Applications of Four-Colour Fluorescent Primer Extension Technology for SNP Analysis and Discovery

Abstract: 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.