Analysis of methods for PCR products concentrating for subsequent sequencing using methods based on the sorption of nucleic acids
Abstract
Methods based on DNA sequencing have become widespread. Often, sequencing methods require the isolation of nucleic acids from the test material, PCR to amplify the marker fragment of DNA, and their subsequent conversion to DNA libraries for sequencing. Sometimes the concentration of the obtained PCR products is insufficient for sequencing. The aim of this study was the selection of the optimal method for PCR products (amplicons) concentrating using nucleic acid sorption-based methods. It was found that the most optimal method for amplicon concentration is the use of columns with silica and the use of magnetic particles. The concentration of amplicon obtained by the amplification of bacterial DNA increased by 3.14 times when using a kit based on a column with silica, and by 2.74 times when using magnetic particles. The concentration of amplicon obtained by amplification of fungal DNA increased by 4.72 times when using a kit based on the columns with silica, and by 3.70 times when using magnetic particles. The different efficiency of concentration methods in relation to PCR products obtained during amplification of bacterial and fungal DNA may be due to different fragment lengths. The maximum concentration of DNA was obtained when using a column with silica. Magnetic particles allowed obtaining a more homogeneous amplicon than in the case of using a column with silica. It can be important for the subsequent preparation of sequencing libraries and direct amplicon sequencing. The presented methods for amplicon concentrating after PCR reactions based on the sorbing properties of silica and magnetic particles will increase the sensitivity of sequencing methods and allow analysis even in cases where the initial amount of DNA does not allow obtaining the nec-essary amount of amplicon for sequencing
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