A NOVEL GENOME WALKING METHOD FOR CLONING OF UNKNOWN DNA SEQUENCES ADJACENT TO KNOWN SEQUENCE

摘要

The present invention relates to a novel genome walking method for cloning unknown genomic regions adjacent to known sequences by PCR. More particularly, the present invention relates to a method for cloning an unknown nucleotide sequence adjacent to a known nucleotide sequence, comprising the steps of 1) cleaving a genomic DNA that contains a known nucleotide sequence and an unknown nucleotide sequence adjacent thereto using a restriction enzyme; 2) modifying the 3'-OH end of the cleaved DNA with a nucleotide analogue; 3) linking the terminal sequence of the cleaved DNA fragment with a linker, adapter or cassette having a terminal sequence complementary thereto; and 4) amplifying the unknown nucleotide sequence using a known nucleotide sequence-specific primer and a cassette sequence-specific primer. Further, the present invention relates to a composition of gene amplification for cloning the unknown nucleotide sequence, and a gene amplification kit comprising the composition.