chapter  27
8 Pages

Applications of Differential Display in Infectious Diseases

I. Introduction 220

A. Definition and Theoretical Aspects 220

B. Applications in the Area of Infectious Diseases 221

C. Optimization of DD 222

II. Materials and Methods 223

A. Preparation of Total Cellular RNA 223

B. Reverse Transcription 223

C. Differential Display and Isolation of DNA Bands for Sequence Analysis 223

D. Identification of Genes 223

E. Semiquantitative RT-PCR and Real-Time Sybr Greenö RT - PCR 224

III. Differentially Displayed Genes During Dengue Virus Infection 224

IV Conclusions 224

Acknowledgments 225

References 226

I. INTRODUCTION

A. DEFINITION AND THEORETICAL ASPECTS

Differential display (DD) is a powerful method used to detect gene expression in a number of biological systems to distinguish differential expression of mRNAs. DD results are easily reproduced, do not require enzymatic restriction, enzyme digestions, and ligations or cloning, and require only very small amounts of starting total RNA (from 20 ng to 200 ng) which results in a less labor-intense method when compared with similar approaches.1-3 Genefragments from differentially expressed genes can be excised from the DD gel, identified, and used to prepare gene tags for the study of gene expression levels, using semiquantitative and quantitative PCR.