Biology Reference
In-Depth Information
Sometimes variability in PCR assays is due to bad batches of
Taq
DNA polymerase,
but a functional assay can be carried out to test its performance (
Wada et al. 1994
).
False-negative results
can occur for no apparent cause. These may be due to
“interferences between our target DNA and the rest of the genome” (
Baldrich
et al. 1999
). A solution can be to first digest the genomic DNA with a restric-
tion enzyme that cuts outside the target region, followed by electrophoresis of
the digested DNA, followed by recovering the restriction fragments of approxi-
mately the desired size by elution from the agarose gel. These fragments are
then used as the template.
8.3.11 Detecting Primer Artifacts
Artifactual products consisting of low molecular-weight DNA products may be
produced and are most obvious if the PCR is carried out with high primer con-
centrations, too much
Taq
in early cycles, small amounts of template DNA, or
too many cycles. The artifacts may be “primer-dimers” or other artifacts derived
from the primers. Methods have been developed to eliminate primer-dimer
accumulation (
Brownie et al. 1997
).
Primer-dimers
occur when the enzyme makes a product by reading from the 3
′
end of one primer across to the 5
′
end of the other. Because each primer serves
as both primer and template, a sequence complementary to each primer is pro-
duced, which upon denaturation is a perfect template for further primer binding
and extension. As the number of cycles is increased over 30, the probability of
mispriming increases, as does the amount of artifact formed. The accumulation
of a large amount of primer-dimers depletes primers and dNTPs from the reac-
tion mixture and competes for enzyme with the desired target DNA.
If a PCR produces inadequate amounts of product, conducting a second
amplification is a better solution to the problem than increasing the number of
cycles of a single PCR. The second reaction is best done using 1
μ
l of the first
reaction as template and a fresh reaction mixture.
8.3.12 How Many Cycles Does a PCR Need?
The answer is not too many and not too few. The optimum varies with the
starting concentration of the template, the quality of the template, the
amount of inhibitory substances in the reaction, as well as all the other param-
eters (
Table 8.2
).
Too many cycles can increase the amount of nonspecific background products.
Too few cycles will give a low yield that can't be detected upon electrophore-
sis and staining with ethidium bromide. Too much template DNA actually can
