John M. S. Bartlett.pdf - Bio-Nica.info

22 Stirling
which will either not anneal, or if the annealing temperature is low enough, will anneal
promiscuously, yielding multiple products. Simple aliquoting primers into manageable
volumes will reduce both the scope for contamination and degradation. This practice
should also be adopted for dNTP stocks for the same reason.
4. Operator Errors
Anyone involved in teaching molecular techniques hears the same complaint again
and again: “These reaction volumes are too small! . . . I can’t see a microliter!” Even for
those with many years of laboratory experience (perhaps especially for those), it can be
difficult to adjust to dealing with small volume reactions. Although the obvious answer
may be to increase the volume, this has both cost and efficiency implications.
• Use appropriate pipetting devices. A pipettor designed for the 20- to 200-µL range will
not accurately dispense 10 µL.
• The use of master mixes not only reduces the dependence on accurately pipetting small
volumes but also improves the control over reaction contents.
• Practice with the same reaction until consistent results are obtained.
5. PCR-Specific Difficulties
Although much of the above could apply to any analytical laboratory technique,
PCR also is subject to the confounding problem of contamination. Cross contamination
of samples is of concern in any discipline, and good laboratory practice, such as
careful pipetting and the constant changing of disposable pipet tips, will minimize
the opportunity of this occurring. Where PCR differs from most other procedure is in
the production of vast quantities of the analyte during the procedure. The presence of
billions of copies of potential template can create severe problems. These problems can
be minimized by physically separating the pre- and postamplification processes (ideally
in different rooms with different pipettors, etc.); however, they should constantly be
monitored by the inclusion of appropriate controls.
6. Controls
• No DNA. Although it can seem extravagant to constantly set up reactions without template,
this is the best way to monitor for contamination. A separate “no DNA” control should
be set up for each master mix or each individual reaction. If contamination is discovered,
the pipettor should be decontaminated (as per manufacturers guidelines), and the reagent
aliquot should be rechecked or discarded.
• Positive control. PCR is often used simply to detect the presence of specific sequence. In
such circumstances, it is essential to include at least one reaction with a template known
to contain the sequence.
• Internal control. Even when master mixes have been used to ensure consistency of reaction
components, and a positive control is used, there is the possibility that template may be
omitted from individual tubes. This can be addressed by the inclusion within each reaction
tube of primers, which will amplify a target known to consistently be present in the test
DNA (see factor IX in Chapter 47 for example).
7. Regional Quality-Assurance Programs
In addition to the in-house precautions detailed above, there are a growing number
of specialist quality-assurance programs that have been developed for most diagnostic
PCR.