Comparison of three methods including temperature, H2O2/ascorbic acid/sonication, and nitrous acid treatments for overcoming the inhibitory effect of heparin on DNA amplification in realtime-PCR

Abstract

Heparin molecules have an inhibitory effect on DNA amplification by binding to the majority of DNA-interacting proteins. Different physical, chemical, and enzymatic methods have been used to degrade and depolymerize heparins in biomedical investigations. In this study, we aimed to evaluate some heparin degradation methods to eliminate the inhibitory effect of heparin on DNA amplification. Here, we report highly efficient, simple, and convenient methods to eliminate the heparin inhibitory effect on DNA amplification by treatments including temperature, nitrous acid, and H2O2/ascorbic acid/sonication. Further, treatment conditions including temperature degree and duration of treatments, the concentration of ascorbic acid, and intensity of sonication were reviewed. Target DNAs were extracted using the phenol-chloroform method. DNA concentrations and purity were analyzed before and after each treatment by Nanodrop spectrophotometry. DNA amplifications were attempted using a commercially available realtime-PCR mastermix. We found that the inhibitory behavior of heparin was well eliminated after the 85 °C/2 h, 65 °C/2 h, nitrous acid (pH = 3), and H2O2/ascorbic acid/sonication treatments, respectively. The further analyses indicated that the application of nitrous acid in pH = 1.5 and H2O2/ascorbic acid/sonication in higher ascorbic acid concentrations and sonication intensities lead to failure in DNA amplification due to the degradation of target sequences. From our experience, simple heat treatments or at the next level using nitrous acid and H2O2/ascorbic acid/sonication have enabled the detection and quantification of virus infection in heparin blood samples. These approaches may enable researchers to utilize blood taken in heparin tubes for genome amplification and diagnostic purposes.