I am working with haploid ascomycete fungus and I am currently looking for a method of point mutation detection in a target gene of the organism. Based on the preliminary results of sanger sequence of few isolates I found that there are two different point mutations present in populations that confer resistance to fungicides by the fungus. Both these mutations are present in the same codon of the target gene, either the first nucleotide substitution or the second nucleotide substitution. Both these mutations lead to two different aminoacid substitution (e.g., if the mutation is in first codon, then the aminoacid H changes to R, if the mutation in the second codon, aminoacid changes from H to Y).
I need to find the way to fast and cheap screen populations of the fungus on the presence and frequencies of these mutations.
I have tried allele-specific PCR (so far just with two primers, forward and reverse, separately for each mutation), but the one of the primers pair that is supposed to be specific just to one SNP is not specific and amplifies the region in individuals with different mutations.
Now I am reading about tetra-ARMS PCR methodology but cannot find an information if anybody tried it in haploid organisms, and also looking for any commercial products (such as kits etc.) that could help me to detect these mutations using PCR.
Has anybody had a similar issue/task? Or have experience with allele-specific PCR methods that are robust?
I will greatly appreciate any help and suggestions that could help me to develop reliable method.
Thank you!
P.S. RFLP-PCR is not applicable here since there is no restriction site present that could distinguish between these mutations.
I understand that one of your ARMS primers is working so the problem is how to get the other change to work. Have you considered ACRS/DCAPS primers for this one?
DCAPS is where you have a sequence that is not a RE site but for most sequences if you make 2 changes then it will be a RE site about 97% of the time. Your first change comes from the mutation and the second change comes from a deliberately designed mismatch in the pcr primer.
So for instance if your sequence is CAGT and the mutation gives you CAGG then a primer ending with GGC will give a sequence with CCGG only when the mutation is present which cuts the primer off the pcr product with MSP1 There is a programme that designs your mutated primers at http://helix.wustl.edu/dcaps/dcaps.html. I have used this many times and it can work very well and usually comes up with 5 or more changes and generated RE sites to give you a good choice of sequence /cost/commercial availability. Often you add some bases to the 5' end of the primer just so the cut size of the amplimer is well separated from the uncut full length amplimer
Hi Tetra-ARMS PCR would be useful. If its in haploid organism only thing is that you wont get heterzygous mutants, however still you would be able to get ARMS results that would be in concordance with sequencing
- Badges
- Science method