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WO2025024436A1 - Systèmes et procédés pour le stockage d'acides nucléiques - Google Patents

Systèmes et procédés pour le stockage d'acides nucléiques Download PDF

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Publication number
WO2025024436A1
WO2025024436A1 PCT/US2024/039139 US2024039139W WO2025024436A1 WO 2025024436 A1 WO2025024436 A1 WO 2025024436A1 US 2024039139 W US2024039139 W US 2024039139W WO 2025024436 A1 WO2025024436 A1 WO 2025024436A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
carrier
rna
cartridge
permeable membrane
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2024/039139
Other languages
English (en)
Inventor
Jesse Julian WAGGONER
David Myers
Sarah Alexis HERNANDEZ
Dhruv MIGLANI
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Emory University
Georgia Tech Research Institute
Georgia Tech Research Corp
Childrens Healthcare of Atlanta Inc
Original Assignee
Emory University
Georgia Tech Research Institute
Georgia Tech Research Corp
Childrens Healthcare of Atlanta Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Emory University, Georgia Tech Research Institute, Georgia Tech Research Corp, Childrens Healthcare of Atlanta Inc filed Critical Emory University
Publication of WO2025024436A1 publication Critical patent/WO2025024436A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes

Definitions

  • Nucleic acids such as Ribonucleic Acid (RNA) can be extracted from cells or viruses and used in various downstream molecular applications (e.g., transcriptome analysis using nextgeneration sequencing, northern analysis, array analysis, RT-PCR, etc.).
  • RNA Ribonucleic Acid
  • One such use is molecular detection of viral pathogens, where, if present, RNA specific to the virus is amplified and detected. For many viruses, this is the most sensitive and specific diagnostic method. Accurate diagnosis is critical for clinical management and disease containment and relies on extracting high quality RNA from a sample.
  • RNA degrading enzymes RNases
  • Systems and methods disclosed herein relate generally to hardware systems and reagents systems for nucleic acid extraction and/or storage that can be used under ambient conditions for downstream molecular applications. In some examples, this disclosure can relate to systems of nucleic acid extraction and/or storage.
  • the system may include a cartridge.
  • the cartridge may include a first surface, a second surface opposing the first surface; and a chamber.
  • the chamber may be disposed (i) parallel to the first surface and the second surface and (ii) between the first surface and the second surface.
  • the chamber may be configured to receive a blotter substrate.
  • at least one opening may be disposed in the first surface above and interfaced with the chamber.
  • the chamber may be configured to receive a blotter substrate and the opening may be configured to receive a permeable membrane.
  • this disclosure can relate to methods of nucleic acid extraction for storage and/or molecular applications, for examples, using reagents according to embodiments.
  • the methods may include a method that includes contacting a sample containing a nucleic acid with lysis solution providing a lysed solution (lysate).
  • the nucleic acid may be microbial RNA or DNA, e.g., including viral, bacterial, and parasite.
  • the sample may be saliva, blood, nasal swab (e.g., nasopharyngeal, anterior nares, nasal mid-turbinate, etc.), oropharyngeal swab, tissue, wastewater, and/or stool samples.
  • the lysis solution may include sucrose, potassium chloride (KC1), proteinase K, and a nucleic acid carrier, such as a DNA carrier, RNA carrier, or poly A carrier RNA.
  • KC1 potassium chloride
  • proteinase K proteinase K
  • a nucleic acid carrier such as a DNA carrier, RNA carrier, or poly A carrier RNA.
  • proteinase K may be conjugated to beads.
  • RNA, DNA, or both, in the sample may non-specifically adhere to the nucleic acid carrier, e.g., RNA or DNA carrier.
  • the nucleic acid carrier e.g., RNA or DNA carrier.
  • an RNA or DNA virus or other microbe may non-specifically adhere, absorb, or hybridize to the nucleic acid carrier, DNA carrier, RNA carrier, or poly A carrier RNA providing a nucleic acid (RNA or DNA) bound nucleic acid carrier.
  • a nucleic acid carrier, DNA carrier, RNA carrier, or poly A carrier RNA and proteinase K in the lysis solution may be at a ratio by weight of 1 to 2.
  • the duration of contacting may be for an incubation period providing an incubated sample.
  • the incubation period may be between about 10 to 60 minutes.
  • the method may include contacting the incubated sample with a pH buffered solution comprising arginine and ethanol. The method may also include providing a binding buffer solution with the RNA or DNA bound nucleic acid carrier.
  • the binding buffer solution may include arginine and magnesium (Mg) salt and potassium salt.
  • the method may include contacting the binding buffer solution with the RNA or DNA bound nucleic acid carrier with the permeable membrane providing an RNA or DNA bound membrane.
  • the permeable membrane may be pre-washed or pre-treated.
  • Figures 1 A-C show an example of a cartridge according to some embodiments.
  • Figure 1A shows an orthogonal view of the cartridge;
