Efficient cDNA cloning method using solid phase DNA probe.

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.

• Authors: H Tsurui, N Kim, Y Umezawa, S Kato
• Journal: Nucleic acids symposium series (Nucleic Acids Symp Ser) Issue 27 Pg. 155-6 ( 1992) ISSN: 0261-3166 [Print] England
• PMID: 1289800 (Publication Type: Journal Article)

Chemical References

• DNA Probes
• DNA, Bacterial
• RNA, Messenger
• Oxygenases
• Dioxygenases
• Catechol 2,3-Dioxygenase

Topics

• Catechol 2,3-Dioxygenase
• Cloning, Molecular (methods)
• DNA Probes
• DNA, Bacterial (genetics)
• Dioxygenases
• Humans
• Kinetics
• Oxygenases (genetics)
• Pseudomonas putida (enzymology, genetics)
• RNA, Messenger
• Tumor Cells, Cultured