A PCR-based method for site-specific domain replacement that does not require restriction recognition sequences.
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product is amplified by PCR using primers A and B. The final product contains FIX cDNA in which its EGF1 sequence has been replaced by the PCEGF1 sequence.
Zhong
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St. Louis University School of Medicine, MO.
Bajaj
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SP
D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D017931:DNA Primers; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D004815:Epidermal Growth Factor / Q000235:genetics; D005164:Factor IX / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007668:Kidney; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011486:Protein C / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification; D014162:Transfection
D017931:DNA Primers; D011486:Protein C; D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor; D005164:Factor IX; D004262:DNA Restriction Enzymes