I try to design specific primer to distinghuish two speicies. However, their housekeeping gene is highly conserved. It's hard to find polymorphic site. Finally, I found different region.
1. TTCTGAATTC
2. CTCACTATTG
Is it possible to design specific primer using this seqeunce??
if you keep these different seq in the very 3 end of the primer, you will be able to distinguish them. The primer should not be too long. We used to use PCR even to distinguish point mutation by only one base difference.
Thank you for your answer! I will make primer according to your advices. The amplicon size is 550 bp Then, Would it be possible to ask you how to set up PCR conditions? I usually used this conditions
1. 95 ℃ 5 min
2. 95 ℃ 30 sec
3. 58 ℃ 30 sec
4. 72 ℃ 1 min
5. 72 ℃ 7 min
6. 4 ℃ pause
Can I get proper results by using above conditions?
See if you can grab this
Article Allele-specific PCR in SNP genotyping
Article WASP: A Web-based Allele-Specific PCR assay designing tool f...
https://www.nature.com/articles/srep20631
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