A METHOD FOR SELECTIVE PCR-AMPLIFICATION OF GENOMIC DNA FRAGMENTS (SAGF METHOD)

A method is suggested for dividing into individual sets of the complex mixtures of fragments obtained by DNA cleavage with type IIS and IIN restriction endonucleases producing single-stranded termini with diffe rent sequences at the DNA fragment ends. The method is based on the li gation of short double-stranded adapters with single-stranded ends com plementary to termini or the selected set of fragments followed by PCR -amplification with the primer representing one of the adapter chains. Using endonucleases BcoKI and Bli736I, recognizing sequences CTCTTC a nd GGTCTC and producing three- and four nucleotide 5-termini, respecti vely, it has been shown that amplification of a set of fragments occur s only upon attachment of the adapters to the DNA fragments with DNA-l igase. Several possible applications of the SAGF method are suggested: obtaining individual bands in: DNA fingerprinting; reducing the kinet ic complexity of DNA in representative difference analysis (RDA method ) of complex genomes; cataloging of DNA fragments; construction of phy sical genome maps.