JMD TIDES - Oligonucleotide and Peptide - May 18-21, 2008, Las Vegas, NV
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JMD 2005, Vol. 7, No. 2
Copyright © 2005 American Society for Investigative Pathology & Association for Molecular Pathology

Type-Specific Multiple Sequencing Primers

A Novel Strategy for Reliable and Rapid Genotyping of Human Papillomaviruses by Pyrosequencing Technology

Baback Gharizadeh*, Maria Oggionni{dagger}, Biying Zheng{ddagger}, Edit Akom§, Nader Pourmand*, Afshin Ahmadian, Keng-Ling Wallin{ddagger}|| and Pål Nyrén

From the Stanford Genome Technology Center, * Stanford University, Palo Alto, California; the Department of Pathology, {dagger} Unit of Experimental Molecular Pathology, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy; the Université du Québec à Montréal, § Montréal, Canada; the Department of Molecular Medicine, {ddagger} Center for Molecular Medicine, Karolinska Hospital, Stockholm, Sweden; the Department of Biotechnology, Royal Institute of Technology, Stockholm, Sweden; and the Department of Public Health and Clinical Medicine, || Nutritional Research, Umeå University Hospital, Umeå, Sweden

DNA sequencing is the gold standard method for accurate microbial and viral typing. However, DNA sequencing techniques have been facing limitations in typing of human papillomaviruses when the specimen harbors multiple genotypes and yields nonspecific amplification products, resulting in nonspecific and noninterpretable sequence data. To address these limitations we have developed a type-specific multiple sequencing primer DNA-sequencing method. This new strategy is suitable for sequencing and typing of samples harboring different genotypes (co-infections with multiple genotypes) and yielding nonspecific amplifications, thus eliminating the need for nested polymerase chain reaction (PCR), stringent PCR conditions, and cloning. The new approach has also proved useful for amplicons containing low PCR yield or subdominant types, avoiding reperforming of amplifications. We have applied the multiple sequencing primer method for genotyping of clinically relevant human papillomaviruses in a clinical test panel by using a combined pool of seven type-specific sequencing primers for HPV-6, -11, -16, -18, -31, -33, and -45. Furthermore, we introduced a sequence pattern recognition approach when there was a plurality of genotypes in the sample to facilitate typing of more than one target DNA in the sample. The multiple sequencing primer method has proved to be a multifaceted approach for typing of human papillomaviruses by DNA sequencing technologies.




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