Efficient cDNA cloning method using solid phase DNA probe.
We are now developing a novel and efficient method using solid phase DNA probe to isolate a particular recombinant cDNA from single stranded cDNA library. Target clone coding metapyrocatechase (MPC) and cDNA library constructed from mRNA of U-937 (human lymphoma cell line) were converted to single stranded form by superinfection of helper phage (M13KO7). Probe DNA (25 mer) composed of a portion of the target cDNA was synthesized, attached to an HPLC gel and used as a solid phase DNA probe. Hybridization between probe DNA and target clone was performed in an Eppendorf tube within a few hours. Competent cell (JM109) was transformed with about one-twentieth of hybridized and eluted fraction by Hanahan's method. From the mixture of 1 ng of MPC vector and 5 micrograms of cDNA library, we obtained 50 colonies containing MPC gene out of 63 transformed colonies.
Tsurui
H
H
Kanagawa Academy of Science and Technology, Japan.
Kim
N
N
Umezawa
Y
Y
Kato
S
S
D050557:Catechol 2,3-Dioxygenase; D003001:Cloning, Molecular / Q000379:methods*; D015342:DNA Probes; D004269:DNA, Bacterial / Q000235:genetics*; D049308:Dioxygenases; D006801:Humans; D007700:Kinetics; D010105:Oxygenases / Q000235:genetics; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics; D012333:RNA, Messenger; D014407:Tumor Cells, Cultured
D015342:DNA Probes; D004269:DNA, Bacterial; D012333:RNA, Messenger; D010105:Oxygenases; D049308:Dioxygenases; D050557:Catechol 2,3-Dioxygenase