Mismatch oligonucleotide primer

In order to introduce point mutations, a two stage-PCR reaction is carried out using a mismatched oligonucleotide primer containing appropnate base change(s) and the universal Ml3 forward and reverse primers (Fig. 3). 

Although the number and location of mismatched bases will affect PCR amplification, mismatches at the 3 terminus are expected to have the greatest effect on the PCR. Kwok et al.9 reported on the effects of 3 -termi-nal mismatches on amplification of HIV sequence. In their system, A-G, G-A, C-C and A-A mismatches were detrimental while all other mismatches had minimal effect. Most notably, oligonucleotides with a 3 -ter-minal T served efficiently as primers even when mismatched with T, C, or G. The concentration of deoxynucleoside triphosphates (dNTPs) in the reaction mix also affects Taq polymerase extension. For example, whereas most 3 -terminal mismatches with the exception of A-G, G-A, C-C, and A-A amplified efficiently in the presence of 800 fiM dNTPs, only T-G and G-T mismatches and perfectly matched sequences amplified in the presence of 6 fiM dNTPs. Thus, 3 -terminal mismatches can be more efficiently extended if the primers terminate in a T and if amplifications are carried out at higher dNTP concentrations. 

Figure 6.36. Oligonucleotide-Directed Mutagenesis. A primer containing a mismatched nucleotide is used to produce a desired change in the DNA sequence.

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