Suppressor-Subtractive Hybridization (SSH) Library Construction to study gene expression differences (CAT#: STEM-MB-0020-WXH)

Introduction

Subtractive hybridization is an approach that allows comparison of two DNA populations and isolation of a fraction enriched in differentially distributed molecules. Subtractive hybridization is usually employed to identify genes with a differential expression pattern, in particular genes involved in the regulation of basic biological processes.
Suppression Subtractive Hybridization (SSH) is the best known and most utilized subtraction method. It is based on the PCR suppression by inverted terminal repeats (PS-effect). Upon initiation of PCR after the denaturation phase, the single-stranded (ss) DNA fragments flanked by inverted terminal repeats (ITR) may form either self-annealing "pan-like" structures (preventing the primer binding to its complementary binding sites and suppressing the PCR), or DNA/primer hybrid structures. In the latter case, if the primer corresponding to the outer part of ITR is used, the DNA synthesis by Taq-polymerase restores the original structure, ensuring the persistence of suppression during further PCR cycles. In complex mixtures, the use of PCR suppression allows selective amplification of molecules that are flanked by different adapters at opposing termini (asymmetrically flanked molecules).