Unspecific Bands in PCR

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Nebulus's picture
Unspecific Bands in PCR

I am doing multiplex with 5 products. I am getting some unspecific banding and could use some advice on additives.
I have tried DMSO at 0% 2% 5% and 10% with significant inhibition at the 5% and 10% concentrations and no real help with the unspecific bands.
This is my first PCR optimization and do not know the benefits/downfalls to other additives such as formamide and BSA.
My primers are short 18-19bp due to the very close relatedness of the 5 species I am probing for and this made it nearly impossible to make species specific primers with longer sequences. My optimal Tm is 50 degrees for my multiplex reaction after runing trials with gradient PCR, thus can not use higher Tm to remove these unspecific bands without loosing 3/5 of my bands.
I was successful in amplifying every species with my designed primers and they were all specific in singleplex, but with over 1000 predators to screen, multiplex will make my life immensly easier.
Is formamide or BSA worth testing? Is there anything else I can try?
Here is my reaction w/o any DMSO ect.
10uL 10x Buffer
0.2uL Taq Polymerase
19.8uL PCR water
1uL of each for/rev primers X5 species =10uL (25ng/uL)
1uL template for each species x5- 5uL  (25ng/uL)
5uL dNTPs
Total 50 uL
I can't control for template concentration when I do my real experiment so modifying template isn't possible.
Many thanks

Ivan Delgado
Ivan Delgado's picture

Hi Nebulus,
I am sure you are aware that you are pushing the limits of multiplexing PCR. In other words, what you are trying to do is hard. After reading your post my first thought was to reduce the amount of template you are using. I understand that you cannot control template concentration, but can you reduce the amount of template you are adding to the reaction? This may help a little. Alternatively, if possible, could you change your DNA extraction technique? The purity of your DNA can have a very large effect on the specificity of your assays.
Do you know which one of the 5 assays is the one that is giving you the unspecific banding. In my experience it is typically only one of them, so if you can reduce your troubleshooting to that one you could save yourself some work. You've already optimized all these assays by themselves. Did you optimize a duplex and triplex assays? The goal here would be to optimize a duplex and triplex first, and in doing so isolate the assay that is causing the problem. Once you do that, my suggestion would be to re-design the assay that is causing the problems. Trying to optimize five assays together when one of them is causing problems could take a long time. 
Good luck.

Nebulus's picture
I will be using this as a

I will be using this as a tool for molecular gut analysis. I will be screening the guts of wild caught predators and thus cannot know how much DNA of a particular prey species, if any are present. Once I optimized my reaction I will be testing the performance of my primers at various ratios of prey to predator DNA and find the threshold of band detection.
I have been using Qiagen DNAeasy extraction kits to extract my DNA. I don't know if there is a better technique.
I have not tried duplex or triplex assays yet. I will consider it and see where the problem lies. The unspecific bands are pretty weak and was hoping I could get rid of them without redisigning my primers. I don't really have backup primers for multiplex since the sequences were so similar and it took me 4 months to desgin my 5 primer sets for 1 attempt.
Thanks for the advice

Ivan Delgado
Ivan Delgado's picture

As I said earlier, you have a tough situation with not knowing how much DNA you have and not being able to change any of your assays. One thing is for sure: by using the Qiagen DNEasy kits you are using one of the best DNA extraction kits available, so the quality of your DNA should be excellent. 
All the best