Genome Walking Method For Cloning Of Unknown Dna Sequences Adjacent To Known Sequences

Document № Publication date Application №
9260749 February 16, 2016 13/345616

A method and a kit for cloning a nucleotide sequence adjacent to a known nucleotide sequence by PCR are disclosed. The method includes 1) preparing a DNA fragment with cohesive ends by cleaving a DNA that contains the known first nucleotide sequence and the second nucleotide sequence adjacent thereto using a restriction enzyme, 2) modifying the 3? end of the DNA fragment with a nucleotide analog to block further elongation of the DNA fragment, 3) linking the cohesive ends of the 3? end-modified DNA fragment with a linker, adapter or cassette having a cohesive end sequence complementary thereto, and 4) performing PCR using a known first nucleotide sequence-specific primer, and a linker, adapter or cassette sequence-specific primer.