|MadSci Network: Molecular Biology|
Hey Seb! I am assuming that you are also working with samples of herbivorous animal feces or raw plant material. First, plant-derived phenolic compounds will inhibit Taq polymerase, whereas cross-linkages between reducing sugars and amino groups (Maillard reaction) may occur (i.e., if the temperature of the animal gut is high enough) and will inhibit amplification of the DNA. It sounds like you are familiar with the paper by Monteiro et al (1997) [referenced below], but if not that would be an especially pertinent resource to start with. The authors outline a modified protocol based on Qiagen’s QIAamp DNA blood extraction kit, which employs silica gel packed columns to capture the DNA under high salt conditions while proteins and polysaccharides pass freely through the column. Qiagen also offers a QIAamp DNA Stool mini kit designed especially for extracting high quality DNA from feces. However, you may be able to optimize your extraction and eliminate most of the PCR inhibitors with the following approaches: The addition of Proteinase K to your extraction will disrupt contaminating proteins (and glycoproteins?), including DNase and RNase, and protein complexes that could reduce the extracted DNA quality. Qiagen uses Proteinase K in their Stool DNA extraction kit for these reasons. Try adding PVP (polyvinylpyrrolidone) to your extraction buffer or to the PCR reaction itself (see Koonjul et al. reference below). PVP binds polyphenolic compounds typical of plant tissues, facilitating their removal during extraction. The inclusion of certain reducing agents (eg., sodium metabisulfate, dithiothreitol) in the extraction buffer may also be something you want to consider (see Horne et al. reference below). Performing additional chloroform extractions of your samples followed by 1-2 precipitations in cold 70% ethanol would be the most straight-forward and common approach to cleaning up DNA extractions. This will usually remove most of the PCR inhibitors present in plant extractions. I hope these suggestions help you. Good luck and thanks for your question! Sincerely, shireef darwish Monteiro L., Bonnemaison D., Vekris A., Petry K.G., Bonnet J., Cabrita R.V.J., and Me’Graud F. (1997). Complex polysaccharides as PCR inhibitors in feces: Helicobacter pylori model. Journal of Clinical Microbiology, 35(4): 995-998. Koonjul P.K., Brandt W.F., Farrant J.M., and Lindsey G.G. (1999). Inclusion of polyvinylpyrrolidone in the polymerase chain reaction reverses inhibitory effects of polyphenolic contamination of RNA. Nucleic Acids Research, 27(3): 915-916. Horne E.C., Kumpatla S.P., Patterson K.A., Gupta M., and Thompson S.A. (2004). Improved high-throughput sunflower and cotton genomic DNA extraction and PCR fidelity. Plant Molecular Biology Reporter, 22: 83a-83i.
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