DARFA: a novel technique for studying differential gene expression and bacterial comparative genomics.

We have developed a powerful method, named differential analysis of restriction fragments amplification (DARFA), which enables researchers to perform comprehensive transcriptome analysis as well as bacterial DNA fingerprinting. The key feature of this novel technique lies within the usage of a type IIS enzyme, Hpy188III, which cleaves cDNA or genomic DNA at a TC/NNGA recognition sequence. Cleavage at this particular site results in the production of a pool of restriction fragments which can be divided into 120 subsets based on the 2-nt 5'-overhang sequence. Each subset of restriction fragments is then selectively amplified by PCR after ligation with a pair of hairpin adaptors containing 2-nt overhangs which are complementary to those in the subset of fragments that are to be analyzed. The results obtained from the analysis of strain-specific and tissue-specific differences using DARFA and further confirmation by DNA sequencing and Northern analysis have demonstrated that the DARFA technique provides a novel tool for expression profiling, as well as bacterial DNA fingerprinting.