I have two mutations(of interest) on a gene, one of which is in intron (splicing region) and another coding sequence. i want to check two things:
whether the intronic mutation is resulting in a normal cDNA or not? How can I do that? The mutation is in a super long (4700bp) intron 2 of the gene and I checked with PCR and do not see any intronic product. Suggesting either the intron is not present on the cDNA or the mRNA is being degraded? Any thoughts/suggestions?
I also want to test if the two mutations are on same allele or different. Can anyone suggest something? They are about 1000bp apart on cDNA and ~6000bp apart on genomic DNA.
TIA
Answering the question are they on the same allele:
in cdna clone the sequence and sequence one clone. In gDNA design primers with a restriction site that does not exist in the sequence on each primer. amplify the sequence and cut then ligate into circular dna. this brings the mutations very close together. PCR amplify across the 2 mutations in a small amplimer and TA clone that and sequence.
Alternatively design a primer that onlt anneals to each change and run an ARMS pcr. Only one allele will amplify which corresponds to the perfectly matching primer and this can be sequenced at the end which has the other mutation. Repeat with the specific primer for the other forward allele
Assuming you have a live, diploid organism the easiest way to answer your question is old-school classic genetics.
You can use a genetic cross to see if the mutations are on the same DNA strand or not. Cross your organism to a wild type organism. Your loci will either segregate together 100% (same chromosome) or always separate (different chromosomes).
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