Can anyone suggest me about the flourcent primers for Aflp analysis?.
FAM is the common flourcent dye which is used in one of the selective primers of the AfLp,
I want to lable my ECOR1 primer with flourcent dye at 5' end but my Primer start with G base. that will result me in decrease in flourcence.40% quencing.
Even other dyes which is suitable for Aflp all results in decrease in flourcence.
Is it ok to use the dye with decrease in flourcence? Or anyother alternative for it.
Any one please help me.
Thank you.
I do not believe that a 5' G makes that much difference ( and I have personally synthesised many dye labelled oligos) and if it did then just put an A (which does not match your sequence) at the 5'end of the primer and add the FAM to that base. Do not include the A in annealing temperature calculations for pcr. In my experience FAM and TET give good strong signals and you will be diluting your cut product greatly to get a good clean signal on a sequencer so any labelling is good enough. I used to dilute my samples up to 1:80 for loading as too strong a signal often looks like 1 large peak and its exact size is difficult to determine
thank you sir Paul Rutland for your valuable suggestion always.
i have another question, if i will modify my oligo by adding A at the 5' will it effect my polymorphic results?
2) FAM can be also labelled at 3' end without any decrease in florescence. can i label my primers at 3' end?
it will make all of your pcr products 1 base bigger than with the original primers and you should check that it will not cause hairpin loop formation within the primer but if this is the case just use one of the other bases at the 5' end instead
Sir @Paul Rutland how about the 3` modification, if i will label the 3' end with FAM or anyother dye.?
Thank you
Hello Mehvish,
your question is not completely clear to me so 2 answers . You must always label the 5' end of pcr primers because the extension of bases with Taq polymerase always takes place starting from the 3' end of the oligo and labelling at this end of the oligo will interfere with the pcr process. If you meant does it matter which end of the amplimer you want labelled by incorporation of the labelled oligo then the prime consideration is the size of the fragment that you are detecting . Genotyping standard are not good at sizing at very small ( less than 50bases) fragments so you will be labelling the fragment which separates well and is accurately sized when designing your genotyping experiment
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