  • Figure IB shows a side view of the cartridge;
  • Figure 1C shows a top view of the cartridge.
  • Figures 3A-I show another example of a cartridge according to some embodiments.
  • Figure 3A shows a top orthogonal view of the cartridge
  • Figure 3B shows a bottom orthogonal view of the cartridge
  • Figure 3C shows a side view of the cartridge
  • Figure 3D shows a top view of a top member of the cartridge
  • Figure 3E shows a bottom view of the top member of the cartridge
  • Figure 3F shows a front and side views of the top member and a front view of a bottom member of the cartridge
  • Figure 3G shows a side view of the bottom member of the cartridge
  • Figure 3H shows a top view of the bottom member of the cartridge
  • Figure 31 shows a bottom view of the bottom member of the cartridge.
  • Figure 4 shows another example of a cartridge according to embodiments.
  • Figure 5 A shows an orthogonal view of the transfer tool
  • Figure 5B shows a top view of the transfer tool
  • Figure 5C shows a side view of the transfer tool.
  • Figures 6A-C show another example of a transfer tool according to some embodiments.
  • Figure 6A shows an orthogonal view of the transfer tool;
  • Figure 6B shows a top view of the transfer tool;
  • Figure 6C shows a side view of the transfer tool.
  • Figures 7A-D show an example of a basket according to some embodiments.
  • Figure 7A shows an orthogonal view of the basket;
  • Figure 7B shows a side view of the basket;
  • Figure 7C shows a top view of the basket; and
  • Figure 7D shows a bottom view of the basket.
  • Figures 8A-D show another example of a basket according to some embodiments.
  • Figure 8A shows an orthogonal view of the basket;
  • Figure 8B shows a side view of the basket;
  • Figure 8C shows a top view of the basket; and
  • Figure 8D shows a bottom view of the basket.
  • Figures 11 A-D show examples of the results using an example of systems and methods according to embodiments.
  • Figure 11A shows RNA stability in the lysate over 14 days;
  • Figure 11B shows inactivation of chikungunya virus in cell culture after the sample is processed using the current protocol;
  • Figure 11C shows a comparison of viral RNA (dengue virus) stability when stored in on membranes placed in drying tubes at 37C compared to control conditions;
  • Figure 1 ID shows dengue virus rRT-PCR Ct values following extraction with the protocol according to embodiments compared to a commercial extraction robot.
  • the disclosed embodiments relate to systems and methods for nucleic storage and/or extraction that can enable economical storage of nucleic acids at ambient temperatures.
  • the systems and methods for nucleic acid extraction and/or storage may be used for stabilization of primary samples, nucleic acid extraction from a sample for downstream molecular applications (e.g., RT-PCR, sequencing, etc.), and/or ambient temperature storage of extracted nucleic acid.
  • the sample may include but is not limited to a sample isolated from saliva, blood, nasal swab (e.g., nasopharyngeal, anterior nares, nasal mid-turbinate, etc ), oropharyngeal swab, tissue, wastewater, or stool samples.
  • the nucleic acid may include but are not limited to RNA, DNA, or a combination thereof.
  • the cartridge may be made of one or more materials.
  • the one or more materials may include but is not limited to resin, polyamide, polyethylene, polycarbonate, polypropylene, polystyrene, thermoplastic elastomer, among others, or any combination thereof.
  • the cartridge may include a chamber configured to receive a blotter substrate and a (first) opening on a top surface that interfaces with the chamber that is configured to receive a permeable membrane.
  • the permeable membrane may include but is not limited to a glass microfiber (GF/D) membrane.
  • the cartridge may include a (second) opening that interfaces with the chamber on a side surface.
  • the chamber may be configured to allow the blotter substrate to slide in and out of the chamber.
  • the chamber can improve loading and reloading of the membrane.
  • the chamber can eliminate the need for centrifugation to move buffers across the membrane while also containing the used reagents. Thus, this can eliminate hazardous liquid waste thereby preventing contamination of the lab environment.
  • the opening may be configured to allow for drop-in loading and pop-out unloading of permeable membrane.
  • this opening can be configured to guide the buffer through the membrane onto the blotter substrate. Thereby, user experience can be improved while increasing nucleic acid recovery.
  • the openings are shown as circular in Figures 1 A-4, it would be understood that different shape openings may also be shown.
  • Figures 1A-C show an example of a cartridge 100.
  • the cartridge 100 may be a unitary piece.
  • the cartridge 100 may include a first surface 102 (also referred to as “top surface”) and a second surface 104 (also referred to as “bottom surface”) opposing the first surface 102.
  • the cartridge 100 may include a chamber 140 disposed (i) parallel to the first surface 102 and the second surface 104 and (ii) between the first surface 102 and the second surface 104.
  • the chamber 140 may be configured to receive a blotter substrate.
