Multiple-primer DNA sequencing method.
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.
Gharizadeh
Baback
B
Department of Biotechnology, Stockholm Center for Physics, Astronomy and Biotechnology, Royal Institute of Technology, Sweden.
Ghaderi
Mehran
M
Donnelly
Declan
D
Amini
Bahram
B
Wallin
Keng-Ling
KL
Nyrén
Pål
P
D017931:DNA Primers / Q000592:standards*; D015344:DNA Probes, HPV; D004279:DNA, Viral / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D005838:Genotype; D006801:Humans; D009693:Nucleic Acid Hybridization; D027383:Papillomaviridae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D017422:Sequence Analysis, DNA / Q000379:methods*
D017931:DNA Primers; D015344:DNA Probes, HPV; D004279:DNA, Viral