[A method of selective PCR-amplification of genomic DNA fragments (SAGF method)].
A method for separating into definite sets of a complex mixture of fragments obtained by DNA cleavage with IIS- or IIN-types of restriction endonucleases producing single-stranded termini of different sequences at the fragment ends has been developed. The method is based on the ligation of short double-stranded adapters with single-stranded termini complementary to the termini of a selected set of fragments followed by PCR-amplification with the primer which represents a strand of the adapters. Using endonucleases BcoKI and Bli7361 recognizing sequences CTCTTC and GGTCTC and producing three- and four-nucleotide 5'-termini, respectively, it has been shown that amplification of a set of fragments occurs only when the adapters are attached to DNA fragments with DNA-ligase. Several applications of the SAGF-method are suggested: for obtaining individual bands in DNA fingerprinting; for reducing the kinetic complexity of DNA in the representational difference analysis (RDA method) of complex genomes; for cataloguing DNA fragments, and for constructing physical genomic maps.
Zheleznaia
L A
LA
Menzeniuk
O Iu
OIu
Matvienko
N N
NN
Matvienko
N I
NI
D000818:Animals; D001483:Base Sequence; D002642:Chick Embryo; D004247:DNA / Q000235:genetics*; D016172:DNA Fingerprinting; D004262:DNA Restriction Enzymes; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*
D004247:DNA; D004262:DNA Restriction Enzymes
Metod izbiratel'noĭ PCR-amplifikatsii fragmentov genomnoĭ DNK (metod SAGF).