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Accessing Genetic Variation by Microarray Technology
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
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. , p. 65
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1127
Keywords [en]
Medical sciences, microarrays, SNPs, minisequencing, four-colour detection, allele frequency, population study, evolutionary biology
Keywords [sv]
MEDICIN OCH VÅRD
National Category
Medical and Health Sciences
Research subject
Molecular Medicine
Identifiers
URN: urn:nbn:se:uu:diva-1792ISBN: 91-554-5251-5 (print)OAI: oai:DiVA.org:uu-1792DiVA, id: diva2:161390
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
List of papers
1. A system for specific, high-throughput genotyping by allele-specific primer extension on microarrays
Open this publication in new window or tab >>A system for specific, high-throughput genotyping by allele-specific primer extension on microarrays
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2000 (English)In: Genome Research, ISSN 1088-9051, E-ISSN 1549-5469, Vol. 10, no 7, p. 1031-1042Article in journal (Refereed) Published
Abstract [en]

This study describes a practical system that allows high-throughput genotyping of single nucleotide polymorphisms (SNPs) and detection of mutations by allele-specific extension on primer arrays. The method relies on the sequence-specific extension of two immobilized allele-specific primers that differ at their 3′-nucleotide defining the alleles, by a reverse transcriptase (RT) enzyme at optimized reaction conditions. We show the potential of this simple one-step procedure performed on spotted primer arrays of low redundancy by generating over 8000 genotypes for 40 mutations or SNPs. The genotypes formed three easily identifiable clusters and all known genotypes were assigned correctly. Higher degrees of multiplexing will be possible with this system as the power of discrimination between genotypes remained unaltered in the presence of over 100 amplicons in a single reaction. The enzyme-assisted reaction provides highly specific allele distinction, evidenced by its ability to detect minority sequence variants present in 5% of a sample at multiple sites. The assay format based on miniaturized reaction chambers at standard 384-well spacing on microscope slides carrying arrays with two primers per SNP for 80 samples results in low consumption of reagents and makes parallel analysis of a large number of samples convenient. In the assay one or two fluorescent nucleotide analogs are used as labels, and thus the genotyping results can be interpreted with presently available array scanners and software. The general accessibility, simple set-up, and the robust procedure of the array-based genotyping system described here will offer an easy way to increase the throughput of SNP typing in any molecular biology laboratory.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-89695 (URN)10899152 (PubMedID)
Available from: 2002-03-14 Created: 2002-03-14 Last updated: 2017-12-14Bibliographically approved
2. Y-chromosomal SNPs in Finno-Ugric-speaking populations analyzed by minisequencing on microarrays
Open this publication in new window or tab >>Y-chromosomal SNPs in Finno-Ugric-speaking populations analyzed by minisequencing on microarrays
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2001 (English)In: Genome Research, ISSN 1088-9051, E-ISSN 1549-5469, Vol. 11, no 3, p. 471-482Article in journal (Refereed) Published
Abstract [en]

An increasing number of single nucleotide polymorphisms (SNPs) on the Y chromosome are being identified. To utilize the full potential of the SNP markers in population genetic studies, new genotyping methods with high throughput are required. We describe a microarray system based on the minisequencing single nucleotide primer extension principle for multiplex genotyping of Y-chromosomal SNP markers. The system was applied for screening a panel of 25 Y-chromosomal SNPs in a unique collection of samples representing five Finno--Ugric populations. The specific minisequencing reaction provides 5-fold to infinite discrimination between the Y-chromosomal genotypes, and the microarray format of the system allows parallel and simultaneous analysis of large numbers of SNPs and samples. In addition to the SNP markers, five Y-chromosomal microsatellite loci were typed. Altogether 10,000 genotypes were generated to assess the genetic diversity in these population samples. Six of the 25 SNP markers (M9, Tat, SRY10831, M17, M12, 92R7) were polymorphic in the analyzed populations, yielding six distinct SNP haplotypes. The microsatellite data were used to study the genetic structure of two major SNP haplotypes in the Finns and the Saami in more detail. We found that the most common haplotypes are shared between the Finns and the Saami, and that the SNP haplotypes show regional differences within the Finns and the Saami, which supports the hypothesis of two separate settlement waves to Finland.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-89696 (URN)10.1101/gr.156301 (DOI)11230171 (PubMedID)
Available from: 2002-03-14 Created: 2002-03-14 Last updated: 2017-12-14Bibliographically approved
3. Minisequencing on oligonucleotide microarrays: comparison of immobilisation chemistries
Open this publication in new window or tab >>Minisequencing on oligonucleotide microarrays: comparison of immobilisation chemistries
2001 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 29, no 13, p. e69-e69Article 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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-89697 (URN)11433045 (PubMedID)
Available from: 2002-03-14 Created: 2002-03-14 Last updated: 2017-12-14Bibliographically approved
4. Multiplex SNP genotyping in pooled DNA samples by a four-colour microarray system
Open this publication in new window or tab >>Multiplex SNP genotyping in pooled DNA samples by a four-colour microarray system
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2002 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 30, no 14, p. e70-Article in journal (Refereed) Published
Abstract [en]

We selected 125 candidate single nucleotide polymorphisms (SNPs) in genes belonging to the human type 1 interferon (IFN) gene family and the genes coding for proteins in the main type 1 IFN signalling pathway by screening databases and by in silico comparison of DNA sequences. Using quantitative analysis of pooled DNA samples by solid-phase mini-sequencing, we found that only 20% of the candidate SNPs were polymorphic in the Finnish and Swedish populations. To allow more effective validation of candidate SNPs, we developed a four-colour microarray-based mini-sequencing assay for multiplex, quantitative allele frequency determination in pooled DNA samples. We used cyclic mini-sequencing reactions with primers carrying 5'-tag sequences, followed by capture of the products on microarrays by hybridisation to complementary tag oligonucleotides. Standard curves prepared from mixtures of known amounts of SNP alleles demonstrate the applicability of the system to quantitative analysis, and showed that for about half of the tested SNPs the limit of detection for the minority allele was below 5%. The microarray-based genotyping system established here is universally applicable for genotyping and quantification of any SNP, and the validated system for SNPs in type 1 IFN-related genes should find many applications in genetic studies of this important immunoregulatory pathway.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-94313 (URN)10.1093/nar/gnf069 (DOI)12136118 (PubMedID)
Available from: 2006-04-20 Created: 2006-04-20 Last updated: 2017-12-14Bibliographically approved
5. Sex-linked speciation in Ficedula flycatchers
Open this publication in new window or tab >>Sex-linked speciation in Ficedula flycatchers
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Manuscript (Other academic)
Identifiers
urn:nbn:se:uu:diva-89699 (URN)
Available from: 2002-03-14 Created: 2002-03-14 Last updated: 2012-02-29

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