Suppression+subtractive+hybridization+(SSH)

=__**Suppression Subtractive Hybridization (SSH)**__= by Matt Connolly

**Basic Description**
In higher eukaryotes many processes are regulated by differential gene expression, such as different genes being expressed in different cell types in a single organism. To study this regulation, subsets of differentially expressed genes must be identified. This is where suppression subtractive hybridization, or SSH, comes in. SSH is a technique that is now commonly used in cellular biology and biochemistry. This is a PCR-based cDNA subtraction method used to identify genes that are differentially expressed between two samples. A representation of the SSH method is shown in figure 1. In SSH there is a “tester” sample and a “driver” sample. The tester sample has the differentially expressed cDNAs while the driver sample does not. First the tester and driver ds (double strand) cDNAs are digested with four-base restriction enzyme that creates blunt ends. The tester cDNA is then divided into two samples (1 and 2) and a different adapter is added to each sample [3]. Figure 1. A representation of SSH [3]

The samples then enter the first of two hybridizations. First excess driver is added to the samples, then they are heat denatured and allowed to anneal. In this process the ss (single strand) cDNAs of the tester sample (A) are greatly enriched for differentially expressed genes because comm on cDNAs between the tester and driver samples form ds cDNAs heterohybrids (C). The samples then enter the second hybridization process, where the two samples are mixed together. The addition of more denatured driver in this stage further enriches ss cDNAs with differential expressed genes. In this stage remaining ss cDNAs are able to reassociate and for (B), (C), and new hybrids (E). The new (E) hybrids have different adapter sequences at their 5’ ends, one adaptor from sample 1 and one from sample 2. This is important because it distinguishes them from hybrids (B) and (C). This now allows for preferential amplification of the new hybrid (E), using PCR and a pair of primers (P1 and P2) that correspond to the outer part of the adapters (adaptor 1 and 2). Selective amplification is accomplished with a reaction that fills the sticky ends of the molecules before PCR is started [3].

Now, in all of the PCR cycles, amplification can only occur in the type (E) molecules. Type (B) molecules form panhandle-like structures that cannot serve as a template for PCR. Type (A) and (D) molecules do not have primer-binding sites, and type (C) molecules can only be amplified at a linear rate. Type (E) molecules have different adaptor sequences on their ends, which allows them to be amplified at an exponential rate. This is important because type (E) molecules are the differentially expressed genes, allowing them to be isolated from all of the common cDNA [3].

Purpose of Technique
The purpose of this technique is to test two samples to find out what genes are expressed in the tester sample, but not in the driver sample. SSH is able to produce very valuable data that allows for comparison between two DNA populations, showing which genes are differentially expressed. SSH is able to generate organ specific cDNA libraries, which is invaluable data for modern day genetics and cellular biology [3].

Origin and History
This technique was developed by several individuals and was released to the world in a paper in 1996. The paper was published to the //Proceedings of the National Academy of Sciences//, is titled “Suppression subtractive hybridization: A method for generating differentially regulated or tissue-specific cDNA probes and libraries,” and was written by Diatchenko et al. Before SSH, there were several older subtraction methods, but they all required a relatively large amount of RNA, involved multiple subtraction steps and were labor intensive. SSH over came these problems, providing a simpler and more efficient subtraction method [3].

Recent Research
Van der Nest et al., authors of “Gene expression associated with vegetative incompatibility in //Amylostereum areolatum”// used SSH to determine genes that were expressed in //A. areolatum// undergoing vegetative incompatibility versus samples of the compatible fungi interactions. The use of SSH in this experiment allowed made it possible to identify numerous genes that are associated with fungal non-self recognition. Many of these genes are implicated in hyphal fusion, stress and defense responses, programmed cell death, and signaling pathways. This study was the first study to identify genes involved in vegetative incompatibility in Basidiomycetes, and it would not have been possible without SSH [4].

Wang et al. used SSH to create a cDNA library for drought tolerant cotton. This was used to help understand the expression of genes induced by drought. This analysis found many drought induced genes, including genes involved in signal transduction, energy metabolism, protein metabolism, nucleic acid metabolism, photosynthesis, and transmembrane transport. All and all, this experiment was able to create a SSH library for drought resistance, which will help future genetic engineering of drought resistant plants [1].

Li et al. used SSH to create a cDNA library of fertility related genes of sterile male wheat. This experiment was able to identify multiple genes related to male sterility by comparing gene expression in sterile and fertile cDNA libraries obtained by SSH. This now gives some explanation to the molecular mechanism of male sterility [2].