Bypassing successive cloning of two fragments by one step cloning using polymerase chain reaction.

ABSTRACT

The polymerase chain reaction (PCR) was used for bringing together two DNA fragments present in two different plasmids. This helped avoiding the difficulties of two successive subcloning in the same plasmid and uncertainities of obtaining rightly oriented constructs. Two different fragments of DNA present in different plasmid were digested with the same enzyme and then ligated. The ligation mixture was used for the PCR using two oligo primers; one was specific for the 5' end of the other fragment and the other one was for the 3' end of the other fragment. The desired amplified fragment was separated by gel electrophoresis, eluted and was cloned in plasmid pBluescript KS(+). The same procedure is also applicable for one step cloning of more than two fragments in desired orientation.