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Minisequencing on oligonucleotide microarrays: comparison of immobilisation chemistries
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, Molecular Medicine.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Molecular Medicine.
2001 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 29, no 13, e69-e69 p.Article in journal (Refereed) Published
Abstract [en]

In the microarray format of the minisequencing method multiple oligonucleotide primers immobilised on a glass surface are extended with fluorescent ddNTPs using a DNA polymerase. The method is a promising tool for large-scale single nucleotide polymorphism (SNP) detection. We have compared eight chemical methods for covalent immobilisation of the oligonucleotide primers on glass surfaces. We included both commercially available, activated slides and slides that were modified by ourselves. In the comparison the differently derivatised glass slides were evaluated with respect to background fluorescence, efficiency of attaching oligonucleotides and performance of the primer arrays in minisequencing reactions. We found that there are significant differences in background fluorescence levels among the different coatings, and that the attachment efficiency, which was measured indirectly using extension by terminal transferase, varied largely depending on which immobilisation strategy was used. We also found that the attachment chemistry affects the genotyping accuracy, when minisequencing on microarrays is used as the genotyping method. The best genotyping results were observed using mercaptosilane-coated slides attaching disulfide-modified oligonucleotides.

Place, publisher, year, edition, pages
2001. Vol. 29, no 13, e69-e69 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-89697PubMedID: 11433045OAI: oai:DiVA.org:uu-89697DiVA: diva2:161387
Available from: 2002-03-14 Created: 2002-03-14 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Accessing Genetic Variation by Microarray Technology
Open this publication in new window or tab >>Accessing Genetic Variation by Microarray Technology
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Microarray technology is a promising approach for the simultaneous analysis of multiple single nucleotide polymorphisms (SNPs), which are the most abundant form of genetic variation. In this thesis enzyme-assisted microarray-based methods were developed to improve the accuracy and genotype discrimination power of the current methods for SNP genotyping. The improved technology was applied for analysing recessively inherited disease mutations, for Y-chromosomal SNPs in a population study, for an evolutionary analysis of SNPs in flycatchers and for multiplexed quantitative determination of SNP-allele frequencies in pooled DNA samples.

A robust attachment chemistry for immobilising oligonucleotides on glass surface was established, based on an evaluation of eight covalent coupling methods. A four-colour fluorescence detection strategy, which enabled a multiplexed quantitative analysis for as little as 2% of a minority allele frequency in pooled samples was generated.

Twenty-five Y-chromosomal SNPs were screened in a collection of 300 samples from five Finno-Ugric-speaking populations using minisequencing on microarrays. In these populations six distinct haplotypes were defined by the six SNPs that were polymorphic. Data from five microsatellite markers was combined with the SNP data, revealing shared Y-chromosomal haplotypes between the Finns and the Saami, indicating, in accordance with earlier data, at least two founding Y-chromosomal lineages in these populations.

Database screening and subsequent validation of 125 potential SNPs in the highly repetitive type 1 interferon genes and genes coding for proteins in the interferon-related regulatory pathways revealed 25 informative SNPs in the Finnish and Swedish populations. These SNPs were included in a panel for microarray based genotyping that should find a variety of applications in genetic studies due to the important immunoregulatory functions of the IFN family.

The significance of sex-chromosome evolution on speciation was investigated in two naturally hybridising flycatcher species (N=459) by analysing a panel of 20 SNPs using minisequencing on microarrays. A strong selection against gene flow across the species boundary of sex-linked genes was observed, as well as a sex-chromosomal influence on male plumage characteristics that have previously been shown to reinforce isolation in these birds. The results suggest a major role for sex-chromosome-mediated isolation of the two flycatcher species.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2002. 65 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1127
Keyword
Medical sciences, microarrays, SNPs, minisequencing, four-colour detection, allele frequency, population study, evolutionary biology, MEDICIN OCH VÅRD
National Category
Medical and Health Sciences
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-1792 (URN)91-554-5251-5 (ISBN)
Public defence
2002-04-09, Rudbeck hall, Uppsala, 09:15
Opponent
Available from: 2002-03-14 Created: 2002-03-14 Last updated: 2016-08-11Bibliographically approved

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