ABSTRACT
The identification of differentially expressed genes can provide insights into the molecular mechanisms underlying disease progression or biological development. To facilitate the discovery of differentially expressed genes, a variety of methods have been developed in recent years including Differential Display PCR,1 RNA fingerprinting,2 SAGE,3 Real-time Quantitative PCR (TaqMan),4 subtractive hybridization,5 and hybridization to gene arrays of various formats.6,7 Among these methods, subtractive hybridization is unique for being able to preferentially enrich differentially expressed genes. The method was first used by Bautz and Reilly to purify phage T4 mRNA in the mid-1960s.8 Pure subtractive methodologies are of limited use because they require large quantities of mRNA to drive hybridization to completion. Moreover, subsequent cloning is difficult because only a tiny amount of cDNA remains after hybridization. The method was improved when Duguid and Dinauer added generic linkers to the cDNA allowing PCR amplification of tester cDNA between hybridization cycles.9 Advancing further, Lisitsyn et al. introduced a method for selective PCR amplification of double-strand tester DNA formed after subtractive hybridization, eliminating the need to physically separate it from the other components.10 Subsequently, Siebert et al. invented Suppression PCR, in which DNA with long and complementary 3 and 5 ends was “suppressed” during PCR amplification due to the formation of a “panhandle,” a secondary structure which prohibits PCR primer binding.11
