Effects of nicking of ds-DNAs on insert sizes when cloning by dG/dC tailing methods

Abstract

Complementary DNA(cDNA), transcribed in vitro from isolated bovine pituitary poly(A) + RNA and made double-stranded, has been inserted into E.coli plasmid pBR 322 by the poly(dG)/poly(dC) tailing and annealing technique. It was observed that the size of cDNA insert from E.coli transformants revealed by agarose gel analysis was shortened during steps of tailing, annealing and transformation. To monitor the cDNA size deletion, the "internal trailing" which had been suggested as a possible reason for size deletion of insert from transformants, was investigated as follows: Nicking of 1078 base pairs long blunt-ended $\phi \times 174$ DNA fragment digested with Hae III by treatment of DNase I, its tailing under $Co^{2+}$, annealing and transformation, then, the size of that fragment from transformants was considerably shortened also. The original size of 1078 base paris long was reduced on the average of below the 500 base pairs long by the treatment of more than lul of $0.01\mu g/ml$ DNase I to $\sim$400ng of DNA. This result was considered to be mainly due to the internal tailing of DNA. And, it has been shown that TCA precipition method to calculate the numbers of the added nucleotides to 3``-OH termini of DNA and optimize the tailing reaction was not to be used properly to determine the tailing time point at which the maximum numbers of transformants would be obtained. In addition, the tailing reaction rate(may be mainly internal tailing) of nicked DNA was increased when adding trivalent metal complex, $Co^{3+}(NH_3)_6$ which is known to cause the conformational change of DNA as well as $Co^{2+}$ which acts as a corfactor of terminal deoxynucleotidyl transferase. As a result of this experiment, the points that should be somewhat improved through the tailing reaction, important in cDNA cloning, were mentioned and the possible methodology was also suggested.