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efforts were expended or whether the disparity represents true differences in
ease of isolation.
A variety of methods have been developed for isolating microsatellites (Tables
13.1, 13.3). Zane et  al. (2002) reviewed the methods and provide a “fast and
easy protocol that is a combination of different published methods.” Their
goal was to “provide a well established universal protocol” but they recog-
nized that “completely new approaches [may] become available due to a better
knowledge of microsatellite evolution combined with new technical advances.”
Techen et  al. (2010) improved on methods to isolate microsatellite sequences,
and an outline of their methods is presented in Table 13.3 . At present, a careful
evaluation of the experimental strategy has to be carried out for each experi-
ment ( Zane et  al. 2002 ). Goodman (1997) , Cornuet et  al. (1999) , Luikart and
England (1999) , Balloux and Lugon-Moulin (2002) , and Softlinks (2007) reviewed
statistical issues associated with the analysis of microsatellite markers.
13.5.8 RFLP Analysis
RFLP analysis can be used to analyze variation in both mtDNA and nuclear
DNA ( Table 13.1 ). Depending on which restriction enzymes are used and tar-
get sequences analyzed, extensive variation can be discerned. However, RFLP
analyses require relatively large amounts of very clean DNA, which may not be
obtainable from single individuals of small insects. The DNA must be digested,
electrophoresed, blotted, and probed to detect the variation. Probes must be
developed, either as consensus sequences from other species, or after cloning
and sequencing of species-specific DNA. Working with large numbers of individ-
ual insects is relatively time-consuming and expensive ( Table 13.1 ).
13.5.9 PCR-RFLP
A modification of RFLP analysis, called PCR-RFLP , eliminates many of the dis-
advantages of traditional RFLP analysis ( Karl and Avise 1993 , Table 13.1 ). If no
probe is available, a genomic DNA library can be constructed and clones isolated
and sequenced. Alternatively, degenerate primers can be designed and the PCR
products cloned and sequenced. Once species-specific sequences are available,
allele-specific PCR primers can be designed. Subsequently, nuclear DNA can be
amplified by the PCR using these primers and the PCR product can be digested
with appropriate restriction enzymes. The cut DNA is visualized after electro-
phoresis by staining with ethidium bromide.
The advantage of PCR-RFLP is that DNA extracted from a single individual
is sufficient (after PCR amplification) to provide bands that can be visualized.
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