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
