If you know what the nucleotide changes are then you can discriminate the sequences with allelic discrimination rt-PCR. Design two probes (each with different fluorophors, one for each variant) where the variant nucleotide occurs in the middle of the probe. With the appropriate annealing temperature a probe will destabilise and not bind to the alternative template sequence and will therefore not be cleaved. Of course dependent on the GC content and the combination of incompatible bases at the point of variation, some probes are better able to discriminate than others.

Alternatively if you only need a qualitative examination of sample you could design a primer pair where base change(s) occurs right at the 3' one of the primers. The idea is that if the last base pair of the primer isn't bound the polymerase should not be able to extend the sequence. Above a particular annealing temperature therefore you should be able to discriminate between the presence or absence of either sequence.

With both methods you will need homozygous DNA for each allele on which to optimise the primers and/or probe.

Dear Marcus Hanley

I am really appreciated for your kind comments I will check this carefully. When I've checked sequences briefly, homology was relatively high between two genotypes. Let me check again. Thanks a lot

best wish,

Jun

Agree with Marcus on this, in some cases even high resolution melt analysis (HRM) can be sufficient for allelic discrimination, even in the case of class IV SNPs. This works by use of an intercalating dye and the different strand dissociation temperature of the two targets which produces different temperature/fluorescence profiles. The advantages of this is that it is cheap and easy to perform, but is not quantitative nor as specific as the probe based approach.

Dear Benjamin Sanyaolu

Appreciated for your kind comments

I will try with this

Good day

Best wish,

Jun