2 replies to this topic

[Ameya P]

Hi,

I am trying to standardize a protocol for ARMS PCR using primers designed by someone else.
The last time I ran the PCR products on the gel, all samples were seen as heterozygous (although one of them was a positive control and should have shown a band in only one of the lanes).

So I sat down and looked at the primer sequences to find out that the 1 bp difference between the primer pairs is for the last nucleotide of the primer. I think this is the reason why everything turns out heterozygous, irrespective of what it is. Is my conclusion correct???
Also, if I had to redesign these primers, what would be an appropriate placement of different nucleotide from the 3' end???

Thanks  

[Trof]

AFAIK there should be mismatch on the last 3' nucleotide. Other mismatch maybe added to the primer near 3' end to increase specificity. If you getting all samples heterozygous, then you should try to optimise your PCR reaction, use more stringent conditions.

Here is one picture and legend about ARMS.

[Ameya P]

Trof, on 20 September 2010 - 04:45 AM, said:

AFAIK there should be mismatch on the last 3' nucleotide. Other mismatch maybe added to the primer near 3' end to increase specificity. If you getting all samples heterozygous, then you should try to optimise your PCR reaction, use more stringent conditions.

Here is one picture and legend about ARMS.

Actually, the ARMS PCR I have used so far, have just three primers. A common primer and two primers, with a bp difference to screen for the mutation. So depending on the presence/absence of the mutation, I either see/don't see a band in the well. Its just recently that I cam across the true concept of ARMS. I shall optimise the conditions, before I redesign primers.