Why is one species DNA favored in mixed sample PCR using universal primers

[brendan101]

Hello,

I am currently using a conventional PCR with a probe based assay (reverse line blot) to identify species of nematodes. I am using universal primers and I have noticed that some species template DNA is favored over another species in a mixed sample during the PCR even when it is present in far lower concentrations than the non amplified species. I know that one species is being favoured because when I amplify either template on there own I get a positive result on my reverse line blot, but not when they are mixed. The universal primers are an exact match for both species and the amplicon is roughly the same size, at most a 20 bo difference also the sample qualities are very similar in regards the 260/280 ratio.
I have sequenced the different species myself and I am sure that the primers are an exact match for all species
Can anyone help explain why one species template is favored over another when using universal primers in a mixed sample.

B

[danfive]

So you have "truely" universal primer pairs but different PCR efficiencies.

This can be due to several reasons (no special order):
1. PCR conditions (buffer, annealing temp) favor one species over the others.
2. Perhaps one species has significantly higher G-C ratio, thus stronger complimentary base pairing interferes with melting + annealing steps.
3. One species has a significantly smaller PCR product, the shorter product has the advantage and will tend to be amplified a greater number of time.
4. One species tends to have a tendency for self-annealing or hairpin loops in the target region.

[danfive]

Originally Posted by danfive

So you have "truely" universal primer pairs but different PCR efficiencies.

This can be due to several reasons (no special order):
1. PCR conditions (buffer, annealing temp, Mg++ conc'n) favor one species over the others.
2. Perhaps one species has significantly higher G-C ratio, thus stronger complimentary base pairing interferes with melting + annealing steps.
3. One species has a significantly smaller PCR product, the shorter product has the advantage and will tend to be amplified a greater number of time.
4. One species tends to have a tendency for self-annealing or hairpin loops in the target region.

5. Competitive binding from highly homologous DNA. If the DNA is so similar, the self complimentarity between strands may create greater rates of DNA-DNA annealing and crowd out primer annealing. Best solution to this is use less initial genomic DNA, PCR adjuvants like BSA, maybe higher Primer conc'n.

[brendan101]

Thanks a million, I will try that