Insufficient therapeutic effectiveness or unanticipated side effects of drugs shown to be safe and effective in clinical trials is a considerable problem for health care practitioners. Individual response to drugs and susceptibility to disease are assumed to be associated with single nucleotide polymorphisms (SNPs) whose interaction and grouping into a few common haplotypes are predictive of gene function and can have more predictive power than the total genotype or any individual SNP. To move toward making medicine more individualistic, an innovative method for intramolecular amplification over large distances, beginning at a chosen SNP, has been proposed. Current techniques for molecular haplotyping are only practical for short regions (2-5 kb) and would be difficult to automate. The proposed innovative technique answers the challenge of high-throughput, long-distance haplotyping. This is achieved by circularizing genomic DNA molecules to create junctions between distal sequences and isolating small, amplifiable fragments of the circular molecule harboring the junction.
The specific aims are to 1) create a genomic library of high molecular weight DNA molecules, 2) devise a technique to join the ends of the high molecular weight DNA molecules, 3) develop a technique to isolate from the circular molecule short, amplifiable DNA molecules harboring the junction of the ends, 4) develop a method to amplify these short molecules, 5) develop a method to amplify these molecules from specific regions of the genome in an allele-independent manner, 6) and an allele-specific manner, and 7) test the ability of one or more SN P-detection methods to detect amplified alleles. Phase II will consist of the development and testing of the haplotype technique, the scaling of the process to high-throughput, and the proof-of-principle in a demonstration project to ensure robustness. This addresses important marketplace needs of pharmacogenomics as allele-specific template would be provided for haplotyping endeavors associated with clinical trials of drugs. The proposed technique provides allele-specific template of large regions of DNA. By scaling the technology to high-throughput, it is anticipated to result in cost reduction of haplotyping in personalized medicine endeavors.
