Biomedical Engineering Reference
In-Depth Information
routinely target primers and probes to intron/exon boundries. The approach we
routinely use for primer and probe design is described.
1. Design primer and probe sequences using Primer Express software (Applied
Biosystems) as close as possible to the 3
coding region of target gene sequences
obtained from Genbank or other sources.
2. Whenever possible, follow the specifi c primer and probe design criteria suggested
by the manufacturers of TaqMan reagents (Applied Biosystems).
3. Pick sequences with 30-80% GC content and avoid runs of more than three
consecutive Gs in either primers or probes. Pick a
T
m
of 59-61°C for primers
and 66-68°C for probes.
4. Choose probe from the strand with more Cs than Gs, but avoid any probes with
G at the 5
end, as this can exert a quenching effect on the reporter dye
(13)
.
5. Position forward and reverse primers as close as possible to each other without
overlapping the probe, and aim to have fewer than three Cs and Gs in the fi ve
most 3
nucleotides of these primers.
6. Primers and probes were purchased from Applied Biosystems and each probe
was synthesized with the fl uorescent reporter dye FAM (6-carboxyfl uorescein)
attached to the 5
end and a quencher dye TAMRA (6-carboxytetramethylrhoda-
mine) to the 3
end.
3.3.2. Primer/Probe Quality Control
1. Check gene-specifi c primer pairs for effi cient amplifi cation using genomic DNA
in standard TaqMan PCR assays (described in
Subheading 3.4.
) but replacing
the probe with water. If testing primers and probes for splice variants, plasmid
or cDNA templates may be required.
2. Run products on 2% agarose gels to confi rm correct size of product amplifi ed.
3. Once TaqMan probes are obtained, check these in standard TaqMan reactions
using genomic DNA as template. We found this standard genomic DNA test
a valuable way of assessing the relative sensitivity and efficiency of each
primer/probe set from the threshold cycle (
C
t
) values and standard curve slopes,
respectively (
7
;
see also
data analysis in
Subheading 3.5.
). We consistently
found that the
C
t
values for hundreds of primer/probe sets against a standard
concentration of genomic DNA were very similar, refl ecting the tight criteria
used for primer/probe design (
Table 1
).
3.4. TaqMan PCR Assay
In recent years, a number of manufacturers have started to produce specifi c
all inclusive buffers for TaqMan assays. We have primarily used the 2X TaqMan
Universal PCR Mastermix from Applied Biosystems for our studies which
are now described.
1. Whenever possible, prepare duplicate or triplicate TaqMan PCR samples in
96-well optical plates.
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