Biomedical Engineering Reference
In-Depth Information
there are limitations to the use of UV for sterilizing dry laboratory surfaces.
Ultimately, clean laboratory practices and physical separation remain the most
important anti-contamination measures, with UV irradiation providing an
additional margin of protection.
b. Restriction enzyme treatment: restriction enzymes that cleave within the tar-
get sequence for PCR may be used to restrict any contaminating sequence
prior to the addition of the target
(
18
,
19
)
. Following decontamination, the
enzyme is destroyed by thermal denaturation (i.e., 94°C for 10 min; thus,
thermostable restriction enzymes such as
Taq
I should not be used for this
purpose) before addition of the template DNA. In a model system, Furrer et
al. showed that restriction with
Msp
I (10 U for 1 h) reduced contamination by
a factor of 5 to 10 without impairing the efficiency of PCR
(
19
)
.
c. DNase I treatment: this approach is similar to that in
item b
except that DNase
I is used. Furrer et al. showed that prior treatment with 0.5 U of DNase I for
30 min reduced contamination by a factor of 1000 without impairing the effi-
ciency of PCR
(
19
)
.
d. Incorporation of dUTP and treatment with uracil-
N
-glycosylase (UNG): as
the carryover of PCR products from previous amplification experiments con-
stitutes a predominant source of PCR contamination, the ability to selectively
destroy PCR products, but not template DNA, presents one way to reduce
contamination. Such an approach is described by Longo et al., who substi-
tuted dUTP for dTTP during PCR
(
20
)
. Carryover PCR products containing
dUs can then be destroyed prior to subsequent amplification experiments by
incubation with UNG. It should be noted that when dUTP is used instead of
dTTP, the MgCl
2
concentration often must be readjusted: typically, dUTP is
used at 600 m
M
with 3 m
M
MgCl
2
. Following the initial thermal denatur-
ation, UNG activity is destroyed; thus, the newly synthesized PCR products
are not degraded. However, UNG may regain some of its activity when the
temperature is below 50°C, and thus an annealing temperature of over 50°C
should be used
(
21
)
and all completed PCR containing UNG should be kept at
72°C until analysis. UNG treatment has been reported to result in a 10
7
-
(
22
)
to 10
9
-fold
(
23
)
reduction in amplicon concentration. In our experience, there
is a very slight reduction in sensitivity in PCR systems incorporating dUTP
and UNG treatment, although up to a 10-fold reduction in sensitivity has been
described
(
22
)
. This method can also be applied to reverse-transcription (RT)-
PCR because PCR products which contain deoxyribose uracil are digested by
UNG preferentially to ribose uracil-containing RNA with the optimization of
the concentration of UNG and the time and temperature of enzyme digestion
(
24
,
25
)
. However, it should be remembered that this method is only effective
against dU-containing PCR products. Thus, carryover contamination owing to
conventional PCR product lacking dUs cannot be eradicated using this method.
e. Incorporation of isopsoralen compound: Cimino et al. describe adding a pho-
tochemical reagent before PCR and activation after the amplification is com-
pleted
(
26
)
. The reagent will then crosslink the two strands of the PCR product
