A Novel and High Throughput Approach for SNP Screening, Scoring and Association Analysis using Genomic or cDNA Pools

Xinghua Pan, Sherman M. Weissman
Department of Genetics
Boyer Center for Molecular Medicine
Yale University School of Medicine
295 Congress Ave., BCMM 331
and Molecular Staging, Inc.
New Haven, Connecticut 06510, USA
telephone: (203)737-2277
fax: (203)737-2286
email: xhpan@biomed.med.yale.edu
prestype: Platform
presenter: Xinghua Pan

Xinghua Pan1,2 and Sherman M. Weissman1
1Department of Genetics, Boyer Center for Molecular Medicine, Yale University School of Medicine, 295 Congress Ave., BCMM 331
2Molecular Staging, Inc., New Haven, Connecticut 06510, USA.

SNP association studies may be the most powerful approach for identifying the genetic determinants of common disease. Developing a very high throughput and robust approach for screening/scoring the SNPs within one pool, and identifying the differences in SNP quality and quantity between two pools of human DNA samples is a critical enterprise in human genetics. Current techniques for SNPs analysis for the most part rely on discriminating the single base difference (allele) of each SNP with previously known sequence, and on typing all of a panel of DNAs with a limited number of candidate SNPs. These limitations seriously hinder complex trait mapping.

We have been developing an alternative approach that potentially allows one to map all SNPs in a pool of DNA samples or between 2 pools of DNA samples in one procedure. First we denature and reanneal DNA fragments and separate the SNP-containing DNA fragment pool from the non-SNP-containing DNA fragment pool. We then propose to apply these two pools in-parallel to oligonucleotide microarrays, gel display or other techniques to determine the relative allele frequencies within a given sample pool or between the tested sample and its control. Alternatively, another approach, SNPs - Representation Differential Analysis (SNPs-RDA) strategy may be used to distinguish the neutral polymorphism of SNPs from the SNP's in strong linkage disequilibrium with a monogenic trait. MFISH on metaphase chromosomes with the SNP fragment pool is proposed for the physical mapping of a complex trait, besides its genetic mapping. A highly specific as well as highly sensitive technique for separating SNP fragments from non-SNP fragments with immobilized DNA glycosylases has been established. With this tool, 250-500 folds of enrichment for fragments containing a single nucleotide mismatch, or for perfectly matched fragments, was obtained after one cycle of treatment. An efficient technique for reducing a complex DNA pool into full coverage and non-overlapping subsets with different sequences at the two ends of the subset fragments has been developed. A strategy for selective recovery of either one of the homphybrids or the heterohybrids from a mixture of denatured and reannealed DNA samples was also designed. As a first test, we are employing this approach in cDNA pool of a lymphoblast cell line. Initial data will be discussed.

The present approach has the potential to provide a simple, accurate, high throughput and efficient flexible platform for new SNPs discovery and identification, for SNP screening/scoring, and most importantly for whole genome/ cDNA SNP association of a given trait.

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