Primer and Probe Design for Quantitative Assays:
Primer and probe design software from ABI
(ABI PRISM Primer Express) is available at both computer stations in
the DNA Sequencing and the Core Genomics Facilities. When designing only
PCR primer pairs, other general primer design programs could be used
such as: Primer Quest
available free of charge at: http://biotools.idtdna.com/primerquest/
and Primer Select by DNAstar (available
at Core Genomics Facility computer station).
Please refer to the following ABI recommended guidelines when designing
primers / probes for Quantitative assays:
· select the probe first
and design the primers as close as possible to the probe without overlapping
it. Amplicons of 50 to 150 bp are strongly recommended. If absolutely
necessary product size could be increased up to 200-250bp at most
· keep primer/probe GC
content within 30-80%
· avoid runs of identical
nucleotides, this is especially true for guanine, where runs of four
or more Gs should be avoided
When designing primers:
· Tm should be within
58°C to 60°C
· the last 5 bases at the 3 prime end
should have no more than two G's or C's
· keep the annealing
Ts of the primers as close as possible
· select primer pairs
with minimal number of potential primer dimers
and primer hairpins as possible
When designing probes:
· Tm should be within
68°C to 70°C
· no Gs on the 5’
end
· select the strand that
gives the probe more C than G bases
· make the TaqMan MGB
probes as short as possible, without being shorter than 13 nucleotides
Before to proceeding with real time PCR, it is necessary to test the
primers on a PCR reaction to ensure that the primers amplify the gene
of interest at the right size and that the primers are specific (i.e.
no other bands present on the gel except that of the expected size).
In doing this, it is important to run your test PCR under conditions
as close as possible to those for real time PCR (refer to the table
below). Also, the best results are achieved with magnesium concentration
at 3mM and with 0.025 U/ul of Taq.
Table 4: Typical Real Time PCR stage Parameters
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