SSH(suppressive subtrative hybridization)

(From CLONTECH PCR-Select cDNA Subtraction Kit User Manual)

Principle: Subtractive hybridization is a powerful technique that enables researchers to compare two populations of mRNA and obtain clones of genes that are expressed in one population but not in the other. Molecular basis of PCR-Select cDNA subtraction: The latter figure details the molecular events that occur during PCR-Select cDNA subtraction.First,cDNA is synthesized from 0.5-2μg of poly A+RNA from the two types of tissues or cells being compared.The tester and driver cDNAs are digested with Rsa 1.The tester cDNA is then subdivided into two proteins,and each is ligated with a different cDNA adaptor.The two adaptors have stretches of identical sequence to allow anneling of the PCR primer once the recessed ends have been filled in.Two hybridizations are then performed.Then an excess of driver is added to each sample of tester.The samples are then heat denatured and allowed to anneal,generating the type a,b,c,and d molecules in each sample.During the second hybridization,the two primary hybridization are mixed together without denaturing.Now,only the remaiining equalized and subtracted sstester cDNAs can reassociate and from new type e hybrids.These new hybrids are ds tester molecules with different ends,which correspond to the sequences of Adaptor1 an 2R.Fresh denatured driver cDNA is added to further enrich fraction e for differentially expressed sequences.After filling in the edns by DNA polymerase,the type e molecules-the differentially expressed tester sequences-have different annealing sites for the nested primers on their 5' an d 3'ends.Then after primaryPCR and second PCR,we can get large specific sequences.

SSH Overview