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Microarrays for genotyping human group A rotavirus by multiplex capture and type-specific primer extension
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Molecular Medicine.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
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2003 (English)In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 41, no 11, 5153-5158 p.Article in journal (Refereed) Published
Abstract [en]

Human group A rotavirus (HRV) is the major cause of severe gastroenteritis in infants worldwide. HRV shares the feature of a high degree of genetic diversity with many other RNA viruses, and therefore, genotyping of this organism is more complicated than genotyping of more stable DNA viruses. We describe a novel microarray-based method that allows high-throughput genotyping of RNA viruses with a high degree of polymorphism by multiplex capture and type-specific extension on microarrays. Denatured reverse transcription (RT)-PCR products derived from two outer capsid genes of clinical isolates of HRV were hybridized to immobilized capture oligonucleotides representing the most commonly occurring P and G genotypes on a microarray. Specific primer extension of the type-specific capture oligonucleotides was applied to incorporate the fluorescent nucleotide analogue cyanine 5-labeled dUTP as a detectable label. Laser scanning and fluorescence detection of the microarrays was followed by visual or computer-assisted interpretation of the fluorescence patterns generated on the microarrays. Initially, the method detected HRV in all 40 samples and correctly determined both the G and the P genotypes of 35 of the 40 strains analyzed. After modification by inclusion of additional capture oligonucleotides specific for the initially unassigned genotypes, all genotypes could be correctly defined. The results of genotyping with the microarray fully agreed with the results obtained by nucleotide sequence analysis and sequence-specific multiplex RT-PCR. Owing to its robustness, simplicity, and general utility, the microarray-based method may gain wide applicability for the genotyping of microorganisms, including highly variable RNA and DNA viruses.

Place, publisher, year, edition, pages
2003. Vol. 41, no 11, 5153-5158 p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-92071DOI: 10.1128/JCM.41.11.5153-5158.2003PubMedID: 14605152OAI: oai:DiVA.org:uu-92071DiVA: diva2:165023
Available from: 2004-09-15 Created: 2004-09-15 Last updated: 2012-04-13Bibliographically approved
In thesis
1. Methods for Analysis of Disease Associated Genomic Sequence Variation
Open this publication in new window or tab >>Methods for Analysis of Disease Associated Genomic Sequence Variation
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In Molecular Medicine a wide range of methods are applied to analyze the genome to find genetic predictors of human disease. Apart from predisposing disease, genetic variations may also serve as genetic markers in the search for factors underlying complex diseases. Additionally, they provide a means to distinguish between species, analyze evolutionary relationships and subdivide species into strains.

The development and improvement of laboratory techniques and computational methods was a spin-off effect of the Human Genome Project. The same techniques for analyzing genomic sequence variations may be used independent of organism or source of DNA or RNA. In this thesis, methods for high-throughput analysis of sequence variations were developed, evaluated and applied.

The performance of several genotyping assays were investigated prior to genotyping 4000 samples in a co-operative genetic epidemiological study. Sequence variations in the estrogen receptor alpha gene were found to be associated with an increased risk of breast and endometrial cancer in Swedish women.

Whole genome amplification (WGA) enables large scale genetic analysis of sparse amounts of biobanked DNA samples. The performance of two WGA methods was evaluated using four-color minisequencing on tag-arrays. Our in-house developed assay and “array of arrays” format allow up to 80 samples to be analyzed in parallel on a single microscope slide. Multiple displacement amplification by the Φ29 DNA polymerase gave essentially identical genotyping results as genomic DNA. To facilitate accurate method comparisons, a cluster quality assessment approach was established and applied to assess the performance of four commercially available DNA polymerases in the tag-array minisequencing assay.

A microarray method for genotyping human group A rotavirus (HRV) was developed and applied to an epidemiological survey of infectious HRV strains in Nicaragua. The method combines specific capture of amplified viral sequences on microarrays with genotype-specific DNA-polymerase mediated extension of capture oligonucleotides with fluorescent dNTPs.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2004. 89 p.
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1371
Molecular medicine, microarray, molecular medicine, single nucleotide polymorphism, whole genome amplification, breast cancer, endometrial cancer, human rotavirus, Molekylärmedicin
National Category
Medical Genetics
urn:nbn:se:uu:diva-4525 (URN)91-554-6027-5 (ISBN)
Public defence
2004-10-08, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15
Available from: 2004-09-15 Created: 2004-09-15Bibliographically approved

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