PCR inhibitor

Paterson, R. R. M. (2004). The isoepoxydon dehydrogenase gene of patulin biosynthesis in cultures and secondary metabolites as candidate PCR inhibitors. Mycol. Res. 108, 1431-1437. 

Another major problem associated with the extraction of DNA from archaeological specimens is that the procedure often co-extracts impurities that can later complicate, or prevent, the study of the extracted DNA by inhibiting PCR amplification (reviewed by 5). Commonly encountered inhibitory substances found in aDNA extracted from teeth, bones, mummified tissue, and coprolites include humic acids, ftilvic acids, tannins, porphyrin products, phenolic compounds, hematin, and collagen type I (37—42). The formation of Maillard products, commonly encountered in coprolite samples, can also prevent PCR amplification by causing DNA to become inaccessibly trapped in these sugar-derived condensation products (12). As the negative results in many aDNA studies are attributed to the presence of PCR inhibitors, our extraction method outlined below pays particular attention to the problem and offers a simple test for the presence of PCR inhibitors in DNA extracts. 

To facilitate removal of co-extracted PCR inhibitors (5, 44), precipitate DNA from the solution by adding one half volume of room temperature 5 M ammonium acetate and, to this combined volume, one volume of room temperature absolute isopropanol. Store the solution for a minimum of 7 hours at... 

If an aDNA extract frails to PCR amplify it should be tested for the presence of PCR inhibitors. This test requires the availability of an authenticated aDNA sample to be used as a positive control.8 Set up side-by-side PCR reactions containing 1) the template suspected to contain inhibitors, to which is added a volume of the ancient positive control equivalent to that of the template, 2) only the template suspected to contain inhibitors and 3) only the positive ancient control. This side-by-side comparison will allow for the preclusion of PCR failure due to factors other than inhibition (e.g. the stochastic nature of PCR amplification). If the template spiked with the positive ancient control (reaction 1) permits its amplification, while the template suspected of containing inhibitors (reaction 2) fails to amplify, the template is likely free of inhibitors and, therefore, does not contain a sufficient amount of DNA for analysis. Alternatively, if the first PCR reaction fails to amplify, whereas the third reaction does amplify, the template is concluded to contain inhibitors. In this case, the silica extraction should be repeated, as described above, and PCR reattempted. Our studies have shown that as may as four repeat silica extractions may be required to sufficiently remove PCR inhibitor from DNA extracts, despite the inherent loss of DNA yield associated with each repetition of the silica extraction (5). 

As illustrated by the examples above, the analysis of DNA extracted from archaeological specimens can provide unique insights about the past. However, the study of aDNA is methodologically challenging, primarily due to the problematical state of its preservation. As such, protocols developed specifically for the extraction and analysis of ancient DNA have been developed, of which we have highlighted one that we have developed over the past few years. Our protocol offers solutions to two of the most common problems associated with the study of aDNA the possible presence of contamination on the surfaces of samples and/or the co-extraction of PCR inhibitors. We are optimistic that with attention to proper aDNA protocols, data acquisition, and authentication of results, DNA extracted from archaeological specimens will continue to provide a wealth of information about the past. 

In a later report, inhibition of PCR in Si-glass PCR chips was found to be mainly caused by the adsorption of the Taq polymerase, rather than by the adsorption of DNA [299]. It was also found by XPS analysis that the primary PCR inhibitor in a glass PCR chamber was Cr, which was involved in the microfabrication process. The highest Cr concentration that could be tolerated was found to be 0.1 M [932]. 

Hemoglobin and heparin present in blood samples are also PCR inhibitors. In one report, these inhibitors were removed from yeast cells (used as the model for blood cells) after DEP cell retention. The yeast cells were retained by DEP, whereas bovine hemoglobin (1 mg/mL) and heparin (13 pg/mL) were washed away [933],... 

PCR is a resilient process and does not require highly purified nucleic acid. In practice, however, clinical samples may contain unpredictable amounts of impurities that can inhibit polymerase activity. To ensure reliable amplification, some form of nucleic acid purification is often used. The idiosyncratic nature of PCR inhibitors within clinical specimens requires demonstration that the sample (or preparation of nucleic acid purified from it) will allow amplification. A control nucleic acid sequence, usually different from the target, can be added to the sample (or extract from the sample). Failure to amplify this control indicates that further purification of the sample is required to remove inhibitors of the reaction. 

In recent years, several methods for the isolation of DNA from biological material have been developed, and kits are commercially available. The method used depends on the consistency of the biological material, the ratio expected for the amount of DNA per amount of biological material, the potential presence of PCR inhibitors in the biological material, and the instruments or pipetting machines present in the laboratory. 

PCR inhibition control control containing a known amount of positive template DNA added in the same amount of analyte DNA as the reaction (that is to be controlled) (this could be the original target or a spike, e.g., a slightly modified target such as a competitor plasmid). This control allows determination of the presence of soluble PCR inhibitors, particularly necessary in the case of negative amplification. 

Internal positive controls (IPCs) can also be used to identify the presence of PCR inhibitors. IPCs are definitively useful for detecting false-negative results however, they cannot be used to determine a precise measure of inhibition strength when template samples are marginally compromised. Comparing the amplification efficiencies of clean standards with those of unknown samples is a statistically sound method that can be used in conjunction with IPCs when amplifications are successful but are compromised, producing erroneous quantification results. 

Kontanis EJ, Reed FA (2006). Evaluation of real-time PCR amplification efficiencies to detect PCR inhibitors. J Forensic Sci., 51(4) 795-804. 

Extraction of quality DNA, concentration of DNA per reaction, and elimination of PCR inhibitors... 

To ensure the extraction of DNA from foods, mainly from highly processed products and the presence or absence of PCR inhibitors, many assays for food allergens include a universal eukaryotic primer pair (Brezna et al., 2006a, b Brezna and Kuchta, 2008) or plant-specific primer pair used to amplify the noncoding region of chloroplast (Rossi et al., 2006 Watanabe et al., 2007 Yamakawa et al., 2007 Yano et al., 2007). 

Sensitivity of qPCR may also be improved by diluting the DNA template purified from skin samples, which may contain PCR inhibitors (19). Prior to use as qPCR template, we routinely diluted DNA extracts from skin samples 1 10, 1 50, or 1 100 in DNA resuspension buffer, depending on the weight (<5, 5-7, or 7-10 mg, respectively) of the original skin biopsy. 

The reliability of the PCR (polymerase chain reaction) is dependent on many external factors, such as false-negative results caused by uncontrolled PCR inhibitor, but also by contamination with foreign DNA. Special attention has to be given to the breakdown of the PCR by contamination through the air the contamination... 

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