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

Biotechniques. 1993 Nov;15(5):874-8.

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product is amplified by PCR using primers A and B. The final product contains FIX cDNA in which its EGF1 sequence has been replaced by the PCEGF1 sequence.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.
  • Technical Report

MeSH terms

  • Base Sequence
  • Binding Sites
  • Cell Line
  • DNA Primers
  • DNA Restriction Enzymes / metabolism*
  • Electrophoresis, Agar Gel
  • Epidermal Growth Factor / genetics
  • Factor IX / genetics
  • Gene Expression
  • Humans
  • Kidney
  • Molecular Sequence Data
  • Plasmids
  • Polymerase Chain Reaction*
  • Protein C / genetics
  • Recombinant Fusion Proteins / genetics*
  • Recombinant Fusion Proteins / isolation & purification
  • Transfection

Substances

  • DNA Primers
  • Protein C
  • Recombinant Fusion Proteins
  • Epidermal Growth Factor
  • Factor IX
  • DNA Restriction Enzymes