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Multiply-primed rolling circle amplification of human papillomavirus using sequence-specific primers
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics. Uppsala University, Science for Life Laboratory, SciLifeLab.
2012 (English)In: Virology, ISSN 0042-6822, E-ISSN 1096-0341, Vol. 432, no 1, 57-62 p.Article in journal (Refereed) Published
Abstract [en]

Multiply-primed rolling circle amplification (RCA) is a suitable technique for amplification of circular templates and has been used to identify novel human papillomaviruses (HPV). In this study we develop an efficient RCA for whole genome amplification of HPV using HPV-specific primers in clinical samples and establish a protocol for whole genome sequencing using the Sanger method. Amplification of cloned HPV-genomes by RCA was compared using specific primers against random hexamers. Using HPV-specific primers increased the effectiveness on average 15.2 times and the enrichment of HPV relative to human gDNA on average 62.2 times, as compared to using random hexamer. RCA products were sequenced without need for cloning, even when using low-input amounts. The technique was successfully used on 4 patient samples from FTA cards, to generate whole HPV-genome sequences. Degenerated HPV-specific primers for RCA produce DNA of sufficient quality and quantity suitable for sequencing and other potential downstream analyses.

Place, publisher, year, edition, pages
2012. Vol. 432, no 1, 57-62 p.
Keyword [en]
Human papillomavirus, Rolling circle amplification, Degenerated primers, Sanger sequencing
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-181993DOI: 10.1016/j.virol.2012.05.030ISI: 000307027100007OAI: oai:DiVA.org:uu-181993DiVA: diva2:559465
Available from: 2012-10-09 Created: 2012-10-02 Last updated: 2017-12-07Bibliographically approved

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Gustavsson, IngerGyllensten, Ulf

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