Biomedical Engineering Reference
In-Depth Information
and render them unamplifiable. The crosslinking is most effective at 5°C and
under UV intensity of more than 27 mW/cm
2
(e.g., in an HRI-300 chamber)
(
27
)
. This method has been shown to be similar in decontaminating efficiency
to the UNG method, and results in the elimination of at least 10
9
copies of
contaminating PCR products
(
23
)
.
f. Exonuclease digestion: it was demonstrated that certain exonucleases, e.g.,
exonuclease III and T7 exonuclease, when added to fully assembled PCR
reactions, were able to render carryover PCR product molecules non-
amplifiable but would spare identical target sequences in genomic DNA
(
28
,
29
)
. In a model system, a 30-min incubation with exonuclease III was
able to degrade 5
10
5
copies of carryover amplicons
(
28
)
. Several mecha-
nisms for the selectivity against PCR products have been postulated: Zhu et
al. attributed it to the relatively long chain length of genomic DNA, which
might resist degradation by exonucleases better than the comparatively short
PCR products
(
28
)
, and Muralidhar and Steinman, in an ingenious series of
experiments, demonstrated that part of this selectivity has a geometric expla-
nation
(
29
)
. Thus, for any stretch of DNA to be amplifiable by a specific pair
of primers following T7 exonuclease treatment, the primer binding sites
should be situated on the same side with respect to the geometric center of the
molecule. As it is extremely unlikely that a particular genomic target would
straddle the center of any stretch of genomic DNA (essentially produced by
random shearing during DNA extraction), this form of exonuclease treatment
would spare the genomic target. The situation with carryover PCR products,
however, is completely different, as the primer binding sites are located at
opposite ends of the molecules and thus would span the geometric center of
the molecule. Exonuclease treatment for the prevention of PCR carryover,
therefore, possesses the chief advantage of uridine incorporation and
glycosylation in that the completed reaction tubes do not have to be reopened
for the addition of the target and/or
Taq
polymerase. Furthermore, exonu-
clease treatment has the added advantage of being able to destroy even
nonuridine-containing PCR products, and would be very useful in an already
contaminated environment.
×
4. Detection of Contamination
Monitoring for contamination is probably as important as measures to pre-
vent it. It is a reality that contamination will be experienced by most, if not all,
workers using PCR. To facilitate the monitoring of contamination, the follow-
ing measures should be undertaken:
1.
Negative controls should be included in every PCR experiment. To detect spo-
radic contamination, multiple controls are usually required. Different negative
controls, testing the different stages in the PCR process at which contamination
may occur, should be included. PCR reagent controls will only test for contami-
nation of the reagents, but not the sample preparation stage.
