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Site-directed mutagenesis for large insertions by oligonucleotide primers in optimized molar ratios.

Site-directed mutagenesis is a powerful tool to study gene expression and protein structure/function. There are numerous protocols to create deletion, insertion and substitution mutations of a DNA sequence of interest. The conventional method utilizes single-stranded (ss)DNA from M13-based vectors (1,5,6,9) and an oligonucleotide primer containing the desired mutation. This method requires the production of ssDNA, which often entails subcloning of the DNA sequence of interest into special vectors. Polymerase chain reaction (PCR)-based mutagenesis procedures (3,4,8) allow the quick creation of site-directed mutations; however, they either require the class IIs restriction enzymes or are subject to the difficulty of amplifying large plasmids with high efficiencies and the subsequent subcloning steps. The mutagenesis method described by Deng and Nickoloff (2) is a popular method to introduce specific base changes into virtually any doublestranded plasmid. This method relies on simultaneous annealing of two oligonucleotide primers to one strand of the target plasmid and DNA synthesis with T4 DNA polymerase, which does not possess strand-displacement activity. While one primer (mutagenic primer) introduces the desired mutation, the other one (selection primer) mutates a unique nonessential restriction site on the plasmid, thus allowing selection against the parental plasmid by restriction digestion. The efficiency to create single or multiple specific base changes and deletions are generally high, but insertion of a relatively large number of bases (>15 bp) is often associated with high background of plasmids, which do not contain the desired mutations. Insertion of a long DNA stretch requires the DNA-loop formation between the mutagenic primer and parental plasmid. Quite often, the problem of a high background is encountered by the generation of mutant plasmids that have resulted from the annealing of only the selection primer. To overcome this problem, we improved the method described by Deng and Nickoloff (2) by optimizing the molar ratio of mutagenic primer to selection primer. The improved procedure described below greatly increased the efficiencies of inserting a 34-bp sequence in front of the insulin response sequence (IRS) of fatty acid synthase (FAS) promoter in the 5.0-kbp parental plasmid p67-LUC. The mutagenic primer (5′-TGCATGCCTGCAGGTCGACCCCAGTGTCCTCAGTGCAGTTCCCAGTGTGACCAAGCCCATGTGGCGTGGCCGCGC-3′) was designed as a 75mer, which contains a 22and 20-base exact match to the parental plasmid sequence at its 5′ and 3′ ends, respectively, and incorporates the 34-base insertion (underlined) in the middle of the sequence. The selection primer (5′GCTGCAGGAATTCGATGTCGACCTTGGCATTCCGGTAC-5′) was designed from the polycloning-site region of the parental plasmid to mutate the unique HindIII site into a SalI site (underlined). Both primers were purified by reverse-phase cartridges (PE Applied Biosystems, Foster City, CA, USA) and phosphorylated with T4 polynucleotide kinase (Amersham, Arlington Heights, IL, USA) at standard conditions (7). Then the following steps were performed. Step 1. For DNA synthesis, a 20-μL annealing reaction containing 1× annealing buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 50 mM NaCl), 1 μg of parental plasmid p67-LUC, 10 pmol of selection primer and 10, 50 or 100