Simple enzymatic means to neutralize DNA contamination in nucleic acid amplification.
Reverse transcription PCR (RT-PCR) is prone to false positives when contaminating DNA molecules are present at the start of a reaction. Contaminants that derive from earlier work using a given primer pair (carryover PCR products) are of particular concern when those primers are used routinely, as in clinical diagnostics or environmental monitoring. In addition, contamination by genomic DNA can significantly interfere with quantitative and qualitative analysis of RNAs by RT-PCR. Here we describe contaminant restriction (ConR), a method that can be used to neutralize carryover and genomic DNA contamination in RT-PCR studies. Restriction enzymes (REs) added to the amplification cocktail cleave contaminant DNA molecules while sparing the intended target nucleic acid. Restriction, reverse transcription, and amplification steps all take place in the same sealed vessel, thus avoiding any danger of recontamination. ConR eliminates carryover contamination in PCR without compromising target sequence amplification. Because the method is effective against both genomic and carryover contamination, it can be employed routinely in one-step RT-PCR, whatever the RNA target or the nature of the potential DNA contaminant. A variation of this decontamination method, amplicon primer site restriction (APSR), is effective specifically against carryover contamination. APSR, unlike ConR, can be applied during PCR-based amplification of DNA target molecules.
Ashkenas
John
J
Samuel Lunenfeld Research Institute, Toronto, ON, Canada. j.ashkenas@axon-com.com
Dennis
James W
JW
Ho
Chi Yip
CY
D004247:DNA / Q000737:chemistry* / Q000235:genetics*; D004262:DNA Restriction Enzymes / Q000737:chemistry*; D004340:Drug Contamination / Q000517:prevention & control*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D004247:DNA; D004262:DNA Restriction Enzymes