Metal-DNA Interactions Improve signal in High-Resolution Melting of DNA for Species Differentiation of Plasmodium Parasite.

Abstract

The success of high-resolution melting (HRM) analysis for distinguishing similar DNAs with minor base mismatch differences is limited. Here, metal-mediated structural change in DNA has been exploited to amplify HRM signals leading to differentiation of target DNAs in an orthologous gene corresponding to four Plasmodium species. Conserved 26-mer ssDNAs from ldh gene of the four Plasmodium species were employed as targets. A capture probe (CP) that is fully complementary to the Plasmodium falciparum target (FT) and has two base mismatches each, with the targets of Plasmodium vivax (VT), Plasmodium malariae, (MT), and Plasmodium ovale (OT), was considered. The DNA duplexes were treated with metal ions for structural perturbation and analyzed by HRM. Distinct resolution of melting fluorescence signal in otherwise identical HRM profiles for each of the DNA duplexes was achieved by using Ca+2 or Mg+2 ions, where, Ca+2 conferred higher resolution. The increase in resolution for CP-FT versus CP-OT, CP-FT versus CP-VT, CP-FT versus CP-MT, CP-VT versus CP-OT, and CP-MT versus CP-OT with Ca-DNA as compared to control was 67.3-, 20.4-, 22.0-, 10.9-, and 8.3-fold, respectively. The signal resolution was the highest at pH 8. The method could detect 0.25pmol/µl of the target DNA. Structural analysis showed that Ca+2 and Mg+2 ions perturbed the structure of DNA. This perturbation helped to improve HRM signal resolution among DNA targets corresponding to the orthologous gene of four Plasmodium species. This novel approach has potential application not only for Plasmodium species-specific diagnosis but also for differentiation of DNAs with minor sequence variation.