Multiple-primer DNA sequencing method

被引:28
作者
Gharizadeh, B
Ghaderi, M
Donnelly, D
Amini, B
Wallin, KL
Nyrén, P
机构
[1] Royal Inst Technol, KTH, Dept Biotechnol, Stockholm Ctr Phys Astron & Biotechnol, SE-10691 Stockholm, Sweden
[2] Karolinska Hosp, Ctr Mol Med, Dept Mol Med, S-10401 Stockholm, Sweden
[3] Univ Orebro, Dept Caring Sci, Div Biomed, Orebro, Sweden
关键词
human papilloma virus; microorganisms; multiple infections; multiple sequencing primers; pyrosequencing;
D O I
10.1002/elps.200390147
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:1145 / 1151
页数:7
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