chapter  16
10 Pages

Global Analysis of DNA Allelic Variations (GADAV) by Specific Enrichment of Mismatches and Selective Amplification of Heterohybrids

I. Introduction 130

A. Background and Aims 130

B. Outline of the GADAV Strategy 130

II. Materials and Methods 131

A. Materials 131

1. Immobilized hTDG and mTDG 131

2. DNA Templates and Mm and Pm Oligonucleotide Duplexes 131

3. Oligonucleotides for Adapters and Primers 131 4. Other Reagents and Instruments 132

B. Experimental Protocol 133

1. Generation of Heterohybrids and Homohybrids from Two DNA Pools 133 2. Enrichment and Separation of Mm Fragments from Pm Fragments 133 3. Selective Amplification of Heterohybrids from a Mixture of DNA Fragments 134 4. Demonstration and Analysis of the Selected Fragments 134

III. Results 134

A. Enrichment of an Mm DNA Duplex from Digested pBR322 Pm Fragments 134

B. Scanning the Polymorphic Fragments in a Human cDNA Pool 135

IV. Discussion 136

A. Future Developments and Potential Applications 136

B. Summary 137

Acknowledgments 137

References 137

I. INTRODUCTION

A. BACKGROUND AND AIMS

Understanding the associations between genetic and phenotypic variation has been a major task of modern molecular biology.1-5 As an important part of this process a number of methods have been

developed either for rapid detection of known variation in DNA sequence or for detection of previously unappreciated sequence variation.6-8 The latter category of approaches has been difficult to apply to large complex DNA pools on a global scale. E. coli MutHLS proteins have been used to study variations via Identity-by-Descent (IBD) in S. cerevisiae, mouse, and human genomes.9-11 However, we and others have not obtained sufficient specificity with these enzymes to detect single nucleotide mutations consistently. Another method, representative differential analysis (RDA), has been successfully used in scanning short or long fragment deletions12,13 but it isgenerally not sensitive to subtle nucleotide variation.