Aqueous compositions that maintains fidelity of the nucleic acid sequences of a biological specimen
Inventors
Fischer, Gerald W. • Daum, Luke T.
Assignees
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Publication Number
US-10870878-B2
Publication Date
2020-12-22
Expiration Date
Abstract
Disclosed are compositions for isolating populations of nucleic acids from biological, forensic, and environmental samples. Also disclosed are methods for using these compositions as one-step formulations for killing pathogens, inactivating nucleases, and releasing polynucleotides from other cellular components within the sample, and stabilizing the nucleic acids prior to further processing or assay. The disclosed compositions safely facilitate rapid sample collection, and provide extended storage and transport of the samples at ambient or elevated temperature without contamination of the sample or degradation of the nucleic acids contained therein. This process particularly facilitates the collection of specimens from remote locations, and under conditions previously considered hostile for preserving the integrity of nucleic acids released from lysed biological samples without the need of refrigeration or freezing prior to molecular analysis.
Core Innovation
An aqueous composition is provided that maintains fidelity of a nucleic acid sequence contained within a biological sample when mixed with the sample and maintained for a period of time without refrigeration. The composition comprises components that, upon mixing, sterilize the sample, denature proteins, and inactivate nucleases of the sample.
The composition comprises guanidine thiocyanate, tris(2-carboxyethyl) phosphine (TCEP), sodium citrate, N-lauroyl sarcosine, a silicone polymer, tris(hydroxymethyl) aminomethane (Tris), ethylenediaminetetraacetic acid (EDTA), and nuclease-free water. The fidelity of the nucleic acid sequence as determined by PCR is within defined CT tolerance limits, with distinctions for RNA sequences and DNA sequences.
The invention addresses preserving nucleic-acid sequence fidelity from a biological sample without refrigeration while achieving sterilization of the sample and inactivation of nucleases. The compositions maintain nucleic acid sequence fidelity under non-refrigerated conditions for at least 7 days and, in one form, have a pH from about 6 to about 7.
Claims Coverage
The consolidated content includes three independent claims. Across these claims, the inventive features center on a defined aqueous composition for non-refrigerated preservation of nucleic-acid sequence fidelity, with claim-dependent distinctions for CT limits, minimum time, and pH.
Non-refrigerated aqueous composition with sterilization, protein denaturation, and nuclease inactivation
An aqueous composition that maintains fidelity of a nucleic acid sequence contained within a biological sample when mixed with the sample and maintained for a period of time without refrigeration, comprising components that upon mixing sterilize the sample, and denature proteins and inactivate nucleases of the sample.
Defined component set for CT-limited nucleic-acid sequence fidelity
The composition comprises guanidine thiocyanate, tris(2-carboxyethyl) phosphine (TCEP), sodium citrate, N-lauroyl sarcosine, a silicone polymer, tris(hydroxymethyl) aminomethane (Tris), ethylenediaminetetraacetic acid (EDTA), and nuclease-free water in amounts such that the fidelity of the nucleic acid sequence as determined by PCR is within three CTs within the period of time, and the period of time is at least 7 days.
RNA versus DNA CT fidelity performance without refrigeration
The composition comprises the same defined component set, with the fidelity of the nucleic acid sequence as determined by PCR within three CTs for an RNA sequence and within two CTs for a DNA sequence within the period of time, and the period of time is at least 7 days.
Aqueous composition pH window for the defined component set
The composition comprises guanidine thiocyanate, tris(2-carboxyethyl) phosphine (TCEP), sodium citrate, N-lauroyl sarcosine, a silicone polymer, tris(hydroxymethyl) aminomethane (Tris), ethylenediaminetetraacetic acid (EDTA), and nuclease-free water, wherein the pH of the composition is from about 6 to about 7.
The claims collectively define aqueous compositions that, when mixed with a biological sample and held without refrigeration, sterilize the sample, denature proteins, and inactivate nucleases while maintaining nucleic-acid sequence fidelity as determined by PCR within specified CT tolerances. The claims further distinguish RNA and DNA CT limits and recite a pH window of about 6 to about 7 for the defined component set.
Stated Advantages
Sterilize the sample upon mixing.
Denature proteins and inactivate nucleases of the sample.
Maintain nucleic-acid sequence fidelity as determined by PCR within CT tolerance limits.
Maintain fidelity when maintained for a period of time without refrigeration, including at least 7 days.
Provide distinct CT-based fidelity limits for RNA sequences and DNA sequences.
Use of an aqueous composition with pH from about 6 to about 7.
Documented Applications
Preservation of released DNA/RNA from a biological specimen while enabling downstream molecular assays, as supported by comparison to commercial extraction kits and reporting PCR CT performance.
Sterilization/inactivation of microorganisms in specimens while preserving nucleic acids for analysis.
Non-refrigerated storage/handling of preserved nucleic acids at ambient or elevated temperatures for periods of time, as described by preservation over about 30 days at room temperature.
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