Restriction fragment length polymorphism analysis of isotype-labeled polymerase chain reaction-amplified human papillomavirus DNA combines sensitivity with built-in contaminant detection.
The detection of low copy number DNA in archival tissue has been revolutionized by polymerase chain reaction (PCR), but widespread acceptance of this technique for diagnostic purposes has been hampered by problems with contamination. There is a technique, restriction fragment length polymorphism analysis for characterizing PCR-amplified human Papillomavirus (HPV) nucleic acids, that reportedly provides for increased sensitivity in detection of both target and contaminating sequences. This technique is a modification of standard restriction fragment length polymorphism and involves radiolabeled nucleotide incorporation during PCR and restriction enzyme digestion of the products followed by high resolution polyacrylamide gel electrophoresis. The amount of initial target DNA amplified by PCR that can be detected by isotope labeling or ethidium staining was compared, as was the sensitivity of the two methods for analyzing a PCR product of fixed amount. The sensitivity of ethidium staining or autoradiography was comparable for detection of initial target HPV DNA by PCR. However, for subsequent typing of amplified HPV DNA by restriction fragment length polymorphism, isotope-labeled products provided an approximately 125-fold increase in sensitivity over ethidium staining, with a maximum of 625-fold greater sensitivity with a 3-day exposure. The detection of low levels of potential contaminants in PCR-amplified HPV DNA was determined in a serial analysis of cervical biopsies. Contaminating DNA was identified in two gels where multiple samples yielded the same restriction pattern. On re-isolation of genomic DNA, these products of contaminating DNA were not seen. Extrapolating from the above experiments, the contamination of many samples would have escaped detection by ethidium staining alone.(ABSTRACT TRUNCATED AT 250 WORDS)
Zitz
J C
JC
Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts.
McLachlin
C M
CM
Tate
J E
JE
Mutter
G L
GL
Crum
C P
CP
D001483:Base Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis*; D005260:Female; D006801:Humans; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D027383:Papillomaviridae / Q000235:genetics / Q000302:isolation & purification*; D030361:Papillomavirus Infections / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D012680:Sensitivity and Specificity; D014412:Tumor Virus Infections / Q000821:virology; D002583:Uterine Cervical Neoplasms / Q000821:virology*; D002578:Uterine Cervical Dysplasia / Q000821:virology*
D017931:DNA Primers; D004279:DNA, Viral