PCR-Based Cloning and Subsequent Expression of Antigenic Proteins in Escherichia coli

The polymerase chain reaction (PCR), originally introduced by Satki et al. (1) and subsequently automated by Mullis and Faloona (2), has emerged as a powerful tool in molecular genetics for the exponential in vitro amplification of specific sequences of Interest from minute quantrties of DNA or RNA Beside the PCR-based diagnosis of human pathogens, this straightforward amplification system has rapidly established itself as a standard technique in the course of cloning procedures. Scharf et al. (3) first showed that it was quite simple to introduce additional foreign sequences into amplification products merely by attaching these sequences to the 5' end of the oligonucleotides used as PCR primers. Although these 5' ends of the primers are mismatched to the template DNA, they usually show little effect on the overall efficiency and specifity of the amplification process since primer specifity is imparted most significantly by the 3' portion of the oligonucleotide (4). As DNA strands mittated by these "5'-mismatched" primers serve themselves as targets during the further rounds of antisense primer elongation, these artificial sequences become fixed into the termini of the growing population of amplification products. Thus principle-the introduction of individual DNA alterations via the PCR primersis of great utility for the de novo creation of terminal restriction enzyme recognition sequences (restriction sites). As shown in Fig. 1, an artificial restriction site can be added to the 5' end of one or both oligonucleotides used in PCR. Fig. 1. Incorporation of artificial terminal restriction sites in amplification products via 5'-mismatching PCR primers.