Nucleic acid extraction using organic solvents to remove inhibitors
Inventors
Chen, Li • STEVENS, JODIE • SCHWAB, KRISTEN • Boone, James • LYERLY, DAVID
Assignees
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Publication Number
US-9528105-B2
Publication Date
2016-12-27
Expiration Date
Abstract
Nucleic acid amplification tests have been widely used in clinical laboratories. Nucleic acid extraction from biological materials is challenging because different unfavorable substances may co-extract and inhibit downstream applications. The present invention relates to a composition of and a method for treating the sample prior, during or post extraction of nucleic acid. More specifically, the claimed invention relates to a composition of and a method for using low concentrations of common organic solvents to remove inhibitors of nucleic acid amplification. The present invention can be used for extracting nucleic acids (DNA/RNA) from bacteria, viruses, parasites, and other biological materials or matrices, including but not limit to, stool samples, body fluids, plants and cultures. The method is rapid, low-cost, and easy to use in a laboratory setting. The nucleic acid extracted in accordance with the invention can be used for nucleic acid amplification reactions.
Core Innovation
The disclosed invention relates to nucleic acid extraction from a biological sample by treating the sample with an extraction buffer that includes 0.5 to 20 wt. % organic solvent. The method includes obtaining a biological sample comprising one or more of stool, blood, saliva, or urine, diluting the biological sample in the extraction buffer to form a mixture, and incubating the mixture at a temperature from 156 C. to 356 C. for five seconds to thirty minutes, or up to 706 C. in the stool-specific independent claim, to extract nucleic acids for further processing.
After incubation, the method includes separating at least a portion of the extraction buffer and at least a portion of the nucleic acid from the biological sample. The remaining nucleic acid is then used to form an amplification mixture that comprises the nucleic acid, at least a portion of the extraction buffer where applicable, and one or more amplification reaction buffers, followed by incubating the amplification mixture for 1 to 10 minutes at 256 C. to 706 C. for amplification in an amplification reaction.
In a second embodiment, the invention further includes adding an organic solvent to at least a portion of the separated nucleic acid to a concentration of from 0.5 to 20 wt. % to form a treated nucleic acid, and then forming an amplification mixture comprising the treated nucleic acid and one or more amplification reaction buffers. The problem addressed is the extraction of nucleic acids from complex biological samples while reducing inhibitors of nucleic acid amplification that can interfere with nucleic acid amplification tests, including downstream amplification reactions.
Claims Coverage
The provided set includes three independent claims. Across these independent claims, the coverage centers on dilution of biological samples in an extraction buffer containing 0.5 to 20 wt. % organic solvent, incubation to extract nucleic acids followed by separation, and formation of an amplification mixture for nucleic acid amplification with defined inclusion of extraction buffer and/or treated nucleic acid containing organic solvent.
Organic solvent extraction buffer at 0.5 to 20 wt. %
Diluting the biological sample in an extraction buffer comprising 0.5 to 20 wt. % organic solvent to form a mixture, where the biological sample comprises one or more of stool, blood, saliva, or urine, or stool in the stool-specific embodiment.
Incubating to extract nucleic acids in 156 C. to 356 C. or 156 C. to 706 C.
Incubating the mixture at a temperature from 156 C. to 356 C. for five seconds to thirty minutes, or from 156 C. to 706 C. for five seconds to thirty minutes in the stool-specific embodiment, to cause nucleic acid present in the biological sample to be extracted from at least a portion of the biological sample for further processing.
Separating extraction buffer and nucleic acid
Separating at least a portion of the extraction buffer and at least a portion of the nucleic acid from the biological sample.
Amplification mixture includes extraction buffer and nucleic acid
Forming an amplification mixture comprising the at least a portion of the extraction buffer, the at least a portion of the nucleic acid, and one or more amplification reaction buffers, followed by incubating the amplification mixture for 1 to 10 minutes at 256 C. to 706 C.
Treating separated nucleic acid by adding organic solvent before amplification
Adding an organic solvent to at least a portion of the separated nucleic acid to a concentration of from 0.5 to 20 wt. % to form a treated nucleic acid, and forming an amplification mixture comprising the treated nucleic acid and one or more amplification reaction buffers.
Across the independent claims, the inventive coverage is defined by extraction of nucleic acids from biological samples using an extraction buffer containing 0.5 to 20 wt. % organic solvent, incubation at defined temperatures for defined time ranges, separation of nucleic acid from the extraction buffer, and formation of an amplification mixture using either extracted nucleic acid with extraction buffer and amplification reaction buffers, or treated nucleic acid produced by adding organic solvent after separation.
Stated Advantages
Enables rapid nucleic acid preparation using low concentrations of common organic solvents without boiling [procedural detail omitted for safety].
Reduces amplification inhibitors from complex matrices to support downstream nucleic acid amplification tests (PCR and isothermal amplification).
Documented Applications
Preparation of nucleic acids from complex biological samples including stool, blood, saliva, and urine for downstream nucleic acid amplification reactions.
Downstream nucleic acid amplification tests including PCR and isothermal amplification methods such as NEAR, RPA, and LAMP.
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