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Multiplex SNP genotyping in pooled DNA samples by a four-colour microarray system
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.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Molecular Medicine.
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2002 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 30, no 14, e70- p.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.

Place, publisher, year, edition, pages
2002. Vol. 30, no 14, e70- p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-94313DOI: 10.1093/nar/gnf069PubMedID: 12136118OAI: oai:DiVA.org:uu-94313DiVA: diva2:168122
Available from: 2006-04-20 Created: 2006-04-20 Last updated: 2016-08-11Bibliographically approved
In thesis
1. Large-Scale Genotyping for Analysis of the Type I Interferon System in Autoimmune Diseases
Open this publication in new window or tab >>Large-Scale Genotyping for Analysis of the Type I Interferon System in Autoimmune Diseases
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Single nucleotide polymorphisms (SNPs) are the most common form of genetic variation. We developed a novel multiplexed method for SNP genotyping based on four-color fluorophore tag-microarray minisequencing. This method allows simultaneous genotyping of 80 samples and up to 200 SNPs in any allele combination. In study I we set up the method for a panel of SNPs from genes in the type I interferon system, and applied it in study III. In study II we used the technique to genotype SNPs from the coding region of the mitochondrial genome. A panel of 150 SNPs was genotyped in 265 individuals representing nine different populations. We demonstrated that the multiplexed SNP genotyping method for mitochondrial DNA increases the power of forensic identification in combination with sequencing of the hypervariable region of mitochondrial DNA.

In study III we performed a genetic association study of SNPs in genes related to the type I Interferon system in Systemic Lupus Erythematosus (SLE). SLE is a chronic autoimmune inflammatory disease with a complex etiology. The SNPs were genotyped in DNA samples from Swedish, Finnish, and Icelandic patients with SLE, unaffected family members, and unrelated controls. The analysis identified SNPs in two genes, the tyrosine kinase 2 (TYK2) and interferon regulatory factor 5 (IRF5) genes that are highly associated with SLE with p-values <10-7 for joint linkage and association.

Study IV describes the analysis of the TYK2 and IRF5 SNPs in a large Rheumatoid Arthritis (RA) sample cohort. We found that SNPs in the IRF5 gene were significantly associated with RA with a p-value = 0.00008. In contrast, we did not detect an association with SNPs in the TYK2 gene. These findings demonstrate that SLE and RA may have a common genetic background in the case of IRF5, while the TYK2 variants appear to be unique for SLE.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 82 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 138
Keyword
Molecular medicine, Minisequencing, SNP, Microarray, Polymorphism, Mitochondria, Systemic Lupus Erythematosus, Rheumatoid Arthritis, Association study, Type I interferon, Genotyping, Molekylärmedicin
Identifiers
urn:nbn:se:uu:diva-6792 (URN)91-554-6532-3 (ISBN)
Public defence
2006-05-11, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjöldsväg 20, Uppsala, 13:15
Opponent
Supervisors
Available from: 2006-04-20 Created: 2006-04-20Bibliographically approved
2. 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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