PCR-based random mutagenesis using manganese and reduced dNTP concentration.

Procedures to introduce point mutations into specific DNA fragments are important tools to study gene function (2,10). UV light and chemical mutagens have been used in vivo to increase the frequency of random mutagenesis for specific DNA targets carried on plasmid or phage vectors being propagated in growing cells (2,11). Both methods induced mutations in the target DNA as well as in vector and host cell DNA. Chemical mutagenesis in vivo also resulted in a limited spectrum of substitutions at a limited set of hot spots on the DNA (2). Chemical mutagenesis has also been done in vitro for target DNA carried on plasmids (10). While a complete spectrum of substitutions was obtained, the procedure also produced vector mutations and required multiple cloning steps. The polymerase chain reaction (PCR) process produces missense errors at a low rate. Others have reported on PCR modifications to induce mutations in vitro (5–7). This paper describes a simple modification of PCR amplification to enhance the production of mutant clones, the majority of which carry only a single base substitution in a specific target DNA sequence. The procedure relies on manganese-induced mis-insertion of nucleotides by AmpliTaq DNA Polymerase (Perkin-Elmer, Norwalk, CT, USA) (1,4,6,7) as well as reduced concentration of each dNTP and increased number of PCR cycles to decrease the fidelity of PCR amplification. Over 50% of the mutated molecules contained only one mutation per clone. This mutagenesis method operates only on the region of interest (the PCR product) and uses only one cloning step to generate a library of mutations in the targeted region. This method is time-efficient, cost-effective and relatively effortless when compared to other random mutagenesis methods. To prepare wild-type templates for mutagenesis, the PCR amplifications for the 16 exons of the wild-type hMSH2 (8) gene were performed on the GeneAmp PCR System 9600 (Perkin-Elmer) in a 50-μL volume containing 1× PCR Buffer II (10 mM TrisHCl, pH 8.3, 50 mM KCl; PerkinElmer), 3 mM MgCl2 (except for exon 1, which used 1.5 mM MgCl2), 0.2 mM of each dNTP, 10 pmol of each primer, 1.25 U AmpliTaq DNA Polymerase and 10 ng of genomic DNA. Samples were subjected to heating at 94°C for 3 min followed by 35 PCR cycles (20 s at 94°C, 20 s at 55°C, 40 s at 72°C) and finished at 72°C for 10 min. The sequences of these wild-type PCR products (hMSH2 exons) were verified by direct sequencing on a PRISM Ready Reaction Dye Terminator Cycle Sequencing Kit with AmpliTaq DNA Polymerase FS (PE Applied Biosystems, Foster City, CA, USA). These wild-type PCR products were then used as DNA templates for PCR mutagenesis. A library of mutants for each exon of the hMSH2 gene was generated by the PCR mutagenesis procedure using 10 ng wild-type PCR product as template in a 50-μL volume of mutagenesis buffer containing: 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2 mM MgCl2, 0.25 mM MnCl2 (added immediately prior to the PCR), 20 μM of each dNTP, 10 pmol of each primer and 1.25 U AmpliTaq. The PCR mutagenesis conditions were heating at 94°C for 4 min followed by 40 PCR cycles (15 s at 94°C, 15 s at 55°C, 45 s at 72°C) and finished at 72°C for 10 min. This extra extension time promoted the yield of a fulllength product. After PCR mutagenesis, a small aliquot of PCR product was ligated into TA Cloning vector (Invitrogen, San Diego, CA, USA) without any further purification. The 10-μL ligation reaction mixture contained 1× ligation buffer, 50 ng pCR II vector (both from Invitrogen), 1 μL T4 DNA ligase and 2–4 μL of fresh PCR product and was incubated for 5–14 h at 14°C. Then this ligation mixture was used to transform One Shot cells (Invitrogen) following the protocol supplied by the manufacturer. Those cells were spread onto LB plates containing ampicillin, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) and isopropyl-βD-thiogalactopyranoside (IPTG) (9), then grown at 37°C overnight for white colony selection. All white colonies were restreaked on fresh LB/ampicillin/X-gal/IPTG plates to confirm the white colony. To verify the correct insert size, each white colony was subjected to PCR amplification (using the wild-type protocol above) followed by gel electrophoresis. All products of the correct size were purified with the QIAquick 96 PCR Purification Kit (Qiagen, Chatsworth, CA, USA), then subjected