Nucleic Acids Research, 1989, Vol. 17, No. 8 2919-2932
© 1989
MOLECULAR BIOLOGY |
PCR-based cDNA library construction: general cDNA libraries at the level of a few cells
Institute for Genetics, University of Cologne 5000 Weyertal 121, FRG
Received February 14, 1989. Accepted March 14, 1989.
A procedure for the construction of general cDNA libraries is described which is based on the amplification of total cDNA in vitro. The first cDNA strand is synthesized from total RNA using an oligo(dT)-containing primer. After oligo(dG) tailing the total cDNA is amplified by PCR using two primers complementary to oligo(dA) and oligo(dG) ends of the cDNA. For insertion of the cDNA into a vector a controlled trimming of the 3'ends of the cDNA by Klenow enzyme was used. Starting from 10 J558Lµm3 myeloma cells, total cDNA was synthesized and amplified approximately 105 fold. A library containing 106 clones was established from 1/6 of the amplified cDNA. Screening of the library with probes for three genes expressed in these cells revealed a number of corresponding clones in each case. The longest obtained clones contained inserts of 1.5kb length. No sequences originating from carriers or from rRNA was found in 14 randomly picked clones.
*Permanent address: Institute of Molecular Biology, USSR Academy of Sciences, Moscow, USSR
+Permanent address: Schemjakun Institute of Bioorganic Chemistry, USSR Academy of Sciences, Moscow, USSR
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