A PCR-based method for site-specific domain replacement that does not require restriction recognition sequences.

Abstract

We describe a PCR-based method for domain replacement that does not require restriction site sequences. We illustrate the technique in which the first epidermal growth factor (EGF1)-like domain of factor IX (FIX) is replaced by the EGF1-like domain of protein C. The method employs four oligonucleotide primers. Two are external primers (forward primer A and inverse primer B) and contain sequences flanking the FIX cDNA nucleotides. The other two primers (forward primer C and inverse primer D) direct the PCR amplification of the EGF1-like domain of protein C, and they are hybrid primers that contain sequences of protein C gene at the 3' end and of FIX gene at the 5' end. Thus the amplified fragment of EGF1-like domain of protein C (PCEGF1 fragment) is flanked by FIX gene sequences on both ends. When this fragment is mixed with FIX cDNA and subjected to one cycle of PCR, two products are obtained: one containing PCEGF1 fragment linked to FIX cDNA sequence upstream and the other containing PCEGF1 fragment linked to FIX cDNA sequence downstream of its EGF1-like domain. The first product is amplified using primers A and D, and the second product is amplified using primers B and C. Both products contain overlapping sequences, which allow annealing upon mixing. The annealed product is amplified by PCR using primers A and B. The final product contains FIX cDNA in which its EGF1 sequence has been replaced by the PCEGF1 sequence.