Isolation of High-Molecular-Weight DNA for Long-Read Sequencing Using a High-Salt Gel Electroelution Trap
Kalendar R. Ivanov K.I. Samuilova O. Kairov U. Zamyatnin A.A., Jr.
5 December 2023American Chemical Society
Analytical Chemistry
2023#95Issue 4817818 - 17825 pp.
Long-read sequencing technologies require high-molecular-weight (HMW) DNA of sufficient purity and integrity, which can be difficult to obtain from complex biological samples. We propose a method for purifying HMW DNA that takes advantage of the fact that DNA’s electrophoretic mobility decreases in a high-ionic-strength environment. The method begins with the separation of HMW DNA from various impurities by electrophoresis in an agarose gel-filled channel. After sufficient separation, a high-salt gel block is placed ahead of the DNA band of interest, leaving a gap between the separating gel and the high-salt gel that serves as a reservoir for sample collection. The DNA is then electroeluted from the separating gel into the reservoir, where its migration slows due to electrostatic shielding of the DNA’s negative charge by excess counterions from the high-salt gel. As a result, the reservoir accumulates HMW DNA of high purity and integrity, which can be easily collected and used for long-read sequencing and other demanding applications without additional desalting. The method is simple and inexpensive, yields sequencing-grade HMW DNA even from difficult plant and soil samples, and has the potential for automation and scalability.
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Institute of Biotechnology, Helsinki Institute of Life Science (HiLIFE), University of Helsinki, Helsinki, 00014, Finland
Center for Life Sciences, National Laboratory Astana, Nazarbayev University, Astana, 010000, Kazakhstan
Department of Microbiology, University of Helsinki, Helsinki, 00014, Finland
Research Center for Translational Medicine, Sirius University of Science and Technology, Sochi, 354340, Russian Federation
Department of Biological Chemistry, Institute of Biodesign and Modeling of Complex Systems, Sechenov First Moscow State Medical University, Moscow, 119991, Russian Federation
HSE University, Faculty of Biology and Biotechnology, Moscow, 117418, Russian Federation
Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, 119234, Russian Federation
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992, Russian Federation
Institute of Translational Medicine and Biotechnology, Sechenov First Moscow State Medical University, Moscow, 119991, Russian Federation
Institute of Biotechnology
Center for Life Sciences
Department of Microbiology
Research Center for Translational Medicine
Department of Biological Chemistry
HSE University
Faculty of Bioengineering and Bioinformatics
Belozersky Institute of Physico-Chemical Biology
Institute of Translational Medicine and Biotechnology
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