The use of exonuclease III for polymerase chain reaction sterilization.

The carryover of previously amplified sequences (amplicons) into new PCR reactions is a serious problem. Recently Furrer et al. reported a 'pre-PCR' sterilization technique using DNase I or restriction enzymes for contamination control (1). Unfortunately, these methods require the reaction tube to be re-opened to add target DNA and Taq polymerase following the enzymatic treatment (providing an opportunity for subsequent contamination), and correspondingly, do not address possible carryover contamination from these materials. Furthermore, our results indicate that the endonucleolytic activity of DNase I severely degrades primers. To overcome these problems, we have developed two alternative protocols for pre-PCR sterilization which utilize exonuclease m (exo 1). Exom catalyzes the sequential cleavage of 5' mononucleotides from the 3' hydroxyl end of duplex DNA. In Protocol I, double-stranded target DNA and all PCR reagents (including Taq polymerase) are incubated with exo Im (30°C/30 min) followed by the heat inactivation of exo Im (95 C/s min). Protocol I is based on the size difference between the carryover amplicon and the target DNA, with the much smaller amplicon being more readily digested by the exo Im treatment. In Protocol II, the target DNA is first denatured in the presence of the PCR reaction components (100°C/5 min) then quick-chilled on ice. Exo IH and Taq polymerase are then added and the mixture incubated (30°C/30 min) followed by heating (95°C/S min). Protocol H works because exo IH does not degrade single stranded target DNA. Following denaturation, the DNA consists primarily of hybrids between the primers and 1) any contaminating amplicon and 2) the genomic target DNA. Upon treatment with