  • the cartridge 100 may include at least one (top) opening 130 disposed in the first surface 102 above and interfaces with the chamber 140. The opening 130 may be configured to receive a permeable membrane.
  • the cartridge 100 may include a first side surface 112 that is disposed perpendicular to the first surface 102 and the second surface 104.
  • the cartridge 100 may include a second opening 142 disposed in the first side surface 112 and configured to interface with the chamber 140.
  • the second opening 142 may be configured to receive the blotter substrate.
  • the second opening 142 may be used to load/unload the blotter substrate from the chamber 140.
  • the cartridge 200 may include a first side surface 212 that is disposed perpendicular to the first surface 202 and the second surface 204.
  • the cartridge 200 may include a second opening 242 disposed in the first side surface 212 and configured to interface with the chamber 240.
  • the second opening 242 may be configured to receive the blotter substrate.
  • the second opening 242 may be used to load/unload the blotter substrate from the chamber 240.
  • the opening 330 may have a tapered shape like the cartridge 200.
  • the opening 330 may have a first circumference 332 that is larger than the second circumference 334 so that its circumference tapers toward the chamber 340.
  • the second circumference 334 may be sized to correspond to a size of the permeable membrane. The second circumference 334 may be directly above the chamber 340.
  • the blotter substrate may be loaded into the chamber 340 before snap-fitting the members 350 and 360.
  • the permeable membrane may be loaded into the opening 330 before the members 350 and 360 are snapped-fit.
  • the cartridge may include more than one opening for a permeable membrane.
  • a cartridge 400 may include a plurality of openings 430 that interfaces with a blotter pad 470 disposed in the chamber 440 thereby enabling simultaneous extraction of a plurality of samples at a time.
  • the blotter pad 470 may be loaded and unloaded using the opening 442.
  • the cartridge 400 may include any number of openings 430 and is not limited to the number and/or configuration of channels and rows shown.
  • the number of openings 430, including the rows and columns may be based on sample volume.
  • unnecessary waste may be eliminated.
  • the openings 430 may have a circular shape with a uniform and/or tapered circumference, for example, as shown in Figures 1-3. In other examples, the openings 430 may have a different shape.
  • the first section 510 may be a handle.
  • the third section 530 may be configured to interface with the membrane.
  • the third section 530 may be a prong.
  • the system may also include a drying container.
  • the drying container may include a body having a chamber.
  • the chamber may be filled with a drying agent, such as silica beads.
  • the drying container may include a basket that is disposed onto the body above the chamber including the drying agent and configured to removably snap-close the chamber.
  • the basket may include a base surface configured to receive the permeable membrane and to separate the membrane from the drying agent.
  • the drying basket may be reusable so that the process can be more cost efficient.
  • the slots 730 may be disposed in the base surface 720 separated by openings 740 to allow for airflow.
  • FIGS 8A-8D show an example of a basket 800 that includes an opening 830.
  • the basket 800 may include a base surface 820 configured to receive the permeable membrane and to separate the membrane from a drying agent.
  • the base surface 820 may be bounded by a wall 810.
  • the basket 800 may have a circular shape.
  • the baskets may include one or more features configured to be used to remove the basket from the body of the drying container.
  • the baskets may include a pole and/or handle.
  • FIGS 9A-9D show an example of a basket 900 that includes a pole 930.
  • the basket 900 may include a base surface 920 configured to receive the permeable membrane and to separate the membrane from a drying agent.
  • the base surface 920 may be bounded by a wall 910.
  • the basket 900 may have a circular shape.
  • the pole 930 may extend from the base surface 930 past the wall 910 so that it protrudes.
  • the basket 900 may include one or more features, such as openings and/or slots, like those shown and described in Figures 7A-8D, configured to allow for airflow between the permeable membrane and drying agent.
  • the reagents according to the disclosure can provide optimized buffers for stabilization of primary samples, nucleic acid extraction from a sample for downstream molecular applications (e.g., RT-PCR, sequencing), and/or ambient temperature storage of extracted nucleic acid.
  • the reagents may include a binding buffer solution, a lysis solution, an elution buffer, a wash buffer, among others, or any combination thereof.
  • the lysis solution may provide a lysed solution.
  • the lysis solution may include but is not limited to sucrose, potassium chloride (KC1), proteinase K, nucleic acid carrier, DNA carrier, RNA carrier, poly A carrier RNA, among others, or a combination thereof.
  • the nucleic acid carrier, DNA carrier, RNA carrier, or poly A carrier RNA and proteinase K in the lysis solution may have a ratio by weight of 1 to 2.
