DNA Amplification by the Polymerase Chain Reaction: Molecular Biology Made Accessible - Insect Molecular Genetics - page 337

Vanlerberge-Masutti 1994 ). Differences in RAPD-PCR patterns are correlated
with the evolution of different taxa, allowing limited estimates of evolutionary
divergence ( Espinasa and Borowsky 1998 ).
RAPD-PCR products can be cloned ( Comes et al. 1997 ) and sequenced so that
“allele-specific” primers can be developed for future PCR analyses. S equence-
C haracterized A mpliied R egion (SCAR) primers will produce allele-specific PCR
products. Agusti et al. (2000) used a SCAR primer pair to amplify single bands of
310 bp to detect the whitefly Trialeurodes vaporariorum in the gut of the preda-
tor Dicyphus tamaninii .
Two or more primers have been used simultaneously to generate reproducible
RAPD fragments that are different from those obtained with each single primer
( Micheli et al. 1993, Sall et al. 2000 ).
RAPD-PCR has been criticized for its lack of reproducibility ( Ayliffe et al. 1994,
Lamboy 1994, Micheli et al. 1994, Hallden et al. 1996, Jones et al. 1997, Khandka
et al. 1997, McEwan et al. 1998, Perez et al. 1998 ). Different RAPD banding pat-
terns can be obtained if different DNA extraction methods are used, probably
due to the presence of different kinds or amounts of contaminants or different
amounts of template DNA ( Micheli et al. 1994 ). Different DNA polymerases also
may amplify different RAPD products ( Schierwater and Ender 1993 ).
RAPD-PCR is sensitive to both DNA template concentration and quality, so
bands may vary in intensity or even disappear if template concentration is not
controlled or DNA is sheared ( Khandka et al. 1997 ). Reproducibility also can be
poor if different PCR machines or pipettors are used, resulting in different tem-
perature cycling conditions or different concentrations of the PCR mixture ( He
et al. 1994a,b; Schweder et al. 1995 ). Occasionally, heteroduplex molecules that
are formed between allelic sequences can cause artifactual RAPD bands ( Ayliffe
et al. 1994 ). Thus, it is critical that researchers use primers only if they produce
bright, consistent banding patterns in the particular thermocycler used. Likewise,
researchers should obtain a reference profile for their own work rather than
comparing their results to those generated by another ( He et al. 1994a ).
Another criticism of RAPD-PCR is that all bands are inherited as dominant
alleles. This means that heterozygotes cannot be identified unless progeny test-
ing is conducted, although this is not an issue when RAPD-PCR is conducted
on haploid males of arrhenotokous species ( Edwards and Hoy 1993 ). Another
problem is that comigration of similar sized bands with different sequences can
occur, but may not be detected, unless the bands are cloned and sequenced.
Use of RAPD markers to calculate genetic similarity coefficients can result in
false positives and false negatives if RAPD artifacts are present ( Lamboy 1994 ).