ANALYSIS OF DELETION OF THE INTEGRATED HUMAN PAPILLOMAVIRUS-16 SEQUENCE IN CERVICAL-CANCER - A RAPID MULTIPLEX POLYMERASE CHAIN-REACTION APPROACH

A protocol for a rapid physical mapping of the integrated type 16 huma n papillomavirus (HPV16) sequences in biospied and paraffin-embedded a rchival cervical cancer samples is described. The procedure involves t he use of an anchor primer and a mixture of indicator primers in a mul tiplex polymerase chain reaction (PCR). A minimal conserved region of viral integration of 2,745 bp in length has been mapped between nucleo tide (nt) 6102-941, containing the entire regulatory region and the E6 and E7 open reading frames (ORFs). A general deletion domain of 1,465 bp in the integrated viral genome has been defined between nt 1417-28 81,covering most of the El ORF at the 3'-half and 60 bp at the 5' term inus of the E2 ORF. This common deleted sequence contains an ATPase ac tive domain speculated to be associated with a DNA helicase function e ssential for the viral replication, and it also falls within the activ ely spliced E1-E2 segment of the primary RNA transcripts. Detection of the loss of the 3'-half of the El ORF would be an ideal marker for PC R-based rapid determination of HPV integration in cervical cancer cell s. (C) 1994 Wiley-Liss, Inc.