Biology Reference
In-Depth Information
of target DNA simultaneously, which should reduce time, minimize costs and
increase efficiency (
Figure 8.5
). In reality, the yield of each product usually is
reduced in proportion to the number of primer pairs included in the reaction.
Up to eight primer pairs have been used simultaneously before the yield of each
product is too low to be visualized by staining with ethidium bromide on an
agarose gel.
Despite the potential benefits, multiplex PCR can be difficult to develop
because all the primers must have approximately the same melting temperature,
the primers should not interact with one another to produce primer-dimers, and
the amplified products should be of approximately the same size, but still be dis-
tinguished from each other by gel electrophoresis.
Steps to develop multiplex PCR include the following: 1) Determine that all
target DNA can be amplified efficiently using the same PCR temperature pro-
file. 2) Titrate the amount of each primer pair to achieve maximum amplifica-
tion in separate reactions using the same program and reaction conditions.
3) Balance the amount of each primer pair to achieve acceptable levels of
amplification of all targets in the multiplex reaction. One solution to prob-
lems with step 3 usually involves increasing progressively the concentration of
Multiplex PCR
DNA
(bp)
310
Fly 1
S R
Fly 2
S R
Fly 3
S R
kdr
234
194
E7
GAPDH
118
Super-kdr
Figure 8.5
Multiplex PCR allows more than one gene to be sampled in a single reaction. In this
example, three pesticide resistance genes (
kdr
, E7, and
Super-kdr
) were monitored in three hornflies,
Haematobia irritans
. Fly 1 has only susceptible alleles for
kdr
,
Super-kdr
, and E7. Fly 2 has one copy of
the susceptible and one of the resistant alleles of
kdr
, but it has susceptible alleles only for the E7
and
Super-kdr
genes. Fly 3 has only
kdr
alleles (is homozygous resistant), susceptible alleles of E7,
and is heterozygous for
Super-kdr
. Amplification of the GABDH gene provides a control to demon-
strate that the PCR is working correctly. (Photo kindly provided by Felix Guerrero.)
