Usefulness of PCR-SSCP for tracing the pathogenesis of human papillomavirus-associated malignancy

Kim, Jae Weon; Song, Yong Sang; Park, Noh Hyun; Kang, Soon Beom; Lee, Hyo Pyo

Abstract

PURPOSE: To set up more simplified detection method for human papillomavirus (HPV) sequence polymorphism which could be used for the study of HPV-related pathogenesis, route of infection, and many other epidemiologic studies.
MATERIALS AND METHODS
One hundred and thirteen cases of uterine cervical tissues containing HPV 16 DNA confirmed by polymerase chain reaction (PCR) from Korean women were subjected to investigate the URR gene mutations. PCR-amplified products were sequenced by the fluorescent dideoxy termination method and opposite strand sequencing was performed as required. The results obtained from sequencing were analysed to find the most hypervariable segment which contains the greatest number of variants and subjected to PCR-single strand conformation polymorphism (SSCP) analysis.
RESULTS
Among the length of nucleotide position (np) from 7175 to 24, we found 60 sites (60/815=7.36%) of base substitutions. Segment from np 7743 to 24 was the most hypervariable and contain 20 kinds of variants. In this segment, C-to-T mutation at position 24, G-to-A at 7842, and T-to-C at 7781 were more frequently found than other sites. By comparing sequencing results with PCR-SSCP, we found 15 patterns distinguishable each other. All of the mobility shift occurred in the PCR-SSCP pattern could be accounted for by the base substitutions and nearly all of the DNA sequencing results observed were reflected as alterations in the PCR-SSCP patterns.
CONCLUSIONS
We have assigned the hypervariable segment in one portion of URR which could be used as PCR-SSCP analysis. Identification of HPV polymorphism by PCR-SSCP is potentially useful for elucidating a number of epidemiologic questions such as the pathway of viral spread and so on.