CHAPTER 3. NUCLEAR MEDICINE SERVICES
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be avoided. Although false positives can result from sample-to-sample contam-
ination, a more serious source is the carry-over of DNA from a previous ampli-
fication of the same target. Because of the large numbers of copies of amplified
sequences, the carry-over of the smallest quantities of a PCR sample can lead
to serious contamination. It is essential that PCR reagents (primers, Taq
polymerase, deoxyribonucleoside triphosphates (dNTPs), water and buffers)
be stored separately from clinical samples, controls or amplicons as detailed
below in order to avoid costly contamination. Strict adherence to the recom
-
mendations below will minimize the carry-over of amplified DNA.
3.8.2. PCR, contamination and good laboratory practice
Although extraneous nucleic acid from multiple sources may serve as a
template for amplification, the main cause of false positive reactions appears to
be PCR products from previous reactions. Caution should be taken when using
numerous amplifications of the same primer pair system. The following
precautions will eliminate the risk of false positives in the context of diagnostic
assays.
Reactions prior to (Areas 1 and 2) and following (Area 3) amplification
should be separated physically. To prevent the carry-over of amplified DNA
sequences, it is important to set up reactions in a separate room or containment
unit such as an ultraviolet (UV) irradiated hood or a biosafety cabinet. A
further set of supplies and pipetting devices should be dedicated to the specific
use of setting up PCRs. Amplified DNAs (post-PCR products) must never be
brought into this area nor should reagents be taken from an area where
amplicon analyses take place. Similarly, it is unwise to take devices such as
pipettors into the containment area after use on amplified material.
Separate sets of automatic pipettors, disposable pipettes, a microcen-
trifuge, tubes and gloves should be kept in each area.
Positive displacement pipettors and plugged tips, to form an aerosol
barrier, should be used in Areas 1 and 2. Positive displacement pipettes are
recommended to eliminate the cross-contamination of samples by pipetting
devices. In Area 3, normal unplugged tips can be used.
Reagents should be aliquoted to minimize the number of repeated
samplings. All reagents used in PCRs must be prepared, aliquoted and stored
in an area that is free of amplicons. It is advisable to record the reagent lots
used so that if carry-over occurs it can be more easily traced.
Laboratory precautions in the handling of radioactivity should be incor-
porated (Area 3).
A selection of the number and types of controls should be made.
Different controls should be used in each reaction: