MadSci Network: Molecular Biology

Re: PCR inhibitors - how to get rid of them ?

Date: Mon Nov 20 19:09:17 2006
Posted By: Shireef Darwish, Grad student, Department of Plant Science, McGill University
Area of science: Molecular Biology
ID: 1162312400.Mb

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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