Use of the polymerase chain reaction to specifically amplify integrated HPV-16 DNA by virtue of its linkage to interspersed repetitive DNA.
A polymerase chain reaction (PCR) based technique that combines a virus specific primer and a human interspersed repetitive sequence (IRS) specific primer in order to detect integration of human papilloma virus type 16 (HPV-16) is described. Amplification of viral-host DNA junctions occurs when viral integration results in placement of the virus specific primer binding site near (less that 3-4 kb) the primer binding site within a repetitive sequence element. The method relies on enzyme labeled oligonucleotide probes to achieve rapid, specific, and nonradioisotopic detection of viral integration related PCR products since episomal forms of the viral DNA do not lead to exponential accumulation of hybridizable PCR products. The technique is demonstrated for human genomic DNA derived from clinical cervical swab specimens and archival paraffin embedded blocks. Viral integration was detected in 41% of the HPV-16 positive samples (n = 34). In this positive subset, 64% were classified as invasive neoplasias, 29% CIN III and 7% CIN II. Analyzing the positive invasive neoplasias, 6 of 9 (66%) of the fingerprint results were obtained when an HPV primer was paired with an Alu primer. Interestingly, 100% of Alu primed fingerprint results obtained were derived from samples presenting invasive neoplasia (P < 0.025 by chi square).
Carmody
M W
MW
Maine Medical Center Research Institute, South Portland 04106, USA.
Jones
M
M
Tarraza
H
H
Vary
C P
CP
D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D002584:Cervix Uteri / Q000821:virology; D016009:Chi-Square Distribution; D016172:DNA Fingerprinting; D017931:DNA Primers; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics*; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D009361:Neoplasm Invasiveness; D015345:Oligonucleotide Probes; D027383:Papillomaviridae / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid; D002583:Uterine Cervical Neoplasms / Q000473:pathology / Q000821:virology*; D016662:Virus Integration
D017931:DNA Primers; D004279:DNA, Viral; D015345:Oligonucleotide Probes