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Applications of Four-Colour Fluorescent Primer Extension Technology for SNP Analysis and Discovery
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Molecular Medicine.
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Studies on genetic variation can reveal effects on traits and disease, both in humans and in model organisms. Good technology for the analysis of DNA sequence variations is critical. Currently the development towards assays for large-scale and parallel DNA sequencing and genotyping is progressing rapidly. Single base primer extension (SBE) is a robust reaction principle based on four-colour fluorescent terminating nucleotides to interrogate all four DNA nucleotides in a single reaction. In this thesis, SBE methods were applied to the analysis and discovery of single nucleotide polymorphism (SNP) in the model organism Drosophila melanogaster and in humans.

The tag-array minisequencing system in a microarray format is convenient for intermediate sized genotyping projects. The system is scalable and flexible to adapt to specialized and novel applications. In Study I of the thesis a tool was established to automate quality control of clustered genotype data. By calculating “Silhouette scores”, the SNP genotype assignment can be evaluated by a single numeric measure. Silhouette scores were then applied in Study I to compare the performance of four DNA polymerases and in Study III to evaluate freeze-dried reagents in the tag-array minisequencing system.

The characteristics of the tag-array minisequencing system makes it suitable for inexpensive genome-wide gene mapping in the fruit fly. In Study II a high-resolution SNP map, and 293 genotyping assays, were established across the X, 2nd and 3rd chromosomes to distinguish commonly used Drosophila strains. A database of the SNP markers and a program for automatic allele calling and identification of map positions of mutants was also developed. The utility of the system was demonstrated by rapid mapping of 14 genes that disrupt embryonic muscle patterning.

In Study III the tag-array minisequencing system was adapted to a lab-on-a-chip format for diagnostic testing for mutations in the TP53 gene. Freeze-drying was evaluated for storing reagents, including thermo-sensitive enzymes, on the microchip to reduce the complexity of the integrated test. Correct genotyping results were obtained using freeze-dried reagents in each reaction step of the genotyping protocol, both in test tubes and in single polymer test chambers. The results showed the potential of the approach to be implemented in fully integrated systems.

The four-colour chemistry of SBE has been developed further to allow massively parallel sequencing (MPS) of short DNA fragments as in the Genome Analyzer system (Solexa/Illumina). In Study IV MPS was used to compare Nimblegen arrays and the SureSelect solution-based system for targeted enrichment of 56 continuous human candidate-gene regions totalling 3.1 Mb in size. Both methods detected known SNPs and discovered novel SNPs in the target regions, demonstrating the feasibility for complexity reduction of sequencing libraries by hybridization methods.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2010. , p. 72
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 581
Keywords [en]
Single nucleotide polymorphism, Genotyping, Massively parallel sequencing, Gene mapping, Lab-on-a-chip
National Category
Medical Genetics Cell and Molecular Biology
Research subject
Molecular Medicine
Identifiers
URN: urn:nbn:se:uu:diva-129221ISBN: 978-91-554-7855-1 (print)OAI: oai:DiVA.org:uu-129221DiVA, id: diva2:343045
Public defence
2010-09-24, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2010-09-03 Created: 2010-08-09 Last updated: 2018-01-12Bibliographically approved
List of papers
1.
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2. High-resolution, high-throughput SNP mapping in Drosophila melanogaster
Open this publication in new window or tab >>High-resolution, high-throughput SNP mapping in Drosophila melanogaster
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2008 (English)In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 5, no 4, p. 323-329Article in journal (Refereed) Published
Abstract [en]

Single nucleotide polymorphisms (SNPs) are useful markers for genetic mapping experiments in model organisms. Here we report the establishment of a high-density SNP map and high-throughput genotyping assays for Drosophila melanogaster. Our map comprises 27,367 SNPs in common laboratory Drosophila stocks. These SNPs were clustered within 2,238 amplifiable markers at an average density of 1 marker every 50.3 kb, or 6.3 genes. We have also constructed a set of 62 Drosophila stocks, each of which facilitates the generation of recombinants within a defined genetic interval of 1-2 Mb. For flexible, high-throughput SNP genotyping, we used fluorescent tag-array mini-sequencing (TAMS) assays. We designed and validated TAMS assays for 293 SNPs at an average resolution of 391.3 kb, and demonstrated the utility of these tools by rapidly mapping 14 mutations that disrupt embryonic muscle patterning. These resources enable high-resolution high-throughput genetic mapping in Drosophila.

