Biomedical Engineering Reference
In-Depth Information
located in the post-PCR area (
see
item d
). However, for applications in which
the PCR tubes must be opened, e.g., for nested PCR, the PCR machine should
be located at a fourth isolated area separated from sample preparation, PCR
setup, and the post-PCR areas. In nested PCR, a dedicated set of pipets should
be allocated for this purpose.
d. Post-PCR area: this is the area reserved for the analysis of PCR products,
including electrophoresis, restriction analysis, and mass spectrometry. No
items from the post-PCR area should be allowed back into the aforementioned
areas. It is important to note that this includes items such as notebooks
and pens.
2.
Laboratory practice designed to minimize the risk of contamination:
a. All PCR reagents should be aliquoted, and reagents that can be autoclaved
should be so treated.
b. Use and change gloves frequently. Kitchin et al. have advocated the use of
face and head masks, as certain individuals appear more prone to the shed-
ding of contaminants
(
11
)
.
c. Positive displacement pipets or aerosol-resistant pipets should be used.
d. When multiple reactions are needed, it is helpful to set up a master mix to
reduce the number of maneuvers, and thus reduce the chance of possible con-
tamination.
e. The number of PCR cycles should be kept to a minimum, as excessively sen-
sitive assays are more prone to contamination
(
12
)
.
f. When given a choice, disposable items are preferable to items that must be
washed prior to being reused.
g. If possible, different personnel should be allocated to the pre-PCR and post-
PCR parts of the project. If this is not practical, then it is preferable to sched-
ule the project or work week such that the pre-PCR and post-PCR procedures
are performed on different days.
h. The use of closed PCR systems, e.g., the TaqMan
®
system
(
13
)
, which use
fluorescence signals for detecting PCR products, can eliminate the opening of
the amplification vessels and post-PCR sample handling. Therefore, carryover
contamination using these systems is much less of a problem than conven-
tional systems. This is especially important for clinical diagnostic applica-
tions
(
5
)
.
3.
Use of specific anti-contamination measures:
a. Ultraviolet (UV) irradiation: Sarkar and Sommer describe the use of UV irra-
diation to damage any contaminating DNA prior to the addition of DNA tem-
plate
(
14
,
15
)
. As this method relies on the crosslinking of adjacent thymidine
residues, the sequence of the PCR target influences the decontaminating effi-
ciency of the method
(
16
)
. Certain primers appear to be more sensitive to the
damaging effect of UV light, and may need to be added after the irradiation
step. Furthermore, the hydration status of DNA appears to have a great influ-
ence on its susceptibility to UV irradiation in that dry DNA seems much more
resistant to the damaging effect of UV
(
16
,
17
)
. This latter fact means that
