Geometric differences allow differential enzymatic inactivation of PCR product and genomic targets

Abstract

The value of the polymerase chain reaction (PCR) is markedly limited by the ease of carry-over contamination. We predicted that the location of the target sequence which spans the linear center of the molecule in PCR products, but not in genomic molecules, would allow digestion by certain exonucleases (Exo). This would eliminate amplifiable targets specifically from PCR products and do so without the need to control either the size of the genomic molecules or the extent of the digestion reaction. Testing with T7 Exo and model targets in phage λ DNA yielded results consistent with those predicted. By heat inactivating the Exo, complete reaction mixtures could be decontaminated and then amplified in an automatic thermal cycler without reopening the reaction tubes and risking recontamination.