PCR-based cDNA library construction: general cDNA libraries at the level of a few cells

Abstract

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.