METHOD FOR PREPARATION OF GENOMIC LIBRARIES OF LIMITED SELECTIONS OF LOCUSES FROM DEGRADED DNA

摘要

FIELD: biotechnology.;SUBSTANCE: method involves hydrolysis of endonuclease restriction genomic DNA forming fragments with a 5'-sticky end and partial end-blunting by a polymerase reaction in the presence of an incomplete set of deoxyribonucleoside triphosphates. Ligation of fragments with adapters having a sticky end, complementary to the sticky end of a target fragment modified by partial blunting, selection of target fragments together with fragments selection by length and amplification with primers, complementary to the adapters. Sequential hydrolysis of genomic DNA can be carried out by two restriction endonucleases that form various sticky ends of fragments, first the endonuclease restriction sensitive to nucleotides chemical modification, then its isoschizomer insensitive to modification. During library fragments amplification, primers are used that include: a site complementary to the adapters, a site complementary to the rest of the restriction endonuclease recognition site, and a site of a specific 1-4 nucleotide extension at the 3' end of the primer to limit the selection of library loci.;EFFECT: invention allows to obtain genomic libraries of degraded DNA samples in a short time and with high selectivity with respect to the target fragments.;2 dwg