Can you scan it?

Could you send me the gel image?

In ARMS PCR, primer designing is a critical step and many failures are due to weak primer design.

The following items must be considered while designing ARMS primers:

1- Always use a control primer (for normal allele) beside the ARMS primer.

2- In many cases a single nucleotide mismatch in the 3' of ARMS primer is not enough to detect the mutated allele from normal allele, and both could be amplified with this kind of primer very well. In general 2 consecutive mismatches at the 3' end gets you a satisfactory result.

3- Although in designing ARMS PCR primer, user is limited to maneuver on the one primer and on the ARMS primer only the length could be adjusted by user, but you should always run a BLAST for designed primers to ensure non specific binding is not happened.

4- Design a primer pair with higher Tm as you can.

5- Using Allele specific primer designing programs such as Beacon Designer could be helpful. This program gives you the optimal counterpart of your ARMS primer. You need only to specify the mutated base and some general parameters, and the software gives you the best possible result.

6- Initially setup your ARMS PCR with the normal allele to check the specificity of your work then go to the next step.

7- Always use a validated Control for both normal and mutated allele beside your work to ensure all conditions and reagents work very well.

Dear prasanata

how is you primer design ?please give us more information.one reason of ARMS mismatch is about primer design. do u observe it with mutant primer?

to detect the β-thalassaemia mutation (G->C), the 3' nucleotide of the ARMS primer is G in order to base pair with the substituted C in the mutant DNA. The primer forms a G-G mismatch with normal DNA, but this is a weak mismatch and will not prohibit extension of the primer by itself.

Only strong mismatches (C-C, G-A and A-A) reduce priming efficiency to zero or below-5%.

to prevent amplification, a further mismatch with the target sequence had to be introduced at the second, third or fourth nucleotide from the 3' end of the primer .

As a general rule for ARMS primer design, if the 3' terminal mismatch is a weak one, a strong secondary mismatch is engineered. If it is a strong one, a weak secondary mismatch is introduced.

after putting the mismatch at the second nucleotide you should test the primer for specificity and generation of product. if non-specific bands are observed (the strength of the mismatch increased) or if the primer does not work , the position of the mismatch can be altered .

The strength of mismatch pairings are; maximum, GA, CT, TT; strong, CC; medium, AA, GG; Weak, CA, GT; none, AT, GC.

may be you can find better prinmer design if you check mutation-specific ARMS primers used in the Oxford laboratory to diagnose the 25 most common β-thalassaemia mutations, plus the hemoglobin variants HbS, HbC and HbE, All are 30 bases long so that they can all be used at a single high annealing temperature (65 oC).

Dear Prasanata

You ought to use Tetra ARMS PCR which is more reliable than general ARMS. In line with this premise, you are able to design your specific primers by evoking http://cedar.genetics.soton.ac.uk/public_html/primer1.html. If you're NOT interest about it, Please show us one of your Gel image.