Multiple-primer DNA sequencing method
2003 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 24, no 08-jul, 1145-1151 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2003. Vol. 24, no 08-jul, 1145-1151 p.
human papilloma virus, microorganisms, multiple infections, multiple sequencing primers, pyrosequencing, ribosomal database project, human-papillomavirus dna, cervical-cancer, prevalence, women, association, carcinoma, infection, lesions
IdentifiersURN: urn:nbn:se:kth:diva-22479ISI: 000182693400002OAI: oai:DiVA.org:kth-22479DiVA: diva2:341177
QC 201005252010-08-102010-08-10Bibliographically approved