Method for extracting nucleic acids from biological sample
Inventors
Min, Jun Hong • BAEK, Chang Yoon
Assignees
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Publication Number
US-11618895-B2
Publication Date
2023-04-04
Expiration Date
Abstract
The present invention relates to a method for extracting nucleic acids from a biological sample, and the extraction method presents a novel method for effectively extracting nucleic acids. When nucleic acids are extracted from biological samples in the related art, various impurities present in the biological samples are not properly removed, such that the purification rate is low, but the present invention provides a method for extracting nucleic acids from a biological sample of which the bacteria, virus and nucleic acid recovery rates are enhanced, by adding a surfactant and a sodium sulfate (Na2SO4) solution in a biological sample disruption step and a purification step, thereby enabling pathogens to be detected more sensitively and accurately.
Core Innovation
The invention relates to methods for extracting nucleic acids from a biological sample in which nucleic-acid recovery from complex samples is improved. A biological sample is disrupted by bead-beating a biological sample-bead solution prepared by adding a buffer solution, a surfactant, and beads to the biological sample, and the biological sample-bead solution is not subject to heat treatment during the bead-beating disruption.
After disruption, the method separates disrupted material into a supernatant or filtrate and solid impurities or filtered material. A sodium sulfate (Na2SO4) solution is added to the supernatant or filtrate, and the treated supernatant or treated filtrate is centrifuged or heat-treated to form impurity-containing portions and a liquid portion.
Impurities-containing portions are then separated from a repurified solution by transferring the selected liquid portion to a second vessel. Nucleic acids are extracted from the repurified solution, and the workflow is supported for biological samples including stool, blood, and soil.
Claims Coverage
Two independent claims are identified. Each independent claim recites a multi-step nucleic-acid extraction workflow centered on non-heat bead-beating, sodium sulfate treatment, and repurification by separating impurity-containing portions.
Bead-beating without heat treatment and supernatant impurity separation
Disrupting a biological sample by bead-beating a biological sample-bead solution prepared by adding a buffer solution, a surfactant, and beads to the biological sample, wherein the biological sample-bead solution is not subject to heat treatment; centrifuging the disrupted biological sample-bead solution to form a supernatant and solid impurities; and separating the supernatant from the solid impurities by transferring the supernatant to a first vessel.
Sodium sulfate treated centrifugation and repurified extraction
Adding a 1.25 to 2.5 M sodium sulfate (Na2SO4) solution to the supernatant and mixing to form a treated supernatant; centrifuging or heat-treating the treated supernatant to form a top portion comprising impurities and a liquid bottom portion; separating the top portion from the liquid bottom portion by transferring the liquid bottom portion to a second vessel to form a repurified solution consisting of the liquid bottom portion; and extracting nucleic acids from the repurified solution.
Bead-beating without heat treatment and filtration-based impurity separation
Disrupting a biological sample by bead-beating a biological sample-bead solution prepared by adding a buffer solution, a surfactant, and beads to the biological sample, wherein the biological sample-bead solution is not subject to heat treatment; filtering the disrupted biological sample-bead solution by passing it through an object having a space through which the beads and impurities cannot escape, thereby forming a filtrate and a filtered material comprising said beads and said impurities; and separating the filtrate from the filtered material by transferring the filtrate to a first vessel.
Sodium sulfate treated heat treatment to lift impurities and repurified extraction
Adding a 1.25 to 2.5 M sodium sulfate (Na2SO4) solution to the filtrate and mixing to form a treated filtrate; performing a heat treatment on the treated filtrate to lift impurities; separating the lifted impurities from the treated filtrate without the impurities by transferring the treated filtrate without the impurities to a second vessel to form a repurified solution consisting of the treated filtrate without the impurities; and extracting nucleic acids from the repurified solution, wherein the biological sample is any one selected from stool, blood, and soil.
Across both independent claims, the core claim coverage is directed to nucleic-acid extraction using bead-beating without heat treatment, followed by sodium sulfate treatment and repurification via impurity separation. One claim uses centrifugation to form top/bottom impurity-containing portions, while the other uses filtration and a heat treatment to lift impurities before separation for extraction.
Stated Advantages
Improves recovery of nucleic acids from complex samples compared with commercial kits.
Documented Applications
Extracting nucleic acids from biological samples including stool, blood, and soil.
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