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Cloning Based on Efficient Three-Fragment Assembly,,,DNA Ligation

endonuclease Sca I, followed by sequencing of all restriction-positive clones using the primer 5'-TGAATTTTCTGTATGAGG-3', the T7 Sequenase 2.0 kit, and 33PdATP from Amersham, according to the manufacturers instructions.

Results and Discussion

Although a T4 DNA ligase-overexpressing E. coli strain that allows cloning of linear DNA molecules was reported recently [4], efficient transformation of E. coli normally requires circular DNA. However, the formation of a circular structure in a three-fragment assembly ligation is a relatively rare event. At high DNA concentrations, which are crucial for a three-component assembly ligation [1], predominantly intermolecular ligation events leading to linear products occur, while DNA circularization is favored at low concentrations of the DNA fragments [5]. Our case was further complicated by the need to ligate two blunt-ended DNA fragments. Blunt-end ligation is known to be one to two orders of magnitude less efficient than cohesive-end ligation [6]. We chose to optimize the conventional procedure by sequential ligation of blunt- and cohesive DNA ends using different temperatures for the two processes. Therefore, we started the reaction at 26C, which was reported to be the optimal temperature for the joining of bluntended termini by T4 DNA ligase [7]. We followed this with cohesive-end ligation and circularization of our DNA at 14C, the temperature generally regarded as the best compromise to retain sufficient activity of the enzyme while still allowing relatively stable hydrogenbonded association of cohesive termini [8]. This strategy led to a more than 10-fold increase in the number of transformants obtained compared with a control ligation performed at single temperature (Table 1).

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