Identification of Differential Genes by Suppression Subtractive Hybridization: VI. Differential Hybridization with Tester and Driver DNA Probes
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. This protocol describes a method for screening a subtracted cDNA library by differential hybridization using (1) a tester-specific subtracted probe (forward-subtracted probe), (2) a driver-specific subtracted probe (reverse-subtracted probe), (3) a cDNA probe synthesized directly from tester mRNA (or tester genomic DNA), and (4) a cDNA probe synthesized directly from driver mRNA (or driver genomic DNA).
