Biology Reference
In-Depth Information
Table 8.7: Some Entomological Problems and Potential PCR Protocols.
Problem
PCR technique(s)
Amplify ancient DNA
Standard allele-specific PCR
Amplify mRNA
Reverse transcriptase PCR
Chromosome walking
Inverse PCR; Long PCR
Cloning a gene
Blunt-end cloning; sticky-ended cloning; anchored
PCR; PCR with degenerate primers; Long PCR
Constructing a genetic map
AP-PCR; RAPD; inverse PCR
Constructing a phylogeny
Standard PCR with primers having polylinkers
for cloning/sequencing; asymmetric PCR and
sequencing; PCR-RFLP; multiplex PCR
Detecting gene expression
RNA PCR; TaqMan PCR
Detecting mutations
Standard PCR; RAPD; AP-PCR; PCR-RFLP
Detecting pathogens in arthropod vectors
Standard PCR; Long PCR
Detecting transgenic arthropods
Standard PCR
Engineering DNA
Introduce restriction sites into DNA fragments
Attach sequences to 5
′
end of primers and conduct
standard PCR probes, or isolating DNA strands on
a column
Label DNA with
32
P or biotin for sequencing
Assemble overlapping DNA segments to make
synthetic DNA
Alter primer sequence when synthesizing, then
standard PCR
Introduce substitutions, deletions, or insertions in
product DNA
Evolutionary analyses
Standard PCR; RAPD; AP-PCR; DNA sequencing;
PCR-RFLP
Identify species
Standard PCR; RAPD; AP-PCR; PCR-RFLP
Identify strains, races, or biotypes
Standard PCR; RAPD; AP-PCR; PCR-RFLP
Identifying upstream/downstream sequences
Inverse PCR; single-specific primer PCR (SSP-PCR)
Monitoring dispersal of individuals
Standard PCR; RAPD; AP-PCR
Sequencing a gene
Asymmetric PCR to produce ss DNA;
dideoxynucleotide chain-termination sequencing
method with
Taq
polymerase; cycle sequencing;
direct sequencing
There are three steps in AFLP-PCR: 1) the DNA is digested by restriction
enzymes and oligo “adapters” are ligated to the digested DNA; 2) sets of restric-
tion fragments are selectively amplified using the adapter and restriction site
sequences as target sites for primer annealing; and 3) the amplified fragments
are analyzed by electrophoresis.
The number of fragments produced in a single AFLP-PCR can be determined by
selecting specific primer sets. Annealing conditions can be stringent in AFLP. AFLP-
PCR allows analysis of closely related populations and species. For example, popu-
lations of an introduced pest in Europe, the grape phylloxera, were compared by