In-frame cloning of synthetic genes using PCR inserts.

Abstract

Because many genes of biological interest are larger than the maximum size that current synthetic oligonucleotide synthesizers can produce (approx 110 bases), there is a need for methods that allow rapid production and expression of genes constructed from multiple synthetic DNA fragments. A number of synthetic genes have been generated using the recursive polymerase chain reaction (PCR) method, but problems concerning primer design and incorrect final gene sequence, especially with large synthetic genes, are a concern (-). The cloning method described here follows a series of steps in which multiple PCR products or synthetic duplex oligonucleotides are positionally cloned into a plasmid vector (). A synthetic gene of practically any sequence or length can be built using the in-frame cloning method. Genes are assembled such that open reading frames are maintained by linking DNA fragments through the use of six basepair blunt-end restriction sites. Each cloning step uses an anchored sticky-end restriction site and a variable blunt-end restriction site that result in specific insert orientation and high cloning efficiencies. The overall strategy of in-frame cloning allows the researcher total control over nucleotide sequence, codon usage, promoter and other regulatory elements, and the placement of unique restriction sites throughout the recombinant construct. One advantage of the in-frame cloning method described here is that it allows for flexible yet precise construction of synthetic genes using standard recombinant techniques. Another advantage is that it employs inexpensive, readily available materials.