engleski

Removal of PCR inhibitors by powdered activated carbon

Bloodstains are common forensic samples, which can often be problematic for genotyping. Besides hematin naturally occurring in bloodstain extracts, components of the substrate on which forensic evidence is found can affect DNA amplification as powerful polymerase chain reaction (PCR) inhibitors. The aim of this study was to overcome the PCR inhibition by using powdered activated carbon (PAC) purification. We evaluated the effectiveness of PAC-purification at 37˚C with 60 minutes contact time vs. ultrafiltration using Amicon®Ultra-4 unit (100K) in removing hematin and soluble humic substances. Genomic DNA from bloodstains on peat soil substrate and from blood without substrate was extracted using Chelex®100 and purified by PAC or Amicon®Ultra-4. In addition, for some samples purification was not performed. Real-time PCR was used for DNA quantity estimation and AmpFlSTR®SGM Plus® kit for amplification. Moreover, concentrations of residual inhibitors were determined by UV/VIS spectrometry and compared to unpurified samples. PAC more effectively removed hematin, without DNA loss and provided high DNA quality. Our results show that purification significantly decreased humic substances content. Conversely, both purification methods significantly increased average DNA yield and provided equivalently good DNA quality. In conclusion, this study provides the first insight into a new application of PAC as a well-known adsorbent, based solely on temperature and contact time without introducing any reagents that may interfere with subsequent downstream analysis and may provide the basis for its further application in forensic casework.

PCR inhibition ; activated carbon ; hematin ; humic substances

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