Optimization of Binding, Washing and Elution Buffer for Development of DNA Isolation Kit

Abstract

Abstract DNA isolation is one of the most crucial part in DNA analysis and is reflected by the abundance of ready-to-use DNA isolation kits available in the market. However, the chaotropic salts used in conventional kits during the binding step has been known to inhibit the downstream process of PCR and deteriorate when exposed to air. This study aims to design a better and faster DNA isolation process with better DNA isolation performance to replace the conventional one. This study aims to replace the chaotropic salt in binding buffer with organic acids or salt and improve the buffer used during the wash step. Sodium perchlorate and several other salts and acids were chosen as candidates for the binding buffer. Simultaneously, 10Mm NaCl and 10Mm Tris-Cl with varying concentrations of organic solvents were selected as candidates for the wash buffer. The performance of the selected buffers was then compared to the readily available commercial kit. Organic acid B was among the best candidates for binding buffer with 81.91% and 83.20% recovery rates. For wash buffer, it was observed that the DNA recovery increases with an increasing organic solvent concentration in 10Mm NaCl and 10Mm Tris-Cl. Wash buffer with 90% organic solvent shows the best compromise of DNA yield and purity compared to 70%, 80%, and 100% organic solvent concentration in 10Mm NaCl and 10Mm Tris-Cl. A combination of organic acid B in binding buffer and 90% organic solvent A in wash buffer were tested against a commercial DNA extraction kit. The combination of organic acid B and 90% organic solvent yielded 72.81 ng/ul compared to 28.46 ng/ul by the commercial kit. The combination of the binding buffer organic acid B and 90% organic solvent in 10Mm NaCl and 10Mm Tris-Cl can replace the current commercial kits without the problems posed by the presence of chaotropic salt.