Biomedical Engineering Reference
In-Depth Information
most serious source of contamination. When such large amounts of PCR prod-
ucts are generated repeatedly over a period of time, the potential for contami-
nation becomes increasingly high. This is further compounded by the fact that
many diagnostic applications require PCR to perform at its highest sensitivity,
namely, at the single-molecule level. Under these circumstances, even one of
the billions of molecules generated from a single reaction is enough to gener-
ate a false-positive result. The second source of contamination is cloned DNA
previously handled in the laboratory. The third type is sample-to-sample con-
tamination. This source of contamination is most detrimental to samples that
require extensive processing prior to amplification. The fourth source is the
ubiquitously present template DNA in the environment from the laboratory
personnel and reagents used for DNA extraction and PCR
(
8-10
)
.
3. Principles of Contamination Avoidance
Like many problems, avoidance is better than cure, and PCR contamination
is no exception. The main principles of contamination avoidance in PCR are:
1.
Strict physical separation of individual PCR-related maneuvers: we recommend
the use of three distinct areas for the sample preparation stage, the PCR setup
stage, and the post-PCR stage. This applies as much to the performance of labo-
ratory procedure as to equipment. Thus, every piece of equipment, no matter how
small, should be restricted to each area. This applies to laboratory notebooks,
which should not be carried between different areas. If transfer of items is essen-
tial, then the direction should be from the pre-PCR area to the post-PCR area and
never the reverse.
a. Sample preparation area: this area is for the processing of sample materials,
such as the extraction of DNA and RNA. No PCR products should ever be
allowed into the area. Dedicated equipment and reagents should be reserved
solely for sample preparation purposes, including pipetting equipment and
laboratory coats. Gloves should be worn at all times and changed frequently.
In general, the simpler the sample processing is, the less chance there is of
introducing contamination. Dedicated storage facilities, e.g., freezers, should
be available for sample preparation alone.
b. PCR setup area: it is recommended that the setting up of PCR reactions be
performed in a laminar flow hood. The defined area of the hood facilitates
the maintenance of cleanliness of the area. Dedicated equipment and storage
facilities should be available near the PCR setup area. A separate area should
be available for the addition of samples to the PCR reagents. DNA or RNA
samples should never be allowed inside the PCR setup hood.
c. PCR machine: the location of the PCR machine depends on the exact amplifi-
cation requirements. For PCR applications involving a single round of PCR
and in which it is not required that individual PCR tubes be opened for the
addition or sampling of reagents prior to analysis, the PCR machine may be
