Preventing false positives: Quantitative evaluation of three protocols for inactivation of polymerase chain reaction amplification products

Abstract

False-positive results because of carryover contamination by previously amplified nucleic acids are currently the greatest impediment to routine implementation of nucleic acid amplification protocols. We evaluated three methods for inactivation of a 156-bp Borrelia burgdorferi polymerase chain reaction (PCR) product: (i) post-PCR cross-linking with isopsoralen (IP), (ii) pre-PCR treatment of a dU-containing PCR product with uracil N- glycosylase (UNG), and (iii) post-PCR alkaline hydrolysis (primer hydrolysis) of PCR products synthesized by using primers containing 3' ribose residues. The sensitivities of the PCR performed under the conditions of each protocol were comparable. Inactivation of amplified DNA was highly efficient for all three protocols; the IP and UNG protocols eliminated at least to 3 x 109 copies of the product. The primer hydrolysis protocol varied in efficiency depending on the number and position of the 3' ribose residues, but inactivation ranged from 104 to 109 copies. We conclude that with some modifications, all three systems are effective for eliminating amplified DNA products. Routine implementation of at least one method should help to avoid false-positive results because of carryover contamination.