Suppression Subtractive Hybridization
Suppression subtractive hybridization (SSH) is a widely used method for separating DNA molecules that distinguish two closely related DNA samples. Two of the main SSH applications are cDNA subtraction and genomic DNA subtraction. To our knowledge, SSH is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. It is based primarily on a suppression polymerase chain reaction (PCR) technique (described narrowly in Chapter 3) and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of DNA fragments within the target population, and the subtraction step excludes sequences that are common to the populations being compared. This dramatically increases the probability of obtaining low-abundance differentially expressed cDNAs or genomic DNA fragments and simplifies analysis of the subtracted library. SSH technique is applicable to many comparative and functional genetic studies for the identification of disease, developmental, tissuespecific, or other differentially expressed genes, as well as for the recovery of genomic DNA fragments distinguishing the samples under comparison. This chapter provides an insight into SSH practical use and contains detailed protocol for generation of subtracted cDNAs (which is the most frequent SSH application) and differential screening of the resulting subtracted cDNA library. As shown in many examples, the SSH technique may result in over 1000-fold enrichment for rare sequences in a single round of subtractive hybridization. Finally, we discuss the characteristics of cDNA-subtracted libraries, the nature and level of background nondifferentially expressed clones in the libraries, as well as procedure for rapid identification of truly differentially expressed cDNA clones.
KeywordsDifferentially regulated genes suppression polymerase chain reaction (PCR) effect enrichment hybridization time high complexity false positive mirror orientation selection mirror-oriented selection (MOS) protocol cap switch tagging RNA 5ʹ -ends highly abundant cDNA primer annealing site adapter sequence physical separation mathematical model background restriction endonuclease heat denaturation reannealing target sequence technical comments differential screening efficiency of SSH Northern blot level of enrichment random clones removal of the adapter sequences size of cDNA fragments disadvantage drawback equalization
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