Method for Transferring Mutations between Plasmids

The identification and isolation of protein fragments that are responsible for specialized functions such as DNA binding, transactivation, and enzymatic activity (3,4,6) is a common technique for dissecting protein structure-function relationships. Subsequent functional analysis of protein fragments by mutational studies frequently aids the investigator in determining critical regions and residues of the fragment that support the specific function (1,2). However, the ultimate test of biological significance requires that the functional consequences of mutations be assessed in the context of the full-length native protein inside a living cell. Therefore, transferring mutations from the initial vector expressing the protein fragment to other vectors expressing either the full-length or alternative fragments of the protein is often necessary. Common procedures for such transfer include subcloning (5), recombination (7), and oligonucleotide-mediated site-directed mutagenesis of the target expression vector (8). However, each method has limitations: subcloning requires convenient restriction sites, recombination is limited by specific vector requirements for selection, and sitedirected mutagenesis requires a unique oligonucleotide primer pair for each mutation to be transferred. Especially when the mutations are naturally occurring or generated by random procedures, the synthesis of numerous pairs of site-directed mutagenic oligonucleotides for transfer would be costly. As an alternative to the traditional approaches for mutation transfer, we developed a simple method for transfer that utilizes a single set of oligonucleotide primers. This technique involves (i) generating a 300–400 bp PCR product from the originally mutated template plasmid (the mutation donor) using a set of nonmutagenic primers that flank the region of interest and (ii) using this initial large PCR product as the mutagenic primer set for amplification of the mutation recipient plasmid that contains the same sequence of DNA without the mutations. In essence, the second round of PCR is similar to typical site-directed mutagenesis, except it uses a much longer primer pair rather than the standard synthetic oligonucleotides. The major advantage of this technique is that a series of mutations in a protein region can each be transferred using a single flanking primer set rather than developing and purchasing individual mutagenic primers sets for each mutation to be moved. This is both more economical and a considerable time saver when several random or naturally existing mutations in a defined region of DNA are to be mobilized. We developed this method as a quick and easy way to transfer randomly generated mutations from the bovine papillomavirus E1 DNA binding domain (E1DBD) region expressed in a yeast plasmid into the full-length E1 gene in a mammalian expression vector. Using this method, we made 25 mutation transfers and isolated 21 mutants on the first round of DNA sequencing. This result represents a high first-pass mutagenesis efficiency of 84%. While our studies were confined to a single donor/recipient plasmid set, this method should function with any two appropriate plasmids and, thus, has general applicability to any situation where mutations need to be transferred from one vector to another. The exact protocol we use is as follows, and a diagram is shown in Figure 1. The initial PCR contains 25 ng mutation-containing template plasmid, 3 μL each flanking primer (5 μM stocks), 0.5 μL 10 mM dNTPs, 2.5 μL 10× Pfu buffer, and 0.5 μL Pfu DNA polymerase (3 U/μL) (Promega, Madison, WI, USA) in a 25-μL reaction volume. PCR cycling conditions include initial denaturation at 94°C for 1 min, followed by 35 cycles of 94°C for 3 s, 48°C for 30 s, 72°C for 2 min, and a final dwell at 4°C. The initial PCR product is isolated by electrophoresis on a 1% TBE agarose gel and extraction with a QIAex® II Gel Extraction Kit (Qiagen, Valencia, CA, USA) according to manufacturer’s protocol. The second PCR contains 25 ng wild-type plasmid to be mutated, 250 ng purified PCR product from first reaction in a volume of 10 μL, 0.5 μL 10 mM dNTPs, 2.5 μL 10× Pfu buffer, and 0.5 μL Pfu DNA polymerase (1.5 U) in a 25-μL reaction volume. PCR cycling conditions include initial denaturation at 94°C for 1 min, followed by 17 cycles of 95°C for 30 s, 50°C for 30 s, and 72°C for 2X min where X is the length of plasmid in kb, and a final dwell at 4°C. Five units of DpnI are added directly to the finished reaction, which is then incubated at 37°C for 1 h. DpnI diBenchmarks