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 human papillomavirus (HPV16) sequences in biopsied 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.
Department of Medical Research, Veterans General Hospital, Taipei, Taiwan, Republic of China.
C M
CM
Chen
M P
MP
Shyu
L C
LC
Au
H W
HW
Chu
W T
WT
Cheng
K B
KB
Choo
D002874:Chromosome Mapping; D005260:Female; D005814:Genes, Viral; D005819:Genetic Markers; D006801:Humans; D016366:Open Reading Frames; D027383:Papillomaviridae / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D030361:Papillomavirus Infections / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods; D017384:Sequence Deletion; D014412:Tumor Virus Infections / Q000821:virology*; D002583:Uterine Cervical Neoplasms / Q000821:virology*; D016662:Virus Integration / Q000235:genetics
D005819:Genetic Markers