Use of multiple displacement amplification in the investigation of human papillomavirus physical status.
OBJECTIVE
The investigation of human papillomavirus (HPV) physical status in pre-invasive cervical lesions has been restricted by the small amounts of tissue available for study. Multiple displacement amplification (MDA), a phi29 DNA polymerase based whole genome amplification technique, has the potential to help resolve this problem by yielding large amounts of high molecular weight DNA from tiny starting quantities.


METHODS
Firstly, a comparison was made of restriction endonuclease fragment patterns of DNA from seven different HPV types and corresponding MDA products. Secondly, E6/E7 and LCR sequencing data from HPV16 recombinant plasmid and MDA copy DNA were correlated. Thirdly, DNA and MDA products from cervical cell lines (CaSki, HeLa, and SiHa that contain integrated HPV) and an invasive cervical carcinoma were analysed by Southern blot hybridisation. Fourthly, MDA product from CaSki cell DNA mixed with HPV18-plasmid DNA was tested for the demonstration of both episomal and integrated HPV. Finally, MDA products from HPV16 positive abnormal cervical cytological samples were assayed for integration by Southern blot hybridisation.


RESULTS
DNA templates and MDA products yielded analogous data. Episomal and integrated HPV DNA were successfully detected by Southern blot assay of the cell line/HPV-plasmid model, and in MDA products of clinical samples.


CONCLUSIONS
These data show that MDA has considerable potential to assist in the investigation of HPV physical status; abundant (>40 microg) DNA can be generated with high fidelity from minuscule (50 ng) starting quantities, and both episomal and integrated HPV DNA are distinguishable in MDA products from solid tumours and cytological materials.
Evans
Mark Francis
MF
Department of Pathology, University of Vermont College of Medicine, Burlington, Vermont, USA. mark.evans@uvm.edu
Adamson
Christine Stewart-Crawford
CS
von Walstrom
Genevieve Montagu
GM
Cooper
Kumarasen
K
D001483:Base Sequence; D015139:Blotting, Southern / Q000379:methods; D004279:DNA, Viral / Q000032:analysis; D005260:Female; D052162:Human papillomavirus 16 / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D027383:Papillomaviridae / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D014407:Tumor Cells, Cultured; D002583:Uterine Cervical Neoplasms / Q000821:virology*; D016662:Virus Integration
D004279:DNA, Viral