chapter  24
11 Pages

Single-Molecule PCR—Basic Protocols and Applications

I. Introduction 195

II. Basic Tool Kit for Single-Molecule PCR 196

A. Expressing Amounts and Concentrations of DNA 196

B. Some Simple Statistics 196

C. Contamination Control 197

D. Preparation and Handling of Template DNA 197

E. Basic Single-Molecule Amplification by Nested Two-Phase PCR 197

F. Hemi-Nested Amplification 199

G. Highly Multiplex Single-Molecule PCR 199

H. PEP and Other Nonspecific Amplification Methods 200

III. Applications and Theory 201

A. Reference Reagents, Validation and Quantitation of Single-Molecule Amplification 201 1. Preparation of a Reference Template 201 2. Precise Quantitation of Reference Template 202 B. Determination of Copy Numbers 203

1. Results Expected and Interpretation 203

C. Happy Mapping 204

IV. Discussion and Further Applications of Single-Molecule PCR 204

References 204

I. INTRODUCTION

With the invention of the PCR1 in the late 1980s, molecular biologists were able to easily amplify a defined segment of DNA from a complex mixture for simple detection or for further analysis. Although Southern blotting, cloning, and other methods had been able to fulfill some of these roles for many years,2 PCR provided unparalleled speed, selectivity, and versatility. It quickly became apparent that it could achieve outstanding sensitivity too, amplifying chosen targets from minute samples of genomic DNA by virtually unlimited factors.3 This great sensitivity has made possible the fingerprinting of forensic samples containing only hundreds or tens of cells, the sequencing of the few surviving DNA molecules in ancient biological specimens, and the diagnosis of genetic abnormalities in biopsy samples or preimplantation embryos. Several of these applications are covered in other chapters in this book.