Thnx for getting back to me.. Well, I really regret now, why dint I keep a screenshot of that. Moving on the topic, let me make myself clear.
Lets say, you are aligning three sequences (1,2,3) and want to design three different sets of LAMP primers, specific for the three sequences aligned. In other way, a specific set of primers (designed for seq 2) should not amplify other sequences (seq 1 and 3). So, the logical way of designing such primer would be, compare "sequence 2" with respect to the aligned file and design a primer (say F2) for "Seq 2" such that the mismatch is included towards the 3' end of F2. Then, that primer would amplify only the "Seq 2" and would fail to amplify Seq 1 and 3. Am I clear?
What I feel is this mismatch should be located at the extreme 3' end of F2 or within 6 bases from the 3' end of F2 (Eiken's concept). But I have seen in many cases that PrimerExplorer reports some primers which includes mismatches beyond the 6th base (say at 8th or 9th base from 3' end) and the other bases towards 3' end are in conserved region on the aligned file. My question lies here. Will the mismatch located at 8th or 9th base impart specificity to the primer (F2)? Can this F2 primer also amplify Seq 1 and 3, since its 3' end region (last 6 bases from 3' end) is designed on conserved region?
I will surely attach screenshot if i get to see such scenerio again.