Primer Design

Question

[Koustav Maiti]

I have to make a specific primer, but it has only one nucleotide difference with others (like to make any primers of HLA allele); so in normal designing, I could not make any specific primer. Is there any tool or website available to make this specific primer?

Answer 1

[Antonio R. Franco]

If Taq polymerase is used (or any other without proof-reading abilities), you can leave the modified base as the last base in the 3' end. This will avoid efficient priming and amplification

[Koustav Maiti]

Sorry sir, I could not get it. Can you please elaborate.

[Antonio R. Franco]

You mention a nucleotide difference. Leave the primer to end in its 3' with any of these variable bases

If the genome sequence has a T and you ended the primer with an A in its 3' end, the primer will work and you will get amplification. But if you ended the primer with a C, G or T, the primer will not work because Taq polymerase lacks 3'5' exonuclease proofreading activity. It will not replace the wrong base with the right one

In addition. Look for LCR PCR https://en.wikipedia.org/wiki/Ligase_chain_reaction