chapter  36
9 Pages

In Vitro Evolution through PCR-Mediated Mutagenesis and Recombination

I. Introduction 293

A. Mutagenic PCR 294

B. In Vitro Recombination via DNA Shuffling 294

II. Materials And Methods 296

A. Mutagenic PCR 296

1. Mutagenesis by Polymerization in the Presence of Unbalanced dNTP Concentrations and Manganese 296 2. Mutagenesis via the Incorporation of Modified Nucleotides 298 B. In Vitro Recombination via DNA Shuffling 298

1. Gene Fragmentation via DNase I Digestion 298

2. Reassembly of DNA Fragments by PCR 299 3. Amplification of Fragment Libraries 299

III. Results and Discussion 300

Acknowledgments 300

References 300

I. INTRODUCTION

Natural evolution occurs via two major mechanisms: random mutation followed by selection and recombination of the selected mutations. In vitro evolution has frequently proven to be an excellent tool for engineering functional nucleic acids and proteins with optimized or novel properties, and it should come as no surprise that laboratory techniques rely on the same basic mechanisms as does nature. However, because of the control which can be exercised over DNA replication in vitro, the number and type of mutations that can be introduced and recombined vastly exceeds the number typically seen during natural selection. The laboratory evolution of functional nucleic acids and proteins is correspondingly much faster than in nature. An excellent demonstration of this was the evolution of antibiotic resistance elements.1 While many of the same resistance mutations were found following “test tube” evolution, as had previously been observed in nature, the mutations arose much more quickly and were recombined into one highly resistant gene within just a few generations.