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Multiple-primer DNA...
Abstract
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- 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 m ore 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(TM) 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.
Keyword
- human papilloma virus
- microorganisms
- multiple infections
- multiple sequencing primers
- pyrosequencing
- ribosomal database project
- human-papillomavirus dna
- cervical-cancer
- prevalence
- women
- association
- carcinoma
- infection
- lesions
Publication and Content Type
- ref (subject category)
- art (subject category)
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