PCR inhibition

When analysing forensic samples a problem that can be encountered is inhibition of the PCR [29]. DNA extraction methods do not produce pure DNA, some chemicals will co-purify and in some cases inhibit the Taq polymerase. Potent inhibitors include haem compounds from blood [30-32], bile salts and complex polysaccharides from faeces [33,34], humic substances from soil [35] and urea from urine [36]. High concentrations of ions, in particular calcium and magnesium, can also act as potent inhibitors of the Taq polymerase [37]. EDTA is used in high concentrations for the isolation of DNA

Figure 5.4 (a) The target sequence on the template DNA molecule has been amplified using two different primer pairs: using primers a + c generates a 460 bp product and the a + b primer pair leads to a 260 bp product. (b) Following amplification of template DNA in two separate reactions the products were separated on a 2 % agarose gel and stained with ethidium bromide. The amplified products can be seen in lane 2 (460 bp) and lane 4 (260 bp). Lane 1 contains a 100 bp ladder and lanes 3 and 5 contain negative controls

Figure 5.4 (a) The target sequence on the template DNA molecule has been amplified using two different primer pairs: using primers a + c generates a 460 bp product and the a + b primer pair leads to a 260 bp product. (b) Following amplification of template DNA in two separate reactions the products were separated on a 2 % agarose gel and stained with ethidium bromide. The amplified products can be seen in lane 2 (460 bp) and lane 4 (260 bp). Lane 1 contains a 100 bp ladder and lanes 3 and 5 contain negative controls from bone and will inhibit PCR unless removed as it binds ions such as magnesium ions that are essential for PCR [38]. In a forensic science context, the blue dye in clothing such as denim, called indigo, has an inhibitor effect on PCR [39].

Extraction methods have been developed to remove commonly encountered PCR inhibitors and, for example, the silica binding methods that are commonly used in forensic analysis are effective at removing most inhibitors whereas the methods that produce a cruder extract such as the Chelex® resin are more prone to inhibition. When it is not possible to remove all the potential inhibitors from a DNA extract, the addition of the protein bovine serum albumin (BSA) to the PCR can in many cases prevent or reduce the inhibition of the Taq polymerase. The BSA acts as a binding site for some inhibitors and can competitively remove or reduce the concentration of the inhibitor [30, 38]. The action of inhibitors can be detected, for example by spiking a PCR with a known amount of DNA, this alerts the analyst that further purification steps are required [40].

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