METHOD OF TARGET GENE DISRUPTION BASED ON CLONING THROUGH RESTRICTION ENZYME CUTTING

摘要

A method for disrupting a target gene based on cloning through restriction enzyme cutting is provided to show improved accuracy, convenience and increased transformation efficiency compared to conventional methods, thereby being useful for genetic engineering and bio-industries. In the method for disrupting a target gene by inserting a nucleotide sequence fragment(c) which includes a nucleotide sequence fragment(a) located at the upper portion of 5' terminal at an open leading frame starting point of the target frame, and a nucleotide sequence fragment(b) from an open leading frame starting point of a selective marker to a certain point of the inside of the open leading frame starting point of the selective marker in sequence from the 5' terminal side; and a nucleotide fragment(f) which includes a nucleotide sequence fragment(d) from the starting point formed to have an overlapped portion with the nucleotide sequence fragment(b) to an open leading frame ending point of the selective marker and a nucleotide sequence fragment(e) located at the lower portion of 3' terminal at the open leading frame ending point of the target gene in sequence from the 5' terminal side into a host, the nucleotide sequence fragment(c) is obtained from the nucleotide sequence fragment(a) inserted via a restriction enzyme site and a vector(A) including all gene sequence of the selective marker and the nucleotide sequence fragment(f) is obtained from the all gene sequence of the selective marker and a vector(B) including the nucleotide sequence fragment(e) inserted via the restriction enzyme site.