  • the wash buffer may contain glycerin hydrochloride at a pH below 5, 6 or 7.
  • the pH buffer may be added after the sample is loaded onto the membrane.
  • the binding buffer solution may be a pH-buffered solution.
  • the pH-buffered solution may include but is not limited to arginine, ethanol, among others, or a combination thereof.
  • the pH-buffered solution may provide a binding buffer solution.
  • the binding buffer solution may include but is not limited to arginine, magnesium (Mg) salt, potassium salt, among others, or a combination thereof.
  • the elution buffer may include but is not limited to a TE buffer.
  • the TE buffer may have a pH of about 8.0.
  • the wash buffer may be a low pH buffer.
  • the wash buffer may include glycine hydrocholoride.
  • this disclosure relates to methods of nucleic acid extraction for storage and/or molecular applications.
  • Figure 10 shows an example of a method 1000 for nucleic acid extraction and/or molecular applications using the reagents and hardware disclosed herein. It will be understood that the methods using the disclosed reagents is not limited to the hardware disclosed herein and may be used with different hardware.
  • the method 1000 may include a step 1010 of contacting a sample containing a nucleic acid with a lysis solution providing a lysed solution, or a lysis sphere, for example, as shown in 1012.
  • the nucleic acid may be viral, bacterial, or other microbial RNA or DNA.
  • the sample may be saliva, blood, nasal swab (e.g., nasopharyngeal, anterior nares, nasal mid-turbinate, etc.), oropharyngeal swab, tissue, wastewater, or stool samples.
  • the lysis solution may include one or more lyophilized lysis reagents that, upon reconstitution with the sample, can form a lysate.
  • the lysis solution may include but is not limited to sucrose, KC1, proteinase K, a nucleic acid carrier, DNA carrier, RNA carrier, or poly A carrier RNA, among others, or any combination thereof.
  • the proteinase K may be conjugated to beads.
  • the viral bacterial or other microbial RNA in the sample may hybridize to the RNA carrier or poly A carrier RNA providing an RNA bound nucleic acid carrier or the RNA may non-specifically absorb or adhere to the RNA carrier, or poly A carrier RNA.
  • the RNA carrier or poly A carrier RNA and proteinase K in the lysis solution may be at a ratio by weight of 1 to 2.
  • the DNA in the sample may hybridize to the DNA carrier providing a DNA bound nucleic acid carrier or the DNA may non-specifically absorb the DNA carrier.
  • the DNA carrier and proteinase K in the lysis solution may be at a ratio by weight of 1 to 2.
  • the contacting may be for an incubation period providing an incubated sample.
  • the incubation period may be between about 10 to 60 minutes.
  • the method 1000 may include a step 1020 of contacting the incubated sample with a pH buffered solution to provide a binding buffer solution, for example, with the RNA or DNA bound nucleic acid carrier.
  • a pH buffered solution may include arginine and ethanol.
  • the binding buffer solution may include arginine and Mg salt and potassium salt.
  • the method 1000 may include a step 1030 of contacting the binding buffer solution with the RNA or DNA bound nucleic acid carrier with a permeable membrane (e g., glass fiber membrane) providing an RNA or DNA bound membrane.
  • a permeable membrane e g., glass fiber membrane
  • the permeable membrane may be provided within a cartridge according to embodiments, for example, as shown in 1032.
  • the cartridge may be the cartridge as disclosed and described with respect to Figures 1A-4.
  • the method 1000 may include a step 1040 of washing the membrane with a washing solution.
  • the method 1000 may include a step 1050 of removing the RNA or DNA bound membrane from the cartridge according to embodiments.
  • the method 1000 may include a step 1060 of placing the membrane onto the base surface of the basket of the drying container filled with a drying agent according to embodiments, for example, as shown in 1062.
  • the basket may be the basket as disclosed and described with respect to Figures 7A- 9D.
  • the transfer tool may be used to remove the membrane and transfer it to the basket.
  • the transfer tool may be the transfer tool as disclosed and described with respect to Figures 5A-6C. In other examples, other tools may be used.
  • it may be washed with a wash buffer solution or ethanol.
  • the RNA or DNA bound membrane may be stored in the drying container for a period of time under ambient conditions.
  • the method 1000 may include a step 1070 of preparing the sample for one or more molecular applications.
  • the step 1070 may include transferring the RNA or DNA bound membrane from the drying container to a container with an elution reagent, for example, as shown in 1072.
  • one or more molecular applications may be performed on the eluates in the container.
  • Figures 11 A-D shows A) RNA stability in the lysate over 14 days, B) inactivation of chikungunya virus in cell culture after the sample is processed using the current protocol, C) a comparison of viral RNA (dengue virus) stability when stored in on membranes placed in drying tubes at 37C compared to control conditions, and D) dengue virus rRT-PCR Ct values following extraction with the current protocol compared to a commercial extraction robot.

