clinical samples will demand new methods that improve the specificity of PCR by eliminating ..... Plenum Press, New York. 12. Syvanen, A.-C. 1992. From one to ...
Vol. 31, No. 9
JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1993, p. 2361-2365
Dependence of Polymerase Chain Reaction Product Inactivation Protocols on Amplicon Length and Sequence Composition MARK J. ESPY, THOMAS F. SMITH,* AND DAVID H. PERSING Division of Clinical Microbiology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905 Received 17 March 1993/Returned for modification 29 April 1993/Accepted 11 June 1993
Specific diagnostic test results generated by polymerase chain reaction (PCR) depend upon control of amplicon contamination in the clinical laboratory. We compared photochemical (isopsoralen [IP]) and enzymatic (uracil N-glycosylase [UNG]) methods for their ability to prevent carryover of amplicons generated from genomic targets of five viruses. PCR products (amplicons) (herpes simplex virus, 342 bp; cytomegalovirus, 250 bp; Epstein-Barr virus, 240 bp) exposed to UV light in the presence of various concentrations of IP compound 10 (IP-10) resulted in apparent increased molecular sizes of the products, as indicated by migration patterns after gel electrophoresis, and were predictive of inactivation by the agent. For amplicons of < 100 bp, IP-10-induced electrophoretic shifts were related to the guanidine-cytidine (G+C) content of the PCR product; no apparent shift and no inactivation were observed for a 92-bp herpes simplex virus amplicon (G+C content, 65%), whereas the 100-bp human papillomavirus product (G+C content, 42%) showed a concentrationdependent shift (25 to 100 jg/ml) in electrophoretic migration and was partially inactivated. UNG effectively controlled amplicon carryover for target DNA of >240 bp; however, this treatment did not inactivate the two amplicons of
