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Analysis of Nucleotide Variations in Non-human Primates
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Molecular Medicine.
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Many of our closest relatives, the primates, are endangered and could be extinct in a near future. To increase the knowledge of non-human primate genomes, and at the same time acquire information on our own genomic evolution, studies using high-throughput technologies are applied, which raises the demand for large amounts of high quality DNA.

In study I and II, we evaluated the multiple displacement amplification (MDA) technique, a whole genome amplification method, on a wide range of DNA sources, such as blood, hair and semen, by comparing MDA products to genomic DNA as templates for several commonly used genotyping methods. In general, the genotyping success rate from the MDA products was in concordance with the genomic DNA. The quality of sequences of the mitochondrial control region obtained from MDA products from blood and non-invasively collected semen samples was maintained. However, the readable sequence length was shorter for MDA products.

Few studies have focused on the genetic variation in the nuclear genes of non-human primates. In study III, we discovered 23 new single nucleotide polymorphisms (SNPs) in the Y-chromosome of the chimpanzee. We designed a tag-microarray minisequencing assay for genotyping the SNPs together with 19 SNPs from the literature and 45 SNPs in the mitochondrial DNA. Using the microarray, we were able to analyze the population structure of wild-living chimpanzees.

In study IV, we established 111 diagnostic nucleotide positions for primate genera determination. We used sequence alignments of the nuclear epsilon globin gene and apolipoprotein B gene to identify positions for determination on the infraorder and Catarrhini subfamily level, respectively, and sequence alignments of the mitochondrial 12S rRNA (MT-RNR1) to identify positions to distinguish between genera. We designed a microarray assay for immobilized minisequencing primers for genotyping these positions to aid in the forensic determination of an unknown sample.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2007. , p. 41
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 263
Keywords [en]
Molecular genetics, SNP, genotyping, primate, chimpanzee, whole genome amplification, multiple displacement amplification, minisequencing, microarray, Y-chromosome, mitochondria
Keywords [sv]
Genetik
Identifiers
URN: urn:nbn:se:uu:diva-7904ISBN: 978-91-554-6904-7 (print)OAI: oai:DiVA.org:uu-7904DiVA, id: diva2:170322
Public defence
2007-06-01, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 13:15
Opponent
Supervisors
Available from: 2007-05-10 Created: 2007-05-10 Last updated: 2013-09-25Bibliographically approved
List of papers
1. Multiple displacement amplification for generating an unlimited source of DNA for genotyping in nonhuman primate species
Open this publication in new window or tab >>Multiple displacement amplification for generating an unlimited source of DNA for genotyping in nonhuman primate species
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2006 (English)In: International journal of primatology, ISSN 0164-0291, E-ISSN 1573-8604, Vol. 27, no 4, p. 1145-1169Article in journal (Refereed) Published
Abstract [en]

We evaluated a whole genome amplification method-multiple displacement amplification (MDA)-as a means to conserve valuable nonhuman primate samples. We tested 148 samples from a variety of species and sample sources, including blood, tissue, cell-lines, plucked hair and noninvasively collected semen. To evaluate genotyping success and accuracy of MDA, we used routine genotyping methods, including short tandem repeat (STR) analysis, denaturing gradient gel electrophoresis (DGGE), Alu repeat analysis, direct sequencing, and nucleotide detection by tag-array minisequencing. We compared genotyping results from MDA products to genotypes generated from the original (non-MD amplified) DNA samples. All genotyping methods showed good results with the MDA products as a DNA template, and for some samples MDA improved genotyping success. We show that the MDA procedure has the potential to provide a long-lasting source of DNA for genetic studies, which would be highly valuable for the primate research field, in which genetic resources are limited and for other species in which similar sampling constraints apply.

Keywords
Alu-SINE, minisequencing, multiple displacement amplification, short tandem repeat, single nucleotide polymorphism
National Category
Medical and Health Sciences Biological Sciences
Identifiers
urn:nbn:se:uu:diva-95932 (URN)10.1007/s10764-006-9067-7 (DOI)000241400100012 ()
Available from: 2007-05-10 Created: 2007-05-10 Last updated: 2017-12-14Bibliographically approved
2. Sequence quality is maintained after multiple displacement amplification of non-invasively obtained macaque semen DNA
Open this publication in new window or tab >>Sequence quality is maintained after multiple displacement amplification of non-invasively obtained macaque semen DNA
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2006 In: Biotechnology Journal, ISSN 1860-7314, Vol. 1, no 4, p. 466-469Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-95933 (URN)
Available from: 2007-05-10 Created: 2007-05-10Bibliographically approved
3. A microarray system for Y chromosomal and mitochondrial single nucleotide polymorphism analysis in chimpanzee populations
Open this publication in new window or tab >>A microarray system for Y chromosomal and mitochondrial single nucleotide polymorphism analysis in chimpanzee populations
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2008 (English)In: Molecular Ecology Notes, ISSN 1471-8278, E-ISSN 1471-8286, Vol. 8, no 3, p. 529-539Article in journal (Refereed) Published
Abstract [en]

Chimpanzee populations are diminishing as a consequence of human activities, and as a result this species is now endangered. In the context of conservation programmes, genetic data can add vital information, for instance on the genetic diversity and structure of threatened populations. Single nucleotide polymorphisms (SNP) are biallelic markers that are widely used in human molecular studies and can be implemented in efficient microarray systems. This technology offers the potential of robust, multiplexed SNP genotyping at low reagent cost in other organisms than humans, but it is not commonly used yet in wild population studies. Here, we describe the characterization of new SNPs in Y-chromosomal intronic regions in chimpanzees and also identify SNPs from mitochondrial genes, with the aim of developing a microarray system that permits the simultaneous study of both paternal and maternal lineages. Our system consists of 42 SNPs for the Y chromosome and 45 SNPs for the mitochondrial genome. We demonstrate the applicability of this microarray in a captive population where genotypes accurately reflected its large pedigree. Two wild-living populations were also analysed and the results show that the microarray will be a useful tool alongside microsatellite markers, since it supplies complementary information about population structure and ecology. SNP genotyping using microarray technology, therefore, is a promising approach and may become an essential tool in conservation genetics to help in the management and study of captive and wild-living populations. Moreover, microarrays that combine SNPs from different genomic regions could replace microsatellite typing in the future.

Keywords
chimpanzee, conservation genetics, microarray, minisequencing, population genetics, sex-linked SNPs
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-110437 (URN)10.1111/j.1471-8286.2007.02000.x (DOI)000254810300007 ()
Available from: 2009-11-16 Created: 2009-11-16 Last updated: 2017-12-12Bibliographically approved
4. A microarray-system for forensic identification of primate species subject to bushmeat trade
Open this publication in new window or tab >>A microarray-system for forensic identification of primate species subject to bushmeat trade
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(English)Manuscript (Other academic)
Identifiers
urn:nbn:se:uu:diva-95935 (URN)10.3354/esr00191 (DOI)
Available from: 2007-05-10 Created: 2007-05-10 Last updated: 2012-02-29

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