A PCR-mediated gene synthesis strategy involving the assembly of oligonucleotides representing only one of the strands.
A modification of PCR-mediated gene synthesis strategy is introduced. This modification enables the synthesis of a gene from oligonucleotides comprising only one of the two strands. Bridging oligonucleotides (approximately 20-mers in length) complementary to the junctions of template strand oligonucleotides and two outer primers are also needed for PCR. A two-step PCR containing a first step of 10 cycles, followed by a second step of 20 cycles, differing only in the annealing conditions was used. A single-step PCR combining the two different cycle conditions could also be used successfully. Optimal conditions for gene synthesis (and amplification) are described. Human and porcine colipase genes (297 and 309 bp, respectively) have been successfully synthesized, cloned into the Invitrogen TA cloning vector and sequenced. There was absolutely no error in the clones that were sequenced.
Jayaraman
K
K
Triplex Pharmaceuticals, The Woodlands, TX 77387-7307.
Puccini
C J
CJ
D000818:Animals; D001483:Base Sequence; D001709:Biotechnology; D003001:Cloning, Molecular; D003089:Colipases / Q000235:genetics; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D005796:Genes; D006801:Humans; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D013552:Swine
D003089:Colipases; D009841:Oligonucleotides; D004247:DNA