Dealing with contamination: enzymatic control of carryover contamination in PCR.

Life Technologies, Incorporated, Gaithersburg, Maryland 20884-9980 PCR produces an abundance of product DNA from traces of input DNA. It was apparent early that because it is so sensitive, PCR is especially susceptible to contamination. In a research setting (e.g., cloning a gene, preparing a probe), contamination is generally not a concern. However, if a primer pair is used many times, if the PCR is designed to be very sensitive, or if the presence or the absence of amplification of a target sequence will have diagnostic implications, then possible contamination must be eliminated for the PCR results to be meaningful. Contaminating DNA can originate from three sources: DNA from other test samples, DNA from experimental materials such as recombinant clones, or DNA generated by previous PCR amplification of the same target sequence. This last source of contamination, often called contamination, has proven to be the most troublesome source. Early users of PCR noted that carryover contaminat ion could be a significant problem owing to the abundance of DNA generated by PCR and the ease with which such DNA can be reamplified. (1'2) Detecting carryover contamination, e.g., by including negative control reactions, is essential. Prevention is clearly preferred, however, because correcting the problem can be costly and testing of samples will probably need to cease until a thorough clean-up can be effected. This will most likely mean discarding all suspected reagents, and cleaning, or even replacing, equipment. A last resort, one not always possible, is to change to a different primer pair, so as to amplify a different region of the target DNA. Two general approaches may be taken to control carryover contamination: physical isolation of PCR products from newly assembled reactions (see also Setting up a PCR Laboratory, pp. $2-$9) and design of the PCR so that products of a first PCR are not efficiently reamplifiable in a subsequent PCR. Physical measures to control carryover contaminat ion are listed in approximate order of difficulty and cost: