A simple and rapid amplification procedure for cDNA cloned in dephosphorylated plasmid.

The preparation of cDNA libraries usually involves multiple steps, such as isolation of poly A mRNA, synthesis of cDNA, cloning of cDNA into plasmid, and amplification of cloned plasmid. The last step, the amplification of insert DNA fragment after cloned in dephosphorylated plasmid vector, is done in either of two ways. The recombinant DNA can be extracted after amplification in host cells, or the amplification of insert DNA can be done by the PCR using primers complementary to both ends of the insertion site of the vector. However, the ligation of dephosphorylated nicks or nick translation beyond the primer sites is essential in the latter case prior to the PCR (see step 1 in Figure 1 (B)). Taq DNA polymerase, by virtue of its 5' to 3' exonuclease activity (Ref 1), may be utilized for nick translation instead of E. coli DNA polymerase 1. This would simplify the amplification of insert cDNA by doing nick translation and PCR steps successively, controlling each reaction condition. We examined the conditions for nick translation by Taq DNA polymerase. The reaction mixture, 20 /tl containing 25 yCi ^PdNTPs (600 Ci/mmol), 0.5 ng RF-M13mpl8 DNA, 0.02 ng pancreatic DNase I, and 2.5 units of Taq DNA polymerase was incubated at 37, 50 and 65°C and the incorporation of PdNTPs into acid-insoluble fraction was measured. Radioactivities incorporated were 167 k, 323 k, and 286 k DPM, respectively, compared with 82 k DPM by a standard nick translation reaction at 12°C using DNA polymerase I and DNase I. These results indicated that the nick translation reaction by Taq DNA polymerase worked normally at all temperatures examined, although the incorporation was reduced at 65°C. Then we amplified insert DNA by nick translation reaction