Biology Reference
In-Depth Information
Table 8.2: (Continued)
Table 8.2: Optimizing Standard PCR Protocols Involves Optimizing Reaction Components.
PCR component
Issues to consider
Annealing
1.
Annealing temperature depends on length and GC content of primers; 55 °C is
good for primers 20 nt long (50% GC).
2.
Higher annealing temperatures may be needed to increase primer specificity.
Cycle number
1.
Optimum number varies with starting concentration of template DNA, and all of
the above-mentioned parameters.
2.
Too many cycles increases amount of nonspecific product, whereas too few results
in a low yield that can't be detected by gel electrophoresis.
3.
If additional product is required, it is better to reamplify, using an aliquot of the
first reaction as the template, than to increase the number of cycles.
Many of the modifications of the PCR have involved modifying the number, size,
and specificity of the primers used, as described in Section 8.4.
What is a primer and where do you get them? A primer is a short (10 to
≈
30
nucleotides) single-stranded polymer of oligonucleotides. The standard (allele-
specific) PCR requires that the specific sequence of the DNA targeted for ampli-
fication be known in order to synthesize a pair of primers. Thus, information
is required about the gene/DNA to be amplified. Primers anneal to the target
DNA by complementary base pairing, with A annealing to T, and C to G. The
primers determine the length, specificity, and nature of the DNA fragment
amplified.
Allele-specific
(
standard
)
PCR
requires a pair of primers to flank the target
DNA to be amplified; extension (copying of the single strand of template DNA)
occurs from each 3
′
-OH end of the primer, so that the 5
′
ends of the primers
define the ends of the amplified DNA. As a result, the length of the DNA gener-
ated is equal to the lengths of the two primers plus the length of the template
DNA (
Figure 8.1
).
Most primers are synthesized to order on a DNA synthesizer. Many commer-
cial suppliers provide this service, with the price determined by the number of
bases in the primers. Primers may be called 10-, 20- or 30-mers, based on their
length. Primers can be constructed that contain extensions so that restriction
enzyme sites, regulatory codons, or labels can be added to the target DNA.
These sequences will be incorporated into the 5
′
end of the target sequence,
making the products more easily cloned or sequenced.
