Biology Reference
In-Depth Information
undesirable, and unnecessary, to use a positive control after the PCR has been
optimized.
By contrast, multiple
negative controls
always
should be included in the PCR
experiment because they will allow you to detect contamination (
false posi-
tives
) if used consistently and in adequate numbers. Negative controls consist of
all reagents, but lack template DNA. A small number of contaminating template
DNA molecules in the negative controls could lead to
sporadic
false-positive results.
Thus, it is important to carry out multiple negative controls
each time
so that rare
contaminants can be detected. How many negative controls should be used? There
currently is no standard number, but a statistician might say that having
more
neg-
ative controls than experimental units is desirable (although expensive).
In conclusion, although UV irradiation has been recommended as effective
in inactivating contaminating DNA and has been widely used, it should not be
counted on as the only method to prevent contamination (
Dwyer and Saksena
1992, Frothingham et al. 1992
). Furthermore, autoclaving may not eliminate
previously amplified PCR products (
Dwyer and Saksena 1992
). Thus, meticulous
attention to the entire set of procedures, including the physical separation of
DNA isolation, PCR amplification, and PCR analysis, is critical in minimizing con-
tamination or carryover problems.
8.4 Some Modifications of the PCR
Up to now, the discussion has described allele-specific PCR for which primers can
be designed because sequence information is available. What can you do if you
want to amplify DNA from an arthropod for which little genetic information is
available? What if you want single-stranded DNA rather than double-stranded
DNA as a product? What if you want to find the sequence of DNA upstream or
downstream from a specific gene? Some solutions to these, and other, problems
have been achieved by modifying the types and numbers of primers used in the
PCR (
Table 8.6
).
Table 8.7
outlines some of the questions that can be answered by
modifications of the PCR.
8.4.1 AFLP for DNA Fingerprinting
A
mpliied
F
ragment
L
ength
P
olymorphism (AFLP) provides a method for devel-
oping DNA fingerprints that eliminates some of the problems inherent in RAPD-
PCR, AP-PCR, and DAF (described below) (
Savelkoul et al. 1999
). AFLP has its
own limitations, namely that it generates dominant rather than codominant
markers (
Mueller and Wolfenbarger 1999
), and it requires absolutely clean tem-
plate DNA in consistent quantities.