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Abstract

La présente invention concerne des systèmes et des procédés pour le stockage d'acides nucléiques pouvant être utilisés dans des conditions ambiantes de manière économique. Dans un mode de réalisation, le système peut comprendre une cartouche. La cartouche peut comprendre une première surface et une deuxième surface opposée à la première surface. La cartouche peut comprendre une chambre agencée (i) parallèlement à la première surface et à la deuxième surface et (ii) entre la première surface et la deuxième surface. La chambre peut être conçue pour accueillir un substrat de buvard. La cartouche peut également comprendre au moins une ouverture située dans la première surface au-dessus de la chambre et qui fait interface avec cette dernière. L'ouverture peut être conçue pour accueillir une membrane perméable.
PCT/US2024/039139 2023-07-24 2024-07-23 Systèmes et procédés pour le stockage d'acides nucléiques Pending WO2025024436A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202363515172P 2023-07-24 2023-07-24
US63/515,172 2023-07-24
US202463573890P 2024-04-03 2024-04-03
US63/573,890 2024-04-03

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WO2025024436A1 true WO2025024436A1 (fr) 2025-01-30

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5496562A (en) * 1988-10-05 1996-03-05 Flinders Technologies Pty Ltd Solid medium and method for DNA storage
US20080026451A1 (en) * 2006-06-15 2008-01-31 Braman Jeffrey C System for isolating biomolecules from a sample
US20140179909A1 (en) * 2010-11-30 2014-06-26 Quantumdx Group Limited Microfluidic device for nucleic acid extraction and fractionation
US20210008552A1 (en) * 2017-06-15 2021-01-14 Lexagene, Inc. Nucleic acid extraction and purification cartridges
CN214907317U (zh) * 2021-04-02 2021-11-30 青岛市市立医院(青岛市临床医学研究所、青岛市医学影像中心) 一种用于核酸采样的护理盘

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5496562A (en) * 1988-10-05 1996-03-05 Flinders Technologies Pty Ltd Solid medium and method for DNA storage
US20080026451A1 (en) * 2006-06-15 2008-01-31 Braman Jeffrey C System for isolating biomolecules from a sample
US20140179909A1 (en) * 2010-11-30 2014-06-26 Quantumdx Group Limited Microfluidic device for nucleic acid extraction and fractionation
US20210008552A1 (en) * 2017-06-15 2021-01-14 Lexagene, Inc. Nucleic acid extraction and purification cartridges
CN214907317U (zh) * 2021-04-02 2021-11-30 青岛市市立医院(青岛市临床医学研究所、青岛市医学影像中心) 一种用于核酸采样的护理盘

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HERNANDEZ ET AL.: "Simple and Economical Extraction of Viral RNA and Storage at Ambient Temperature", MICROBIOL SPECTR., vol. 10, no. 3, 1 June 2022 (2022-06-01), pages 1 - 9, XP093130226, DOI: 10.1128/spectrum.00859-22 *
MCFALL SALLY M., NETO MÁRIO F., REED JENNIFER L., WAGNER ROBIN L.: "Filtration Isolation of Nucleic Acids: A Simple and Rapid DNA Extraction Method", JOURNAL OF VISUALIZED EXPERIMENTS, UNITED STATES, vol. 114, no. 114, 1 August 2016 (2016-08-01), United States, pages e54289 - 7, XP009560604, ISSN: 1940-087X, DOI: 10.3791/54289-v *

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