SUBTRACTIVE CLONING:Past, Present, and Future

2010-03-05 13:21 · Byron

C. G. Sagerstr¨om,1 B. I. Sun,1 and H. L. Sive2 Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge,Massachusetts

C. G. Sagerstr¨om,1 B. I. Sun,1 and H. L. Sive2

Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge,Massachusetts 02142

KEY WORDS: differential gene representation, subtraction, positive selection, hybridization,reassociation kinetics

ABSTRACT Subtractive cloning is a powerful technique for isolating genes expressed or present in one cell population but not in another. This method and a related one termed positive selection have their origins in nucleic acid reassociation techniques. We discuss the history of subtractive techniques, and fundamental information about the nucleic acid composition of cells that came out of reassociation analyses. We then explore current techniques for subtractive cloning and positive selection, discussing the merits of each. These techniques include cDNA library–based techniques and PCR-based techniques. Finally, we briefly discuss the future of subtractive cloning and new approaches that may augment or supersede current methods.

CONTENTS

INTRODUCTION:752

The Basic Idea:752

THE PAST—ORIGINS AND APPLICATIONS OF REASSOCIATION ANALYSIS 753

Origins:754

Factors Affecting Reassociation Rate:754

Equations Describing Driver Excess Hybridizations:756

Measuring Reannealing: : 756

Useful Information Derived from Hybridization Kinetics757

Relevance of Renaturation Studies for Subtractive Cloning 762

THE PRESENT—USEFUL TECHNIQUES: 762

Strategies for Subtraction763

Tracer and Driver Preparation: : 763

The Hybridization Step: : 765

The Subtraction Step: Removing Driver-Tracer Hybrids and Excess Driver : 765

Positive Selection : 769

Monitoring Subtraction and Isolating Differentially Represented Genes 773

Choosing a Subtraction Method : 775

Success Stories : 775

Comparing Subtraction, Positive Selection, and Other Methods for Isolating

Differentially Represented Genes 776

THE FUTURE—EASIER TECHNIQUES? : 777

Problems to Overcome : 777

Solutions : 778

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