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The self-complementary octanucleotide dT-A-G-T-A-C-T-A has been synthesized and its sequence confirmed by two-dimensional fingerprinting. Under conditions used for the T4 polynucleotide ligase reaction, this oligonucleotide forms a dimeric duplex which shows a Tm of 18 degrees C. The optimal rate of joining of the 32P-labeled duplex occurs between 12 and 15 degrees C. The rate is highly concentration dependent, as expected for a bimolecular process. Polyacrylamide gel electrophoretic analysis of this reaction shows the presence of products up to 120 nucleotides in length. In a denaturing gel, each product appears as a double band due to the presence of its 5'-adenylylated activated intermediate. Substrates larger than eight base pairs are utilized more rapidly than the eight base pair duplex, indicating that the T4 ligase has a higher affinity for longer substrates. The low level of nicked intermediates suggests that the joining of both strands requires two steps, the rates of which must be similar.
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