Site-Directed Mutagenesis by the Cassette Method

Abstract

Cassette mutagenesis is a technique for altering a protein sequence at the DNA level by replacing a section of genetic information with an alternative sequence, normally provided by a synthetic DNA duplex. First, the gene contained in a suitable vector is cleaved with two restriction enzymes. This releases a small section of DNA from the gene. The prerequisite for this method to be practical is that these restriction enzymes cut at sites that flank the area of DNA to be changed and are unique in the gene/vector system. In this way only the desired cutting occurs. A synthetic duplex is then ligated in place of the released cassette and the resultant construct is sequenced through the cassette and the reformed restriction sites to check that the mutated gene now has the intended sequence. This technique can be used to make single or multiple amino acid changes to the protein sequence and to insert sequences, or indeed, delete them from the protein structure. The changes are only limited by the available size of the synthetic DNA cassette. With current DNA synthesis technology and the expertise of the average DNA synthesis service, duplexes of up to 100 bp are readily available. This methodology has been used extensively in this laboratory to perform site-directed mutagenesis experiments on a number of synthetic genes including bovine pancreatic DNase 1 (1). Synthetic genes are normally designed with a number of unique restriction sites in the sequence, making cassette mutagenesis particularly applicable in these cases. In addition to performing amino acid changes to the protein sequence, the technique can be utilized to alter the noncoding portion of genes to increase expression levels (2). When designing synthetic genes, it is useful to flank the Shine-Dalgarno sequence and the first few codons at the beginning of the gene by restriction sites. This enables the Shine-Dalgarno sequence, its spacing to the ATG start codon, and the initial coding nucleotides to be easily altered. This often has a dramatic effect on expression levels (3). We routinely mutate genes cloned in M13mp18/19 before subcloning into an expression vector for the following reasons: