Biology Reference
In-Depth Information
During the DNA extraction process negative control extractions must always be
carried out to monitor for contamination; positive controls that involve extracting
material similar to the casework samples, for example buccal swabs or bloodstains,
can be carried out to monitor that the extraction and amplification procedures are
working efficiently. The PCR set-up introduces another positive and negative control:
the positive control involves setting up a PCR with DNA of known origin and
whose profile is known. Successful analysis demonstrates that the PCR worked. In
the negative control PCR, water replaces the DNA to monitor for contamination
in the reagents or introduced during the PCR set-up.
Post-PCR
The most potent source of contamination is previously amplified PCR products. Fol-
lowing a PCR there are millions of copies of the target sequence that can potentially
contaminate subsequent reactions. Each time a PCR tube is opened there is some
aerosol spray, and a single droplet of aerosol will contain thousands of copies of the
amplified target, resulting in transfer of some of the amplified product. The funda-
mental feature of any laboratory that engages in PCR analysis is that there must be
physical separation of the pre-PCR and the post-PCR analysis to minimize the pos-
sibility of contaminating DNA extractions and PCR set-ups with amplified material.
In addition to the two physical spaces, there should also be dedicated equipment,
protective clothing and reagents for each area. There must be a unidirectional work
flow through the laboratory; PCR products must never be brought back into the pre-
PCR part of the laboratory. There must also be temporal separation of tasks; it is not
possible for a scientist who has been working in the post-PCR to then work in the pre-
PCR area without the possibility of introducing contamination; an overnight break
before returning to the pre-PCR area is normally recommended. Larger laboratories
will have scientists who are dedicated to only the pre- or the post-PCR analysis.
Further reading
Dieffenbach, C.W. and Dveksler, G.S. (2003)
PCR Primer: A Laboratory Manual
, 2nd edn, Cold
Spring Harbor Laboratory Press.
WWW resources
Primer3 - PCR primer design software: http://primer3.sourceforge.net/
OligoCalc - oligonucleotide properties calculator: http://www.basic.northwestern.edu/biotools/
oligocalc.html
References
1. Mullis, K., Faloona, F., Scharf, S., Saiki, R., Horn, G. and Erlich, H. (1986) Specific enzymatic
amplification of DNA in vitro - the polymerase chain-reaction.
Cold Spring Harbor Symposia
on Quantitative Biology
,
51
, 263 - 273.
