chapter  33
8 Pages

Cloning of Unknown Virus Sequences by DNase Treatment and SequenceIndependent Single Primer Amplification

I. Introduction 267

II. Example 269

A. Key Reagents 269

1. Adapters 269

2. Enzymes 269

B. Experimental Protocol 270

1. Sample Pretreatment 270

2. DNA Protocol: Extraction and Synthesis of a Second Strand of DNA 270 3. RNA Protocol: Extraction and cDNA Synthesis 270

4. Restriction Digestion and Amplification 271 5. Analysis of PCR Products 272 6. Sequence Analysis 272

C. Interpretation of Data 272

1. Contamination 272

2. Nonviral sequences 273

3. Viral Sequences 273

4. Sequences with No Homology to Database Sequences 273

5. Disease Association 273

III. Discussion 273

References 274

I. INTRODUCTION

For many human diseases infection by unrecognized viruses is a possible etiology. Moreover, the identification of new viral species is important for understanding our normal physiology and microbiological environment. Traditionally, tissue culture has been the basic tool for isolation and characterization of viruses. However, many viruses are difficult or impossible to grow in cell culture.