Strategy for typing human papillomaviruses by RFLP analysis of PCR products and subsequent hybridization with a generic probe.
Genital human papillomaviruses (HPV) were detected by PCR using L1 consensus primers MY09 and MY11. To determine the underlying HPV type(s). PCR products were subsequently analyzed employing a combination of restriction fragment length polymorphism (RFLP) and hybridization with a generic oligonucleotide probe that binds to a conserved region located close to the MY11-binding site within the PCR products. Using computer-assisted sequence analysis, the lengths of the corresponding BamHI, DdeI, HaeIII, HinfI and PstI restriction fragments hybridizing with the generic probe were calculated, revealing distinct patterns for each of the 45 mucosal HPV types. This method is superior to RFLP analysis since it is not impaired by large amounts of restriction fragments resulting from nonspecific PCR products. Moreover, considering clinical specimens containing two or three different HPV types, direct sequencing of PCR products will be inconclusive, and the increased number of restriction fragments will complicate interpretation of RFLP patterns. Subsequent hybridization with the generic probe, however, results in the appearance of, at most, 2 or 3 bands per restriction enzyme digest and thus facilitates identification of the underlying HPV types.
Meyer
T
T
Institute of Applied Immunology and Biotechnology, Hamburg, Germany.
Arndt
R
R
Stockfleth
E
E
Flammann
H T
HT
Wolf
H
H
Reischl
U
U
D001003:Anal Canal / Q000821:virology; D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004279:DNA, Viral / Q000032:analysis*; D015245:Deoxyribonuclease BamHI; D015252:Deoxyribonucleases, Type II Site-Specific; D005835:Genitalia / Q000821:virology; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D027383:Papillomaviridae / Q000145:classification* / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D017422:Sequence Analysis, DNA
D004279:DNA, Viral; D015345:Oligonucleotide Probes; D004262:DNA Restriction Enzymes; D015245:Deoxyribonuclease BamHI; C027908:endodeoxyribonuclease DdeI; C024388:CTGCAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; C032229:GANTC-specific type II deoxyribonucleases; C024403:GGCC-specific type II deoxyribonucleases