Design Tips For Allelic Discrimination Assays - Applied Biosystems 7900HT User Manual

Design Tips for Allelic Discrimination Assays

Discrimination by
Multiple Probes
TaqMan Probe
Design
Guidelines
Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide
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).
When designing probes, it is important to consider probes from both
IMPORTANT!
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
Avoid probes with a guanine residue at the 5´ end of the probe.
1
A guanine residue adjacent to the reporter dye will quench the reporter
fluorescence, even after cleavage.
Select probes with a Primer Express software–estimated T
2
Make the TaqMan MGB probes as short as possible, but no fewer than
3
13 nucleotides in length.
Avoid runs of an identical nucleotide.
4
This is especially true for guanine, where runs of four or more should be
avoided.
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
5´
N N N N N N N N N N N N N N N N N N N N N
Design Tips for Allelic Discrimination Assays
First, try to position the polymorphic
site in the central third of the probe.
Polymorphism
DRAFT
September 1, 2004 11:39 am, App_P&PDesign.fm
of 65–67 °C.
m
Do not place
it here.
If necessary, place the
polymorphism here.
3´
B-5