A general method of polymerase-chain-reaction-enabled protein domain mutagenesis: construction of a human protein S-osteonectin gene.
Polymerase chain reaction (PCR) amplification was employed to construct a mosaic gene consisting of the propeptide region of protein S and the glutamic acid-rich domain of osteonectin. The strategy is straightforward, results in large amounts of material, and is universally applicable for the generation of protein domain chimeras. In some cases 10% dimethyl sulfoxide aided the amplification. Four base CCGC "clamp" sequences adjacent to BamHI restriction sites at the ends of the PCR products were used to enhance the ligation of products. A hybrid inverse complement oligonucleotide primer composed of sequences containing 20 nucleotides of protein S and 16 nucleotides of osteonectin was used in the first round of PCR. An additional osteonectin sequence was added to the initial amplified product by performing PCR using a second "boot-strap" primer containing 18 nucleotides of osteonectin. Primers used to amplify osteonectin encompassed the 146-aminoacid NH2-terminal half of osteonectin. The double-stranded first-round fragments of protein S-osteonectin and osteonectin were subsequently mixed together and one elongation cycle of PCR was performed. Annealing occurred as the result of the 34-base-pair overlap region composed of osteonectin sequence. Taq polymerase was used for elongation with subsequent recombinant DNA synthesis. After elongation, external primers were added to amplify the protein S-osteonectin gene construct. The protocol we have developed allows noncoding and coding segments of DNA to be linked, GC-rich areas of DNA to be amplified, hybridization temperatures to be increased, annealing times to be reduced, and PCR of products to be subcloned.
Villarreal
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Department of Biochemistry, University of Vermont, Burlington 05405.
Long
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D001483:Base Sequence; D003001:Cloning, Molecular; D004259:DNA-Directed DNA Polymerase; D006023:Glycoproteins / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009841:Oligonucleotides / Q000138:chemical synthesis; D015676:Osteonectin / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011487:Protein Conformation; D017293:Protein S; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D019914:Taq Polymerase
D006023:Glycoproteins; D009841:Oligonucleotides; D015676:Osteonectin; D017293:Protein S; D011993:Recombinant Fusion Proteins; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase