Site-directed mutagenesis using a double-stranded DNA fragment as a PCR primer. Nucleic Acids Res

The polymerase chain reaction (PCR) (1) has found wide application in identifying gene mutations in patients with genetic diseases. It is often useful to test the effect of specific mutations on gene expression in vitro. We describe a PCR protocol to rapidly and efficiently introduce specific mutations found in patient material into cloned DNA using double-stranded PCR fragments containing the mutations of interest. The procedure uses a double-stranded PCR product amplified from an M13 clone containing a mutation as one primer and an oligonucleotide derived from another exon of the gene as a second primer to generate a PCR product that can then be easily cloned into the desired vector. We identified two mutations in exon 4 of the tyrosinase gene in patients with tyrosinase-negative oculocutaneous albinism (unpublished data). To test the effect of these mutations on tyrosinase enzyme activity we introduced them into a tyrosinase cDNA for in vitro expression. Exon 4 (182 bp) was amplified by PCR from DNA (30 ng) of two M13 exon 4 subclones containing either of the two mutations in 100 ^1 volumes of 10 mM TrisHCl pH 8.3, 50 raM KC1, 1.5 raM MgCl2, 200 yM of each dNTP, 100 /tg/ml gelatin, 100 pmoles of both exon 4 primers (derived from the very 5 ' and 3 ' ends of exon 4), and 2.5 U of Taq DNA polymerase (Perkin-Elmer Cetus, Norwalk, CT) using an automated thermal cycler (Coy Laboratory Product