Digital PCR DNA Preparation
DNA preparation is a crucial step for the success of your digital PCR assay. Template extraction, its quality control, and its storage are important parameters to monitor.
Template extraction
Digital PCR has been reported to be compatible with DNA & RNA templates extracted by various extraction methods used in laboratory- from phenol chloroform method to modern extraction kits1,2,3,4,5,6
Quality control
For optimal digital PCR performance, you should check the purity and the quality of your template. Using relevant methods such as spectrophotometry, you will be able to verify the good absorbance (A260/230 and A230/260) ratios of your extracted DNA solution. The presence of contaminants and/or potential inhibitors should be avoided.
Storage
The storage of your extracted DNA template is also an important factor to monitor. Indeed, for example, you need to avoid base degradation such as cytosine deamination and 8-Oxo-2′-deoxyguanosine formation due to oxidative damage since it may lead to base transversion during PCR amplification.
Buffering and temperature conditions have an impact on the quality of your sample. Storing at -20°C in Tris-EDTA is the standard condition, but please do not hesitate to take a look at the extraction kit manufacturer’s recommendations.
Other parameters, inherent to the sample itself, might have an impact on your digital PCR experiment and should be verified:
DNA with high GC content
If your template contains a GC-rich region, this might conduct to an incomplete amplification. Indeed, GC bonds are highly stable. Thus, in order to obtain a better denaturation of your template, you can try to add DMSO or betaine in your PCR mix.
High molecular weight DNA
High molecular weight DNA or plasmids have been reported by some manufacturers as problematic for partition generation. Thus, we recommend DNA shearing using chemicals or enzymatic methods prior to performing digital PCR. Please note that the digestion step might be performed directly in the PCR mix.
Finally, before starting your digital PCR assay you will have to proceed on the:
Conversion of mass of DNA in number of copies
If you are a qPCR user, you may be used to thinking in terms of mass of DNA input. In digital PCR, you will need to think in terms of the number of copies of genes or genomes in your reaction volume. In order to proceed on the conversion, the only thing you will have to know is the mass of the studied genome. Then, simply apply this formula: Number of copies in reaction volume = mass of DNA in reaction volume (in ng)/ mass of the studied genome (in ng) For more details about how to perform this calculation, refer to Part 3a of Rare Mutation Detection Tutorial.
Online calculators are also available :
- http://cels.uri.edu/gsc/cndna.html
- https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/dna-copy-number-calculator.html
Bibliography
1 Cai, Y., Li, X., Lv, R., Yang, J., Li, J., He, Y., & Pan, L. Quantitative Analysis of Pork and Chicken Products by Droplet Digital PCR. BioMed Research International, 2014, 810209. http://doi.org/10.1155/2014/810209. PMID: 25243184
2 Pérez-Barrios, C., Nieto-Alcolado, I., Torrente, M., Jiménez-Sánchez, C., Calvo, V., Gutierrez-Sanz, L., Palka, M., Donoso-Navarro, E., Provencio, M., Romero, A. Comparison of methods for circulating cell-free DNA isolation using blood from cancer patients: impact on biomarker testing. Transl Lung Cancer Res. 2016 Dec; 5(6):665-672. doi: 10.21037/tlcr.2016.12.03. PMID: 28149760
3 Demeke, T., Malabanan, J., Holigroski, M., Eng, M. Effect of Source of DNA on the Quantitative Analysis of Genetically Engineered Traits Using Digital PCR and Real-Time PCR. J AOAC Int. 2017 Mar 1;100(2):492-498. doi: 10.5740/jaoacint.16-0284. Epub 2016 Dec 22. PMID: 28118137
4 Holmberg, R.C., Gindlesperger, A., Stokes, T., Lopez, D., Hyman, L., Freed, M., Belgrader, P., Harvey, J., Li, Z. Akonni TruTip® and Qiagen® Methods for Extraction of Fetal Circulating DNA-Evaluation by Real-Time and Digital PCR. PLoS One. 2013 Aug 6;8(8):e73068. doi: 10.1371/journal.pone.0073068. Print 2013. PMID: 23936545.
5 Rajasekaran, N., Oh, M. R., Kim, S.-S., Kim, S. E., Kim, Y. D., Choi, H.-J., Byum, B., Shin, Y. K. Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines. Journal of Visualized Experiments : JoVE, 2015, (104), 53190. Advance online publication. http://doi.org/10.3791/53190. PMID: 26484710.
6 Devonshire, A. S., Whale, A. S., Gutteridge, A., Jones, G., Cowen, S., Foy, C. A., & Huggett, J. F. Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency, fragment size bias and quantification. Analytical and Bioanalytical Chemistry, 2014, 406(26), 6499–6512. http://doi.org/10.1007/s00216-014-7835-3. PMID: 24853859
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