A coupled PCR and restriction digest method for the detection and analysis of the SV40 regulatory region in infected-cell lysates and clinical samples.
The polymerase chain reaction (PCR) is an increasingly popular analytical tool for the detection of virus sequences in laboratory preparations as well as in human clinical samples. In studies involving papovaviruses SV40, BK virus (BKV), and JC virus (JCV), one of the primary targets for analysis is the viral regulatory region, as that section of the papovavirus genome is distinct. A primary concern with PCR-based studies is whether amplified DNA sequences may be derived from laboratory contaminants. Recognizing that common sources of PCR contamination are the positive control templates, we devised a facile method to distinguish between real and false-positive PCR-amplified SV40 regulatory region DNAs. SV40 constructs that had been engineered to contain different combinations of 72-basepair (bp) enhancer elements and 21-bp repeats, as well as two introduced unique restriction enzyme sites, were used as positive control templates for PCR amplification. Cleavage of PCR-amplified DNA identifies products from the engineered control plasmids. The procedure is rapid, simple and cost-effective. We also report that primer sets predicted to be specific for the SV40 regulatory region can be used to amplify BKV and JCV regulatory region sequences under conditions of reduced stringency.
Lednicky
J A
JA
Division of Molecular Virology, Baylor College of Medicine, Houston, TX 77030-3498, USA.
Butel
J S
JS
D001739:BK Virus / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004279:DNA, Viral / Q000032:analysis; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D006801:Humans; D007577:JC Virus / Q000235:genetics / Q000302:isolation & purification; D030361:Papillomavirus Infections / Q000473:pathology / Q000821:virology*; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D012045:Regulatory Sequences, Nucleic Acid; D013539:Simian virus 40 / Q000235:genetics / Q000302:isolation & purification*; D014412:Tumor Virus Infections / Q000473:pathology / Q000821:virology*
D017931:DNA Primers; D004279:DNA, Viral; D004262:DNA Restriction Enzymes; C034510:CTCGAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; C029792:GTCGAC-specific type II deoxyribonucleases