Hello,
I'm using the QIAGEN Multiplex PCR Kit and 10 primer pairs (product sizes from ~130 to ~900 bps) to monitor mitochondrial DNA in Candida glabrata, a close relative of Saccharomyces cerevisiae. One primer pair target the genomic DNA, and is a positive control in case the mitochondrial DNA is completely lost (this is possible in yeast). The other nine amplify nine different genes on the mitochondrial DNA. Every product has a distinct and fixed size, and they separate well in a 2% agarose gel.
Every primer pair works fine when run alone, but when run together (for 45 cycles), I only get 7 bands. The 3 missing bands are the genomic DNA product and two of the mitochondrial rRNA subunits (SSU and LSU). The 7 bands that do work are all protein-coding mitochondrial genes, which suggests a pattern, although I don't have an explanation for it.
A number of observations that might be relevant:
1) In C. glabrata, the mtDNA is present in a much larger copy-number than the gDNA - I estimate about 20-50 times more, but I could be wrong. Still, given that PCR is exponential and I'm using 45 cycles, I should still get a band from the gDNA primer pair, even if weak.
2) The LSU and SSU products are quite GC-poor (20-23%), but the other mtDNA products are not much better (~24-32%), so I don't know if that matters.
3) All the primers were designed to have a similar Tm.
4) Running the PCR in a temperature gradient didn't seem to do much.
5) In rho- petites (yeast that completely lost their DNA) I see no bands from the mtDNA primers (as expected) but the gDNA band does appear (also as expected - perhaps because the mtDNA primers don't interfere with the reaction anymore?).
I can start playing around with the primers and their mixes, but I feel there's something theoretical I'm missing. In any case, any and all advice will be appreciated.
Thank you in advance!
Hi,
I have 2 points here and i hope they help.
1. I am assuming that you are using a commercial master mix for PCR. for low GC content template, you might need to have more Mg2+ in your PCR reaction. Maybe you want to try to supplement some MgCl2 in your PCR mix. (I had the problem when I PCR low GC products from Candida albicans genome, and supplementing Mg2+ helped.)
2. Have you checked the primer dimers across the pairs of primers you have? Could it be that some of the primers form strong dimers which resulted in missing of PCR products?
I'd second Jianfeng Li's second point about primer dimer across pairs. When I design multiplex pools I often run MFE-primer to check
Jianfeng Lin , Shaun Clare - thank you for your suggestions! I just checked using MFE-primer, and found no dimers. I am indeed using a commercial kit, the QIAGEN Multiplex PCR Kit. I will try adding MgCl2 to it.
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