Identification of Differential Genes by Suppression Subtractive Hybridization: III. PCR Amplification of Differentially Presented DNAs
This protocol was adapted from “Identification of Differential Genes by Suppression Subtractive Hybridization,” Chapter 22, in PCR Primer, 2nd edition, (eds. Dieffenbach and Dveksler). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.INTRODUCTION
Suppression subtractive hybridization (SSH) is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. This technique can be used to compare two mRNA populations and obtain cDNAs representing genes that are either overexpressed or exclusively expressed in one population as compared to another. It can also be used for comparison of genomic DNA populations. In this protocol, differentially presented DNAs are selectively amplified using PCR. It is strongly recommended that subtractions be performed in both directions for each tester/driver DNA pair. Forward subtraction is designed to enrich for differentially presented molecules present in the tester but not in the driver; reverse subtraction is designed to enrich for differentially presented sequences present in the driver but not in the tester. Therefore, each experiment should have at least four reactions: (1) subtracted tester DNAs, (2) unsubtracted tester control, (3) reverse-subtracted tester DNAs, and (4) unsubtracted control for the reverse subtraction.
