I did single point mutation using Inverse PCR method. But, when the purified plasmid was checked using the mutation and wild-type primers, both primers can amplify. What happens? I think only mutation primer should multiply. Now, I am preparing for sequencing. However, I would like to know how can I check the mutation occurs or not using traditional PCR.
if the mutation was introduced in the middle of one primer then it will have little effect on the annealing temperature of the primer and both normal and mutant primer sets will work, You get more specificity with ARMS pcr by designing a forwards primer with the mutation at , or very near, the 3' end of the primer then at the right temperature only the mutant primer will amplify. You might be better using the DCAPS method where you introduce a second change into a forward primer to create a restriction site which only happens when the first mutant base is present in the sequence. It shows very clearly that the mutant must exist if the enzyme cuts
dcaps id found at http://helix.wustl.edu/dcaps/dcaps.html
if the mutation was introduced in the middle of one primer then it will have little effect on the annealing temperature of the primer and both normal and mutant primer sets will work, You get more specificity with ARMS pcr by designing a forwards primer with the mutation at , or very near, the 3' end of the primer then at the right temperature only the mutant primer will amplify. You might be better using the DCAPS method where you introduce a second change into a forward primer to create a restriction site which only happens when the first mutant base is present in the sequence. It shows very clearly that the mutant must exist if the enzyme cuts
dcaps id found at http://helix.wustl.edu/dcaps/dcaps.html
You may follow Paul's suggestions. However, given the efficiency of PCR mutagenesis techniques, particularly if you use DpnI to destroy the wt template, most of the time picking up a couple of clones for sequencing is sufficient to find at least one clone with the desired mutation.
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