Hi!
im trying to genotype WT and heterozygous mutant zebrafish for a gene that’s only has 1 bp different between the WT and mutant alleles.
I’ve tried designing primers such that at the mutation site, the 3’ site of a forward primer binds to the mutant allele and the 3‘ site of a reverse primer binds to the WT allele, and then have two other forward and reverse primers before and after the mutation on the genome that would give two different fragment lengths.
The predicted primer temperatures for these 4 primers range between 56 and 61 C, so I also tried using touchdown PCR, but I have yet to be successful with seeing correct band lengths on a gel.
Any advice would help!!
Can you reach a real-time PCR instrument? If the answer is yes, you can use high resolution melting curve method (HRM) instead. HRM is more simple with a pair of primers flanked the mutant point, the products should be around 100bp. Maybe you can have a try. HRM works well in our lab.
Have you tried design mismatch-primers? They would introduce a mismatch in the PCR (you can choose where, but I would suggest close to the mutation spot) and then sequence the product via Sanger!
Also, if you're getting aspecific bands, maybe they're targeting a pseudo-gene in zebrafish or something with a similar sequence. At that point you can try to increase the temperature (higher than predicted) or design new primers (and perhaps doing it by hand and not using tools, so you can actually check if there are any SNPswithin primer sequence that could prevent binding).
Hope this could be helpful!
I agree with the above that a qPCR based approach is probably prudent. I've used qPCR to genotype single-bp mutations in the past, but it would take some digging to get the references.
There may be an alternative method, in case running qPCR isn't feasible for you. If the BP difference produces a restriction site, you may be able to PCR around the mutation then digest the products to check for the presence of the mutation (e.g. a normal might have one band, and a mutant might have two smaller bands). You can check whether this is feasbile using NEBcutter real quick.
https://www.labtools.us/nebcutter-v2-0/
Dear Mr. Aaron Baugh!
You can genotype your expected heterozygous by using the SNP-RFLP (in other words PCR-RFLP) if any restriction site is available at the surrounding your target base pair. Just simply you can design primers to amplify target seq including heterozygous base (try to keep variation in terms of PCR product length from the heterozygous base). Then you can do restriction digestion by a specific enzyme and quantification by the gel electrophoresis. For the heterozygous, you will get both digested band and the undigested band as well.
Please go through the following references if needed:
Reference paper:
1. Article Genotyping of single nucleotide polymorphisms using the SNP-...
2. https://www.nature.com/articles/nprot.2007.407
3. To check wether the restriction site is available or not at your target site, you may check from here: http://watcut.uwaterloo.ca/template.php
Wish you all the best!
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