( ...)Cloning Based on Efficient Three-Fragment Assembly,,,DNA Ligation

Abstract

The efficiency of the Rapid DNA Ligation Kit from Roche Molecular Biochemicals was compared with standard and optimized conventional ligation procedures. The reactions generated circular recombinant bacteriophage DNA in a three-fragment assembly DNA ligation. Sequential blunt-end and cohesive-end ligation, including a temperature shift from 26C to 14C, led to a more than 10-fold increase in the number of transformants obtained. Applying a modified protocol, the kit was found to provide a reliable, fast, and remarkably efficient procedure for this complex application.

Introduction

To construct a phage displaying integrin-binding ligands, we needed a reliable and efficient ligation procedure, by which three DNA fragments could be assembled simultaneously, yielding circular DNA. Such a ligation is known to require high concentrations of the reacting DNA molecules [1], especially if blunt-ended DNA is involved [2] and is therefore usually circumvented by subcloning. Because of the absence of appropriate restriction sites in the DNA fragments, subcloning into another suitable vector and subsequent ligation of the two fragments in this construct was impossible in our case. A standard ligation protocol led only to poor transformation results, whereas parallel ligations using either an optimized conventional procedure or the Rapid DNA Ligation Kit from Roche Molecular Biochemicals resulted in efficient and correct formation of the desired cloning vector.

Materials And Methods

The bacteriophage fd-tet which confers tetracycline resistance upon infected cells was donate
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