Figure 1. Schematic diagram of site-directed mutagenesis using Splicing by Overlap Extension (SOE)

To change a cysteine to alanine at position 47 of the myocilin sequence, a 5' fragment (55 bp) was amplified using a forward primer (MYOC-5'; nucleotides 133-155) and a reverse primer (Cy1-3al; nucleotides 156-187). The 5' fragment contains a 2 base substitution (TG to GC) at nucleotides 175 and 176 that changes a cysteine codon to an alanine codon. A 1388 bp 3' fragment was generated using a forward primer (Cy1-5al; nucleotides 164-193) and a reverse primer (MYOC-3'; nucleotides 1528-1551). The 3' fragment also contained a TG to GC substitution at nucleotides 175 and 176. The 5' and 3' fragments containing the 2 base substitution were mixed together and denatured. The top strand of the 5' fragment and the bottom strand of the 3' fragment were annealed by alignment of the 24 base pair overlapping sequence (nucleotides 164-187). This overlapping sequence serves as a primer so that extension with Taq polymerase will create a template of wild type myocilin containing a base pair substitution that replaces the cysteine at amino acid 47 with alanine. In the presence of oligonucleotides MYOC-5' and MYOC-3', a mutant myocilin product will be amplified. The numbers above the 5' and 3' ends of the 5' fragment, 3' fragment, and the final mutant fragment designate the nucleotide location in myocilin.