Research Project
7157108
[unreadable] DESCRIPTION (provided by applicant): Project Summary/Abstract: a) We propose to develop an economical, automated system (BioSprint96) for preparing sub-megabase (250-500kb), and preferably megabase-size genomic DNA from blood or buccal swabs. b) Similar protocols on the same system will enable the targeted extraction of specific, haploid gene regions through Haplotype-Specific Extraction (HSE). This project will result in the development of new protocols for DNA sample preparation on existing instrumentation that significantly improve the length of extracted DNA compared to current methods. The proposed process is expected to be significantly faster and more cost effective than existing methods for the isolation of high molecular weight DNA and sample preparation for individual haplotyping. The completion of the project will result in a generic DNA isolation and molecular haplotype preparation technology with broad applicability in biomedical research, such as association studies, disease prognosis, pharmacogenomics, oncology testing and genomics in general. We will use specific probes and magnetic beads to separate sub-megabase and megabase diploid, genomic DNA into its haploid components based on signature sequences and polymorphic components as small as single nucleotides (SNPs). These very-long-strand, haploid genomic DNA preparations can then directly be used in conventional genotyping processes such as real-time PCR, sequencing or array-based SNP analyses to determine long range molecular haplotype data. We will develop kits for up to 3 common applications. Project Narrative: The goal of the current project of isolating large DNA fragments is to produce extended, haploid gene regions of unaltered genomic DNA. Since the method works on unamplified, genomic DNA of individual patients, it retains the DNA fragments in their original state, for instance with their epigenetic methylation pattern preserved, and does not require statistical inference to derive haplotypes. This will provide an improved tool to characterize potentially complex genetic signatures for organ transplantation (such as genes of the Major Histocompatibility Complex, MHC, and Killer Immunoglobulin-like Receptor, KIR, loci) and genetic disorders. By isolating and separating large regions of DNA into its parental fractions, unique genetic differences contributed by each parent can be used to try and link complex genetic disorders to particular allele combinations or haplotypes. This understanding should lead to a better understanding of the etiology of genetic disorders, which should ultimately result in better treatments and preventive care. [unreadable] [unreadable] [unreadable]
