Hello dears
I want to design primers for validation of alleles in RIL population of two inbreed lines one is sensitive and second is resistance.
I have run the PCR of two lines and got the targeted sequence to identify the SNP and InDel. Now I want to distinguish the favourable allele variation in RILs.
Q1, how to design Primers ?
Hi Javed,
one simple, reliable and not expensive protocole should be for you ARMS PCR. you need to design 3 primers, one common and two designed for both alleles. there is an old paper, take advices in (Article Amplification-Refractory Mutation System (ARMS) Analysis of ...
).fred
You can use melt-curve based genotyping. Include a fluorescent dye in your PCR mix (I used SYBR Greener) and use a real-time PCR machine just for the melt-curve. You can easily detect small InDels and SNPS if your PCR products are SMALL (80-200 bp).
To design the primers, you need to know the DNA sequence that surrounds your SNP and/or Indels. I use Primer3 plus http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi
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