How to genotype eds 5-1 arabidopsis mutant? It's SNP but no idea where it is. Articles were found only with contradictory primer sets.
Every articles are citing the same author and journal (Ausubel, The Plant Cell, Vol. 9, 305-316, March 1997), but I don't find any information where is the SNP mutation. On Arabidopsis.org portal they write only, that it's SNP, but no further data. I found more articles describing the genotyping method regarding this mutant, but if it is only one SNP, how can it be, that the primers are always designed to a different area of the sequence? I have 2 months left until I should figure it out, how to solve this issue. Any suggestions warmly welcomed! Thanks in advance for your help!
Hi Judit,
This article mentions mapping and sequencing of the eds5/sid1 mutant genes and suggests that eds5-1 has a SNP in the 9th exon (see figure 1C). You could amplify the genomic sequence with primers located 200bp upstream and downstream of the beginning of exon 9 and then perform sanger sequencing to identify the SNP, relative to the WT Ler sequence.
Hope that helps,
Johan
http://www.plantcell.org/content/14/1/275.long
Thanks Johan, I had the same idea, just I hoped to find a way without sequencing.
Hi,
if you already know the location of SNP, you can use the PCR-based method to genotyped your mutant.
Here the reference:
Article Simple allele-discriminating PCR for cost-effective and rapi...
I try several times and it works well, but probably you need to test several primer sets.
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