title: Dependence of polymerase chain reaction product inactivation protocols on amplicon length and sequence composition.
abstract: 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 < or = 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 concentration-dependent shift (25 to 100 micrograms/ml) in electrophoretic migration and was partially inactivated. UNG effectively controlled amplicon carryover for target DNA of > or = 240 bp; however, this treatment did not inactivate the two amplicons of < or = 100 bp, regardless of the G + C content of the product. Larger products were inactivated efficiently by both methods, regardless of their G + C contents. We concluded that both IP and UNG effectively inactivated PCR amplicons but not short amplicons of < or = 100 bp. We recommend that with the adoption of PCR technology in clinical laboratories, primers should be designed to produce amplicons of at least 240 to 350 bp (depending on G + C content) and that at least one effective method of controlling carryover contamination should be incorporated into each PCR protocol.
authors.affiliation: Division of Clinical Microbiology, Mayo Clinic, Rochester, Minnesota 55905.
authors.forename: M J
authors.initials: MJ
authors.lastname: Espy
authors.forename: T F
authors.initials: TF
authors.lastname: Smith
authors.forename: D H
authors.initials: DH
authors.lastname: Persing
mesh_terms: D001482:Base Composition; D001483:Base Sequence; D002460:Cell Line; D045647:DNA Glycosylases; D017931:DNA Primers; D015342:DNA Probes; D011564:Furocoumarins; D006564:Herpesviridae / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000494:pharmacology; D027383:Papillomaviridae / Q000235:genetics*; D010777:Photochemistry; D016133:Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays; D051981:Uracil-DNA Glycosidase; D014777:Virus Diseases / Q000175:diagnosis
chemical_list: D017931:DNA Primers; D015342:DNA Probes; D011564:Furocoumarins; C011659:angelicin; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase