Point Mutation Primer Calculator

Mutation Miner

Compare the Melting Temperature of PCR Primers with Point Mutations.

Mutation Miner is a bioinformatics program that has been developed to compare the melting temperatures (Tm) of two different primers that have had point mutations introduced. Primer sequences and target DNA sequences can be inputted to calculate percent of mismatches present.

The melting temperature (Tm) is a function of three variables: %GC, N, and %mismatch.

%GC is the percentage of the total number of nucleotides in the primer that are either guanine (G) or cytosine (C). These two nucleotides have a higher melting temperature than the other two nucleotides (adenine (A) and thymine (T)), so a higher %GC will result in a higher Tm
N is the length of the primer in bases, or nucleotides. As the length of the primer increases, the Tm will generally decrease
%mismatch is the percentage of nucleotides in the primer that do not match the target sequence. A higher %mismatch will result in a lower Tm

Notes:

Primers should aim for a minimum GC content for 40%
Primers should aim for a minimum length of 7 nucleotides
Desired mutation must be in the middle of the primer with >/= 15 bp on both sides of the mutation/insertion/deletion
3’-end of primers should end in 1 or more G or C

Mutation Miner

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