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PubMed · 12707905

Multiple-primer DNA sequencing method.

Baback Gharizadeh·Mehran Ghaderi·Declan Donnelly·Bahram Amini·Keng-Ling Wallin·Pål Nyrén
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Abstract

A multiple-primer DNA sequencing approach suitable for genotyping, detection and identification of microorganisms and viruses has been developed. In this new method two or more sequencing primers, combined in a pool, are added to a DNA sample of interest. The oligonucleotide that hybridizes to the DNA sample will function as a primer during the subsequent DNA sequencing procedure. This strategy is suited for selective detection and genotyping of relevant microorganisms and samples harboring different DNA targets such as multiple variant/infected samples as well as unspecific amplification products. This method is used here in a model system for detection and typing of high-risk oncogenic human papilloma viruses (HPVs) in samples containing multiple infections/variants or unspecific amplification products. Type-specific sequencing primers were designed for four of the most oncogenic (high-risk) HPV types (HPV-16, HPV-18, HPV-33, and HPV-45). The primers were combined and added to a sample containing a mixture of one high-risk (16, 18, 33, or 45) and one or two low-risk types. The DNA samples were sequenced by the Pyrosequencing technology and the Sanger dideoxy sequencing method. Correct genotyping was achieved in all tested combinations. This multiple-sequencing primer approach also improved the sequence data quality for samples containing unspecific amplification products. The new strategy is highly suitable for diagnostic typing of relevant species/genotypes of microorganisms.

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DNA PrimersDNA Probes, HPVDNA, ViralElectrophoresis, Agar GelGenotypeHumansNucleic Acid HybridizationPapillomaviridaeSequence Analysis, DNAJournal ArticleResearch Support, Non-U.S. Gov't

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Baback Gharizadeh, Mehran Ghaderi, Declan Donnelly, Bahram Amini, Keng-Ling Wallin, Pål Nyrén. 2003. Multiple-primer DNA sequencing method.. https://doi.org/10.1002/elps.200390147

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