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Homogeneous scoring of single nucleotide polymorphisms: The 5’-nuclease ”TaqMan” assay versus Molecular beacon probes
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
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2000 (English)In: BioTechniques, ISSN 0736-6205, Vol. 28, no 4, 732-738 p.Article in journal (Refereed) Published
Abstract [en]

Homogeneous assays based on real-time fluorescence monitoring during PCR are relevant alternatives for large-scale genotyping of single-nucleotide polymorphisms (SNPs). We compared the performance of the homogeneous TaqMan 5'-nuclease assay and the Molecular Beacon assay using three SNPs in the human estrogen receptor gene as targets. When analyzing a panel of 90 DNA samples, both assays yielded a comparable power of discrimination between the genotypes of a C-to-T transition in codon 10 and a G-to-A transition in codon 594 of the estrogen receptor gene. The Molecular Beacon probes distinguished better than the TaqMan probes between homozygous and heterozygous genotypes of a C-to-G transversion in codon 325. The sensitivity of detecting one allele, present as a minority in a mixed sample, varied between the SNPs and was similar for both assays. With the Molecular Beacon assay, the measured signal ratios were proportional to the amount of the minor allele over a wider range than with the TaqMan assay at all three SNPs.

Place, publisher, year, edition, pages
2000. Vol. 28, no 4, 732-738 p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-92066PubMedID: 10769752OAI: oai:DiVA.org:uu-92066DiVA: diva2:165018
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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