Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries.

TLDR

The method builds on suppression PCR, integrating normalization and subtraction into a single procedure. The authors developed suppression subtractive hybridization (SSH) to generate subtracted cDNA libraries. SSH normalizes cDNA abundance and subtracts common sequences, then uses the resulting library to create a testis‑specific cDNA set and as a probe to locate homologous sequences in a human Y‑chromosome cosmid library. SSH enriched rare transcripts over 1,000‑fold, produced a testis‑specific cDNA library, identified testis‑specific Y‑chromosome cosmid inserts, and demonstrates broad applicability for locating differentially expressed genes.

Abstract

A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a recently described technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. In a model system, the SSH technique enriched for rare sequences over 1,000-fold in one round of subtractive hybridization. We demonstrate its usefulness by generating a testis-specific cDNA library and by using the subtracted cDNA mixture as a hybridization probe to identify homologous sequences in a human Y chromosome cosmid library. The human DNA inserts in the isolated cosmids were further confirmed to be expressed in a testis-specific manner. These results suggest that the SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes.