Patent FR-2952071-A1 (FR)
Title: Hybridization process for enriching specific sequence from nucleic acid directory to test directory comprises e.g. adding restriction endonuclease to hybridized DNA of directory and incubating mixture to transform host cells in the mixture
Inventor: BOIZIAU CLAUDINE
IPC: C12N15/63
Filed: 2009-11-03
Abstract: Subtractive hybridization process of enrichment of specific sequences from nucleic acid directory corresponding to a test directory, by comparing with a directory corresponding to a reference directory, comprises: (a) obtaining DNAs from directories; (b) hybridizing the DNAs of the directories; (c) adding one or more restriction endonuclease; (d) incubating the hybrid mixture under conditions; (e) transforming host cells with the mixture and culturing the cells transformed under conditions; and (f) recovering the integrity vectors. Subtractive hybridization process of enrichment of specific sequences from nucleic acid directory corresponding to a state called test directory, by comparing with a directory corresponding to a reference state called reference directory, comprises: (a) obtaining DNAs from directories for comparing in order to placing at least a portion of DNA comprising the entire cassette for each directory, obtaining the test directory in the form of single-stranded circular DNA, and placing antiparallel strand of the test directory from reference directory; (b) hybridizing the DNAs of the directories for comparing obtained in step (a) in conditions and hybridizing and denaturation temperatures greater than the fusion temperature of the areas surrounding the cassette of variable sequence and lower than the fusion temperature of the homohybrids; (c) adding one or more restriction endonuclease where the cleavage site is present in the DNA portion comprising the cassette for each directory that is obtained in step (a); (d) incubating the hybrid mixture under conditions where restriction endonuclease is active for carrying out hydrolysis of homohybrids; (e) transforming host cells with the mixture and culturing the cells transformed under conditions where the vectors containing molecules of the test directory can grow and propagate in the host cells; and (f) recovering the integrity vectors which contain molecules of test directory enriched in specific clones, and optionally concentrating the vectors. Independent claims are included for: (1) kit for the enrichment of specific sequences of a nucleic acid directory corresponding to the given state comprising: a vector capable of expressing the peptides on the surface; pair of primers to obtain in vitro, for example by PCR, DNA comprising part of the cassette of the reference directory; and one or more restriction endonuclease that has a cleavage site in the cassette of test directories and reference for comparison and the buffer required for its optimal activity; and (2) use of two nucleic acid directories corresponding to a test sample and a reference sample, to carry out a comparison by subtractive hybridization, in which directories to be compared contain the same overall cassette, the test directory is in the form of single-stranded circular DNA and the cassette of the reference directory is in an orientation antiparallel to that of the test directory.