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Use of multiple displacement amplification in the investigation of human papillomavirus physical status
  1. Mark Francis Evans,
  2. Christine Stewart-Crawford Adamson,
  3. Genevieve Montagu von Walstrom,
  4. Kumarasen Cooper
  1. Department of Pathology, University of Vermont College of Medicine, Burlington, Vermont, USA
  1. Dr M F Evans, Department of Pathology, University of Vermont College of Medicine, 89 Beaumont Avenue, Burlington, VT 05405, USA; mark.evans{at}uvm.edu

Abstract

Background and Aims: 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 μg) 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.

  • cervical cancer
  • human papillomavirus
  • integration
  • multiple displacement amplification
  • whole genome amplification
  • APOT, amplification of papillomavirus oncogene transcripts
  • CIN, cervical intraepithelial neoplasia
  • HPV, human papillomavirus
  • ICC, invasive cervical carcinoma
  • LCR, long control region
  • LEEP, loop electrosurgical excision procedure
  • MDA, multiple displacement amplification
  • Pap, Papanicolaou

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Footnotes

  • Funding: This research was supported by a grant from the Cancer Research and Prevention Foundation.

  • Competing interests: None declared.

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