I want analyze specific polymorphism of a specific gene with Restriction Fragment Length Polymorphism (RFLP) method. So How can I design primer perfectly? Pls tell me the perfect procedure..
Dear Mamun, I think you are trying to design primers for a specific SNP not for a gene. For a specific SNP, you can easily design primer with Primer 3 plus that is available online.
We can use the Restriction Fragment Length Polymorphism (RFLP) method to analyze some polymorphism, if there has a restriction enzyme site in the polymorphism. However, some polymorphism can not be analyzed by this method.
I'm assuming you have selected your gene and know a restriction enzyme that will cut depending upon the SNP you are interested in. Select the region you are interested in ensuring your restriction site is included. Blast your sequence to ensure it is unique:
https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch
If you have trouble finding unique sequence you may have to considera larger region.
It would probably be useful to repeat mask it too:
http://www.repeatmasker.org/
Once you have good sequence design primers eitherside of tyour restriction site using Primer3:
http://primer3.ut.ee/
You can play about with the picking conditions to design someting that should work for you.
There are currently lot of soft wares are available for primer designing.For example Primer 3 is one among them. you can also calculates the basic criteria for prmer design using oligo calculator.
all the best
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