I am developing a multiplex PCR assay to identify three strains of bacteria (A, B & C) of which two of them (A & B) are very close-related amongst each other. Two pairs of primers were designed for each strain A (red & yellow) and B -not shown-.
Size fragments are the following:
1. Red primers: 256 bp.
2. Yellow primers: 139 bp.
As you can notice, red and yellow primers amplify identical fragments on strains A & B, but do not any with C. On the right hand side of the gel, there is the mixture of primers for multiplex PCR on colour black and is comprised by the red primers for A and a pair of primers for B that generates a fragment size of 159 bp. Therefore, a band of 256 bp on strain A was expected with two observable bands of 256 & 159 on B.
However, cross priming amplification on A is lost and red primers do not amplify anything on B.
Why do you think I am seeing this? Note that there is more unbound primers at the end of the gel on multiplex bands. Am I losing Taq efficiency?
IMPORTANT CONSIDERATION:
a. All primers have same Tm.
MasterMix composition per rxn for single PCR (25 uL): Buffer 1X, MgCl2 2mM, dNTPs 200 uM, Primers 250 nM, Taq Pol 1 U/uL, add water to complete 20 uL + 5 uL DNA.
MasterMix composition per rxn for multiplex PCR (25 uL): Buffer 1X, MgCl2 2mM, dNTPs 200 uM, Primers 250 nM each pair, Taq Pol 1 U/uL, add water to complete 20 uL + 5 uL DNA.
1. Try to double these Buffer 1X, MgCl2 2mM, dNTPs 200 uM, Taq Pol 1 U/uL on multiplex pcr reaction mastermix. Perhaps, the polymerase can not working well because of lacking of these components
2. Have you checked between those of two primer (red and yellow) so they are not forming secondary structure each other?
Did you sort this out German Gomez
There may be structural reasons like a polymorphism under the 3'end of a primer in one sample which stops one primer set from working but one thing to try is the Mg. Both DNA ( especially if it is low quality) and primers remove Mg from the reaction so adding more primer sets may have reduced the available Mg and Taq needs at least 1mM Mg to work so I would try doubling the amount of Mg to the reaction mixture. Sample C could just be either the wrong dNA or poor quality DNA...has it amplified with any other primer set?
Mathew A Beale
Thanks for your reply. I have done some in silico analysis of the primers and as you say, there is cross amplification between primers designed for strain A when swapping pairs. However, there is none when swapping primer pairs for strains A and B.
Paul Rutland, thanks for your reply.
Not yet. I increased the amount of MgCl2 to the reaction, but I obtained a thicker band with a better resolution instead. Apparently, I can discriminate each strain when mixing both primers in the master mix ( I think due to the MgCl2), but lose all specificity when each pair is tested in a separate end-point PCR.
Thank for your reply, Bimo Satrio , I have gradually increased the amount of each reagent, but I only have got a thicker band with the same layout as the gel above. I also have done some in silico analysis and yes, there is some unspecific product between primers for strain A (red and yellow), but none when mixing and swapping primers A with B.
It seems that the primers have a problem then German Gomez in which case possibly keeping the temperature always hot may minimise this so possibly use a hot start enzyme if you are not already doing so
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