44 Landgraf and WolfesTop this mixture carefully with paraffin oil, incubate at 95°C for 3
min, add 1-4 U of Taq polymerase/100 paL reaction vol, and repeat 30
cycles (temperature measurement in the heating block) of:
- 1 min at 74°C;
- 1 min at 92°C; and
- 1 min at 50°C.
End the procedure with an incubation at 74°C for 3 min. Analyze a
10-gL aliquot on an agarose or PAGE gel; stain with ethidium bromide.
3.2. 7. Reliable PCR Experiments
In clinical diagnosis, reliability is the most important criterion for the
application of a test. The extreme sensitivity of the PCR requires high
standards for the conditions of operation. Even minor contaminations
with foreign DNA will render results uncertain. With this caveat in mind,
some precautions are necessary to avert false positive results (30). - Separate all PCR materials and equipment from processes involved in
DNA isolation. - Aliquot all buffer, dNTP, and primer stock solutions.
- Pipet the DNA last.
- Always process controls in parallel: (a) a negative control with all compo-
nents, but lacking the template, and (b) a positive control with a control
template in a concentration similar to the template under investigation. - Perform only the minimum number of amplification cycles necessary
to detect the product. This is crucial for experiments in which the loss
of a band (e.g., demonstration of the deletion of particular a sequence)
has to be detected (45).
Recently, it was reported (46) that irradiation at 254 nm for 5 min
of the PCR mix lacking template and enzyme will reduce DNA con-
tamination by a factor of 105.
- PCR Applications
As an overview, the next paragraphs will deal with the application of
the PCR. For more detailed information, see the literature cited (47--49).
4.1. PCR as a Standard Method
in Clinical Diagnosis
The scope of PCR application in clinical diagnosis ranges from
prenatal diagnosis for sickle cell anemia (2) to postmortem analysis of