Primers base mismatch

Extension of single base mismatches by AMV RTase occurs with varying efficiencies, ranging from 10" for G T (primer/template) to 10 for G G compared with G C. The discrimination in mismatch extension is ascribed primarily to a kinetic effect rather than to the difference in polymerase binding affinity to the 3 termini of the mispaired primer (95). 

Illustration of a general method of mutagenesis using PCR. Primers are represented as short lines with arrowheads pointing in the 3 direction. The bump in primers 2 and 3 and their products represent a mismatched base, a deliberate alteration in base sequence from that present in the starting DNA. Of the four major products resulting from step 3 only D is extendable by DNA polymerase. 

The Allele-Specific Amplification Assay (ASA) assay is based on the fact that Taq polymerase will not initiate amplification from a primer that has a mismatch at the 3 ends. Two primers are designed so that the 3 base of the primer corresponds to the site of the genetic mutation to be tested, with either the normal or the mutant sequence at the 3 base positions. An unknown sample can then be tested for the presence of the mutation by using both the normal and the mutant primers in PCR with a common reverse primer. If the sample contains only normal sequence, a PCR product will only be produced when the normal primer is used, and similarly when the sample contains mutant sequence a product will only result from use of the mutant primer. Like the PCR-restriction enzyme method discussed, the ASA approach has also been applied to the detection of mutations in the CYP2D6 gene (16). 

The 3 -+5 exonuclease activity plays an important role in polymerization in proof reading the base pair formed at each polymerization step. The enzyme checks the nature of each base-paired primer terminus before the polymerase proceeds to add the next nucleotide to the primer. It thus supplements the capacity of the polymerase to match the incoming nucleotide substrate to the template. A mismatched terminal nucleotide on the primer activates a site on the enzyme which results in the hydrolysis of the phosphodiester bond and the removal of the mismatched residue. The function of this 3 - 5 exonuclease activity is therefore to recognize and cleave incorrectly or non-base paired residues at the 3 -end of DNA chains. It will therefore degrade single stranded DNA and frayed or non-base paired residues at the ends of duplex DNA molecules provided they terminate in a 3 -hydroxyl group. 

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. 

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