Efficient cDNA cloning method using solid phase DNA probe.

H Tsurui; N Kim; Y Umezawa; S Kato

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PubMed · 1289800

Efficient cDNA cloning method using solid phase DNA probe.

H Tsurui·N Kim·Y Umezawa·S Kato

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Abstract

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.

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Catechol 2,3-Dioxygenase Cloning, Molecular DNA Probes DNA, Bacterial Dioxygenases Humans Kinetics Oxygenases Pseudomonas putida RNA, Messenger Tumor Cells, Cultured Journal Article

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H Tsurui, N Kim, Y Umezawa, S Kato. 1992. Efficient cDNA cloning method using solid phase DNA probe.. https://pubmed.ncbi.nlm.nih.gov/1289800/

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