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983) Which one of the following statements about controls for polymerase chain reaction (PCR) or reverse-transcriptase polymerase chain reaction (RT-PCR) is true?

When setting up a PCR, one should always add a template to the no-DNA control ӿrst, to avoid accidentally contaminating it. When setting up a PCR, one should add a template to the test samples ӿrst, followed by the no-DNA control, and then add the normal and abnormal controls. When setting up an RT-PCR, in addition to a no-template (i.e., no RNA) control, one should set up a no-RT control for each RNA sample being tested to test for directly amplifiable RNA. When setting up a PCR following an RT reaction, one should set up a separate no-template control in addition to the product of the no-RNA RT reaction. The use of deoxyuridine triphosphate (dUTP) in PCR with uracil-N-glycosylase to prevent PCR contamination is of no value for RT-PCR because RNA contains uracil.

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• Contamination of polymerase chain reaction (PCRs) can result from carry-over contamination of the PCR product, cross-contamination with template, or (especially when dealing with microorganisms) environmental contamination (including from laboratory personnel).

• Physical controls include separate pre- and post-PCR areas with separate sets of instruments. Procedural approaches to minimizing contamination include ultraviolet irradiation of equipment and surfaces, cleaning of surfaces with bleach or other nucleic acid damaging agents, autoclaving when possible, the use of single-use aliquots of reagents, and the use of disposable containers when possible.

• An enzymatic approach to preventing cross-contamination is the use of deoxyuridine triphosphate (dUTP) in PCR to partially substitute for thymidine. The resulting double-stranded DNA product will contain uracil and can be treated with uracil-N-glycosylase so the PCR product will not be able to act as a template.

• In addition, the use of proper negative controls is necessary to detect contamination. This includes using a template control (or multiple controls when testing for microorganisms), which must always be set up and capped last to maximize the chances of detecting contamination occurring at any time during reaction set-up.

• When performing reverse-transcriptase polymerase chain reaction (RT-PCR), a no-RT control is needed to detect DNA ampliӿcation. In addition, a separate negative control is required for the PCR step to detect DNA contamination occurring at this step. The use of single-tube RT-PCR, whenever possible, obviates this issue.

Stirling D: Quality control in PCR.Methods Mol Biol 2003;226:21–24.

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Clinical Pathology: General Principles, Lab Management, Clinical Chemistry, Immunology & Histocompatibility, Genetic Testing

983) Which one of the following statements about controls for polymerase chain reaction (PCR) or reverse-transcriptase polymerase chain reaction (RT-PCR) is true?

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