[17] Gene Splicing by Overlap Extension

Conventional methods of engineering recombinant DNA make use of restriction enzymes to cut molecules apart at specific nucleotide sequences and ligases to rejoin the parts. A significant limitation of this technology is that restriction enzymes are sequence dependent and these recognition sequences appear more or less randomly in DNA. That is, restriction enzymes cut where recognition sites are located and not necessarily at optimal positions along the gene for purposes of genetic engineering. The polymerase chain reaction (PCR) has made possible a sequence-independent engineering method that is referred to as “gene splicing by overlap extension” or “SOE.” This technology is especially useful in complicated constructions that require precise recombination points—such as joining two coding sequences in frame—and it also provides a straightforward way of performing site-directed mutagenesis.