Efficient cDNA cloning method using solid phase DNA probe

Nucleic Acids Symp Ser. 1992:(27):155-6.

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.

MeSH terms

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

Substances

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