A simple and rapid method of high quantity DNA isolation from cervical scrapes for detection of human papillomavirus infection.
Infection with human papillomavirus (HPV) is an important etiological factor in the development of cervical cancer, and detection of the viral genome is of prognostic importance, particularly for preneoplastic lesions. We developed a simple, easy and efficient non-organic method of DNA extraction from cervical scrapes for reliable detection of HPV DNA sequences. The method involves incubation of cell nuclei in higher concentration of proteinase K at 65 degrees C for 2.5 h. Following prolonged incubation at higher temperature, the enzyme is autoinactivated and the DNA isolated can be used directly for analysis without further purification. The recovery of DNA is more than 95% and it can be easily cleaved by restriction enzymes and is suitable for amplification by the polymerase chain reaction (PCR). The whole procedure is carried out in a single Eppendorf tube and a large number of specimens can be processed at a time without any error of handling. DNA extracted from a single smear sample is sufficient to conduct as many as four different molecular biology tests. This provides an opportunity for verification of sensitivity, specificity and reliability of each test for diagnosis of HPV infection without resorting to biopsy.
Gopalkrishna
V
V
Division of Molecular Oncology, Institute of Cytology and Preventive Oncology (ICMR), New Delhi, India.
Francis
A
A
Sharma
J K
JK
Das
B C
BC
D000328:Adult; D001483:Base Sequence; D015139:Blotting, Southern; D002584:Cervix Uteri / Q000382:microbiology*; D004279:DNA, Viral / Q000302:isolation & purification*; D005260:Female; D005821:Genetic Techniques; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D027383:Papillomaviridae / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D014412:Tumor Virus Infections / Q000175:diagnosis* / Q000382:microbiology; D002578:Uterine Cervical Dysplasia / Q000382:microbiology; D002583:Uterine Cervical Neoplasms / Q000382:microbiology; D014626:Vaginal Smears
D004279:DNA, Viral