Biology Reference
In-Depth Information
for most amino acids). Methionine and tryptophan are encoded by a single
codon, but the other amino acids are encoded by two to six different codons.
When designing degenerate primers, it is useful to choose a segment of the pro-
tein in which the amino acids have minimal degeneracy. The lower the degen-
eracy in the primers, the higher the specificity of the PCR. The degeneracy of the
primer may be restricted further by considering which codons are most often
used in a particular species (codon bias), if it is known. Furthermore, degener-
acy may be reduced if primers containing fewer (15-20) nucleotides are used.
Because a single mismatch, especially at the 3
′
end of the primer, may prevent
Taq
from extending, degeneracy at the 3
′
end should be avoided. Empirical test-
ing of primers may be necessary and modifications made to ensure that the
desired product is synthesized.
8.4.6 Hot-Start PCR
A hot-start PCR protocol can optimize the yield of the desired product while
limiting the likelihood of nonspecific amplification. Hot-start PCR is achieved by
leaving an essential component out of the reaction mixture until the mixture
has been heated to a temperature that inhibits nonspecific priming and exten-
sion. Typically, all PCR components are added and held at high temperature
before the DNA polymerase is added.
A modification of this method involves using wax to provide a physical barrier
between the components of the reaction. The primers, Mg
2
+
, dNTPs and buffer
can be mixed at room temperature in the bottom of the reaction tube and then
covered with melted wax that melts at low temperature (53-55 °C). The remain-
ing components are then added on top of the wax barrier. During the first cycle
of the PCR the wax barrier melts during the denaturation step, allowing the
components to combine. The melted wax floats to the top of the mixture where
it acts as a barrier to evaporation. Hot-start
Taq
DNA polymerase is now avail-
able in which the enzyme is activated only after the reaction reaches 94 °C or
higher, allowing all components to be mixed at room temperature.
Hot-start PCR especially is useful when nonspecific amplification is a problem
because there is too little template DNA, the template DNA is complex, or sev-
eral pairs of primers are used (multiplex PCR) (
Sambrook and Russell 2001
).
8.4.7 Inverse PCR
An unknown sequence that flanks a “core” region with a known sequence can
be amplified by
inverse PCR
(
Ochman et al. 1990, Sambrook and Russell 2001
).
Inverse PCR involves digesting the template DNA with a restriction endonuclease
