FIELD: chemistry.
SUBSTANCE: claimed invention relates to field of biotechnology. Claimed is method of constructing libraries of gene deletion derivatives based on PCR with random primer. Single-strand breaks are introduced into investigated gene in form of linear DNA by treatment with pancreatic DNAse I in a series of dilutions. Breaks are extended by treatment with polymerase I from E.coli in absence of nucleotidetriphosphates. After that, random primer, which has on 3'-tail 6-, 11- or 17-membered random sequence, and on 5'-tail - constant site (20 nucleotides), intended for adapter primer annealing is attached to matrix. After that, performed is preparative elaboration of library by PCR method with symmetric adapter primer, which is attached to prepared matrix by treatment with T4 DNA-ligase, which makes it possible to delete dimers of primers, formed due to mutual annealing of random sequences, in efficient way. Banks of deletion derivatives obtained in vivo can be further subjected to screening to select variants for their further expression in vivo. Method can be used for obtaining libraries of gene deletion derivatives for their further application in the field of biotechnology, agriculture and food, pharmaceutical industries and medicine.
EFFECT: method makes it possible to obtain shortened variants of practically important genes with improved biotechnological characteristics.
12 dwg, 1 ex
