Ligation-free gene synthesis by PCR: synthesis and mutagenesis at multiple loci of a chimeric gene encoding OmpA signal peptide and hirudin.
A unique kination and ligation-free method that allows de novo synthesis of a gene through a novel application of polymerase chain reaction (PCR) involving stepwise elongation of sequence (SES) is described. SES-PCR is simple and efficient. Optimal utilization of nucleotides, ability to use only partially purified oligodeoxyribonucleotides, and elimination of kination and ligation of intermediates make SES-PCR-mediated gene synthesis more economical in terms of time, labour and money. Site-directed mutagenesis and/or gene fusion by SES-PCR is not limited by the prior availability of the gene(s) in question. The potentials of this novel method in gene synthesis, mutagenesis at multiple loci of DNA and gene fusion have been demonstrated using a chimeric gene encoding fusion between OmpA signal peptide and hirudin, as an example. The SES-PCR product was cloned and sequencing of positive clones demonstrated the presence of genes with expected sequence and bearing only the desired mutations. A nearly 100% efficiency of mutation was easily achieved by the design of the method.
Majumder
K
K
International Centre for Genetic Engineering and Biotechnology, New Delhi, India.
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D002678:Chimera; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D005813:Genes, Synthetic; D006629:Hirudins / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000379:methods; D021382:Protein Sorting Signals / Q000235:genetics*
D001425:Bacterial Outer Membrane Proteins; D006629:Hirudins; D021382:Protein Sorting Signals