I am doing microvolume extraction which include physical( freeeze and thaw) and chemical lysis followed by a pcr base metagenomic library prep. My samples contain phytoplankton cultures with their microbiome. The mthod worked for most samples and timepoints but did not work for samples of one timepoint with high loads of phytoplankton and bacteria. I tried 3X dilution for direct PCR , bead-clean up and 3X dilution of sample and then bead-cleanup in case some inhibitors were hindering.
Looking forward for your scientific advice!
Thanks.
Strategies to mitigate these challenges... Hope you can use my thoughts..🙂
There are several strategies in my mind....
Sample Dilution: The concentration of inhibitors can be reduced by diluting the sample as attempted. However, this approach may also dilute the target DNA to below detectable levels. The efficacy of this method is contingent on finding an optimal balance between dilution and target DNA concentration.
Optimized Lysis Protocols: To ensure efficient lysis of phytoplankton cells while minimizing the release of inhibitory substances, it is essential to incorporate both physical (freeze-thaw cycles) and chemical lysis methods. To ensure efficient lysis of phytoplankton cells while minimizing the release of inhibitory substances, it is essential to incorporate both physical (freeze-thaw cycles) and chemical lysis methods. The lysis conditions, such as buffer composition and incubation times, should be adjusted accordingly.
Additionally, the use of inhibitor-resistant enzymes, such as certain commercially available DNA polymerases, can improve DNA yield and purity. Using such enzymes can improve amplification success rates in challenging samples significantly (Schrader et al., 2012).
DNA purification is crucial for enhancing DNA quality. DNA purification kits or methods designed to remove polysaccharides, proteins, and other potential inhibitors are recommended. Magnetic bead-based cleanup protocols, as mentioned, are effective but may require optimization for phytoplankton-rich samples.
Alternative extraction methods should also be considered. Considering alternative DNA extraction methods that include steps specifically designed to remove inhibitors can be beneficial. For example, the use of CTAB (cetyltrimethylammonium bromide) in the extraction buffer can help to remove polysaccharides (Doyle & Doyle, 1987).
Implementing qPCR with internal controls can aid in quantifying the extent of PCR inhibition and verifying the amplification efficiency. This method allows for the detection and correction of samples that exhibit significant inhibition prior to library preparation.
Regards,
Phil
Phil Käding Hi, Thank you so much for your comprehensive response. So true dilution of DNA is a very important consideration. I may go for X100 and get some amplification but diversity may not be comparable to the same volume of my original sample. CTAB is a very nice suggestion. I was also thinking about that since I used to use (Doyle & Doyle, 1987) method for my seaweed microbiome extraction, just I was not sure it worked well for enhancing extraction or inhibitor removal , but now I will go for it for sure :) I havent tested qpcr but I had a positive control with different amount of inhibiting samples added to that to see how amplification was affected through intensity of amplified DNA and could see the inhibiting impact but they could not totally inhibit the reaction. Do you think CTAB2% is a nice place to start?
Phil Käding Do you have any recommendation on a specific polymerase? I used the one from phusion kit. I can try KAPA hotstart.
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