that differ by as little as a single nucleotide will exhibit allele-specific cleavage. This
is true even for probes with a reporter on the 5' end and the non-fluorescent quencher
on the 3´ end (Bloch, 1991).
TaqMan Probe
Design
Guidelines
IMPORTANT! When designing probes, it is important to consider probes from both
strands.
Follow the guidelines in the table below for designing TaqMan MGB probes:
Table B-2 Guidelines for Designing TaqMan MGB Probes for Allelic Discrimination
Priority Guideline
1
Avoid probes with a guanine residue at the 5´ end of the probe.
A guanine residue adjacent to the reporter dye will quench the reporter
fluorescence, even after cleavage.
2
Select probes with a Primer Express software–estimated T
m
of 65–67 °C.
3
Make the TaqMan MGB probes as short as possible, but no fewer than
13 nucleotides in length.
4
Avoid runs of an identical nucleotide.
This is especially true for guanine, where runs of four or more should be
avoided.
5
Position the polymorphic site in the central third of the probe.
Note: The polymorphic site can be shifted toward the 3´ end to meet the
above guidelines, however, the site must be located more than two
nucleotides upstream from the 3´ terminus.
The following figure illustrates the placement of a polymorphism in an example
probe (N = Nucleotide).
5´ 3´
N NNNNNNNNN
NNNNNNNNNNN
Polymorphism
First, try to position the polymorphic
site in the central third of the probe.
If necessary, place the
polymorphism here.
Do not place
it here.