Microwave-assisted enzymatic hydrolysis of DNA for mass spectrometric analysis: A new strategy for accelerated hydrolysis

Study of DNA base composition, DNA adducts and modification in its primary structure is a subject of interest in different fields of scientific research. Various methods like immunochemistry, capillary electrophoretic separation, chromatographic separation coupled with mass spectrometric (LC-MS/MS) detection have been developed for DNA analysis. During the past decade LC-MS/MS has emerged as a more sensitive and selective technique and now frequently used for the analysis of DNA. The workflow for the DNA analysis include DNA extraction, hydrolysis of DNA into mononucleosides and analysis. Though high-throughput methods are available for the analysis DNA hydrolysis oftentimes is a rate limiting step. Conventional enzymatic hydrolysis of DNA using a multienzyme mixture will take a minimum of 6-17 h to complete the hydrolysis. In this work, we developed an accelerated enzymatic hydrolysis of DNA using microwave-technology for the first time. 1.00 μg of calf thymus DNA was used to demonstrate the microwave assisted enzymatic hydrolysis of DNA. The resultant mononucleosides were separated on Atlantis T3 (50 × 2.1 mm i.d.; 3 μ particle size) C18 column and analyzed on ABSCIEX QTRAP 5500 LC/MS system. The sample through put and recovery of microwave-assisted digestion were compared with the conventional enzymatic hydrolysis. Efficient digestion of DNA with a performance similar to that obtained by the conventional overnight digestion procedure was attained in just 30 min with a hydrolysis yield of ≥90%. Furthermore, our method was found to be much more accurate and easier to perform. Thus, this new application of microwave technology to DNA enzymatic digestion will facilitate the application of DNA analysis in biological and clinical research.