Direct subcloning of target region from BAC insert using restriction enzymes that produce non-identical cohesive ends.
Type IIS or interrupted palindrome restriction endonuclease digestion produces unique cohesive ends. Utilization of this feature of BglI, SfiI and BstXI has led to direct subcloning of 28.5-kb and 22.9-kb regions, which contains candidate genes for medaka Double anal fin (Da) mutant, from a 229-kb BAC insert. This method does not contain gel-fractionation of the fragments and time-consuming screening process using hybridization. Non-recombinant backgrounds were distinguished from recombinant clones by introducing GFPuv gene into the subcloning vector. This strategy was successfully applicable even when the target region was cut into three fragments by these enzymes.
Ohmi
Yuhsuke
Y
Division of Basic Molecular Science and Molecular Medicine, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa 259-1193, Japan.
Sato
Masahiro
M
Ohtsuka
Masato
M
D000818:Animals; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D009990:Oryzias / Q000235:genetics
D004262:DNA Restriction Enzymes; C047564:CCANNNNNNTGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific