Biology Reference
In-Depth Information
The results indicated that one of the populations was resistant to permethrin
through a different biochemical method, indicating that both PCR and bioas-
say data should be obtained for monitoring resistance-allele frequencies and the
mechanism(s) of insecticide resistance.
8.5.10 Developmental Biology
It is possible to detect the presence of specific mRNAs in tissues or cells by
reverse transcription and DNA amplification by the PCR.
8.5.11 Engineering DNA
DNA can be engineered in several ways by the PCR. Sequences can be added to
the 5
′
end of primers. Such sequence changes are readily accepted, even though
these add-ons do not base-pair with the template DNA. The DNA being synthe-
sized contains the add-on because the primers are incorporated in the synthe-
sized DNA fragment. For example, it is possible to add a restriction site sequence
to DNA being amplified by the PCR by attaching the restriction site sequence
to the primers (
Figure 8.7
). Such restriction sites facilitate subsequent manipula-
tions of the final PCR product.
The T7 promoter located at the 5
′
end of one primer can be added to PCR
products. This promoter allows RNA copies to be generated from the DNA syn-
thesized in the PCR reaction. Although the add-on sequences in the primers
don't base-pair to the template DNA, in most cases they have little effect on the
specificity or efficiency of the amplification. The 3
′
end of the primer apparently
is most important in imparting specificity.
One PCR product strand or both can be tagged with a radioactive, biotin,
or fluorescent label (
Chehab and Kan 1989, Mertz and Rashtchian 1994
). DNA
Figure 8.7
A 5
′
add-on of a restriction site sequence (
Eco
RI) to a primer, which is annealed to a tar-
get DNA sequence. Although this add-on does not specifically match the template DNA, this does
not significantly affect the PCR. The extra bases that are added 5
′
to the
Eco
RI site ensure that the
efficiency of the restriction enzyme cleavage is maintained.
