THE PCR LABORATORY

12 Apr

THE PCR LABORATORY

Figure 5.5 The PCR laboratory is designed so that the work flows through the different processes in one direction starting with sample reception and forensic biology and finishing with the post-PCR analysis. The samples are passed through air-lock hatches to minimize the possibility of any material being transferred from post-PCR to pre-PCR areas. Access to the pre- and post-PCR laboratories is through different changing areas and dedicated staff will work in either pre- or post-PCR areas. Positive air pressure in pre-PCR areas and negative pressure in post-PCR rooms also reduces the possibility of introducing any contamination into the pre-PCR areas efficiently. The PCR set-up introduces another positive and negative control: the pos- itive control involves setting-up a PCR with DNA of known origin and whose profile is known. Successful analysis demonstrates that the reaction 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 contaminatesubsequentreactions.EachtimeaPCRtubeisopenedthereissomeaerosol 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 fundamental 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 possibility of contaminating DNA extractions and PCR set-ups with amplified material. In addition to the two phys- ical 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. Second Edition. Cold Spring Harbor Laboratory Press.

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