Biology Reference
In-Depth Information
PCR-RFLP makes RFLP analysis suitable for studying individual specimens of very
small species, requires no labeled probes, and is faster and less expensive than
standard RFLP analysis. If consensus primers are available, then cloning is not
required. For example,
Simon et al. (1993)
analyzed mtDNA in 13- and 17-year
periodical cicadas using standard primers for the COII-A6-A8-COIII segment
(
Simon et al. 1991
). A disadvantage to PCR-RFLP is that the method requires two
procedures: allele-specific PCR followed by restriction digest.
13.5.10 RAPD-PCR
Hadrys et al. (1992)
noted that RAPD-PCR is a versatile method for molecular
ecology because it can be used to determine biotypes or species identity, assess
kinship, and analyze paternity. It can estimate genetic variation within popula-
tions and can be used to monitor colonization. RAPD-PCR is suitable for studying
insects for which very little genetic information is available, requires very small
amounts of DNA, and can be used with very small insects. It is rapid and rela-
tively inexpensive when compared to RFLP analysis, DNA sequencing, PCR-RFLP,
DSCP, microsatellite, or microarray analysis (
Table 13.1
, see Chapter 8).
Because RAPD-PCR uses short primers of arbitrary sequence (10-mers), it does
not require the investigator to know the sequences of specific genes in order to
develop primers for the PCR.
Haymer (1994)
evaluated the sequences of various
RAPD primers used on insects and listed 55 that had been particularly informa-
tive. RAPD-PCR primers sample both single-copy and repetitive DNA. Although
the repeatability and reliability of RAPD-PCR can be problematic
if care is not
exercised
, RAPD-PCR can provide inexpensive, repeatable and useful data
(
Penner et al. 1993, Edwards and Hoy 1993, MacPherson et al. 1993
).
RAPD-PCR was used to analyze the amount of genetic variation in the para-
sitoids
Trioxys pallidus
and
Diglyphus begini
(
Figure 13.2
). DNA from individual
T. pallidus
males was amplified using 120 different primers (
Edwards and Hoy
1993
). Of the 120 primers, 92 produced a total of 342 scorable bands, of which
118 exhibited presence/absence polymorphisms.
Diglyphus begini
was evalu-
ated with 25 primers, and 17 produced a total of 51 scorable bands in haploid
males. The level of genetic variation detected was greater than any found in
Hymenoptera by using allozymes (
Menken 1991, Packer and Owen 1992
) and
comparable to the variation detectable with RFLPs. The bands considered reli-
able were inherited as dominant Mendelian traits in diploid females. Because
only small amounts of DNA are needed for each RAPD reaction, multiple reac-
tions could be conducted using DNA from a single individual (
Edwards and
Hoy 1993, 1995
). RAPD-PCR can be used to analyze population structure and
gene flow and to monitor establishment and dispersal of particular biotypes
