Identification of Differential Genes by Suppression Subtractive Hybridization: IV. Mirror Orientation Selection (MOS)

INTRODUCTIONSuppression subtractive hybridization (SSH) is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. This technique can be used to compare two mRNA populations and obtain cDNAs representing genes that are either overexpressed or exclusively expressed in one population as compared to another. It can also be used for comparison of genomic DNA populations. The major drawback of SSH is the presence of background clones that represent nondifferentially expressed DNA species in the subtracted libraries. In some cases, the number of background clones may considerably exceed the number of target clones. This protocol describes mirror orientation selection (MOS)--a simple procedure that substantially decreases the number of background clones in libraries generated by SSH. The MOS technique is based on the rationale that after PCR amplification, during SSH, background molecules will be present in one orientation only, relative to the adapter sequences. Genuine SSH clones will be present in both sequence orientations.