Analysis of deletion of the integrated human papillomavirus 16 sequence in cervical cancer: A rapid multiplex polymerase chain reaction approach

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Abstract
A protocol for a rapid physical mapping of the integrated type 16 human papillomavirus (HPV16) sequences in biospied and paraffin‐embedded archival cervical cancer samples is described. The procedure involves the use of an anchor primer and a mixture of indicator primers in a multiplex polymerase chain reaction (PCR). A minimal conserved region of viral integration of 2,745 bp in length has been mapped between nucleotide (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 1417–2881, covering most of the E1 ORF at the 3′‐half and 60 bp at the 5′ terminus of the E2 ORF. This common deleted sequence contains an ATPase active domain speculated to be associated with a DNA helicase function essential for the viral replication, and it also falls within the actively spliced E1‐E2 segment of the primary RNA transcripts. Detection of the loss of the 3′‐half of the E1 ORF would be an ideal marker for PCR‐based rapid determination of HPV integration in cervical cancer cells.