Keywords
Animals, Chromosome Mapping, Drosophila melanogaster/embryology/*genetics, Genome; Insect, Muscle Development/*genetics, Mutation, Polymorphism; Single Nucleotide
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-16561 (URN)10.1038/nmeth.1191 (DOI)000254559400019 ()18327265 (PubMedID)
Available from: 2008-05-28 Created: 2008-05-28 Last updated: 2017-12-08Bibliographically approved
3. Positional cloning by fast-track SNP-mapping in Drosophila melanogaster
Open this publication in new window or tab >>Positional cloning by fast-track SNP-mapping in Drosophila melanogaster
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2008 (English)In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 3, no 11, p. 1751-1765Article in journal (Refereed) Published
Abstract [en]

Positional cloning of chemically induced mutations is the rate-limiting step in forward genetic screens in Drosophila. Single-nucleotide polymorphisms (SNPs) are useful markers to locate a mutated region in the genome. Here, we provide a protocol for high-throughput, high-resolution SNP mapping that enables rapid and cost-effective positional cloning in Drosophila. In stage 1 of the protocol, we use highly multiplexed tag-array mini-sequencing assays to map mutations to an interval of 1-2 Mb. In these assays, SNPs are genotyped by primer extension using fluorescently labeled dideoxy-nucleotides. Fluorescent primers are captured and detected on a microarray. In stage 2, we selectively isolate recombinants within the identified 1-2 Mb interval for fine mapping of mutations to about 50 kb. We have previously demonstrated the applicability of this protocol by mapping 14 muscle morphogenesis mutants within 4 months, which represents a significant acceleration compared with other commonly used mapping strategies that may take years.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-98390 (URN)10.1038/nprot.2008.175 (DOI)000265781600008 ()18948975 (PubMedID)
Available from: 2009-02-20 Created: 2009-02-20 Last updated: 2017-12-13Bibliographically approved
4. Dried reagents for multiplex genotyping by tag-array minisequencing to be used in microfluidic devices
Open this publication in new window or tab >>Dried reagents for multiplex genotyping by tag-array minisequencing to be used in microfluidic devices
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2010 (English)In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 135, no 9, p. 2377-2385Article in journal (Refereed) Published
Abstract [en]

We present an optimized procedure for freeze-drying and storing reagents for multiplex PCR followed by genotyping using a tag-array minisequencing assay with four color fluorescence detection which is suitable for microfluidic assay formats. A test panel was established for five cancer mutations in three codons (175, 248 and 273) of the tumor protein gene (TP53) and for 13 common single nucleotide polymorphisms (SNPs) in the TP53 gene. The activity of DNA polymerase was preserved for six months of storage after freeze-drying, and the half-life of activities of exonuclease I and shrimp alkaline phosphatase were estimated to 55 and 200 days, respectively. We conducted a systematic genotyping comparison using freeze-dried and liquid reagents. The accuracy of successful genotyping was 99.1% using freeze-dried reagents compared to liquid reagents. As a proof of concept, the genotyping protocol was carried out with freeze-dried reagents stored in reaction chambers fabricated by micromilling in a cyclic olefin copolymer substrate. The results reported in this study are a key step towards the development of an integrated microfluidic device for point-of-care DNA-based diagnostics.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-129216 (URN)10.1039/c0an00321b (DOI)000281007300027 ()20668755 (PubMedID)
Available from: 2010-08-09 Created: 2010-08-09 Last updated: 2017-12-12Bibliographically approved
5.
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