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WO2021205470A1 - Composition tampon pour préparation d'échantillons d'acides nucléiques en une étape et stockage à partir d'échantillons biologiques - Google Patents

Composition tampon pour préparation d'échantillons d'acides nucléiques en une étape et stockage à partir d'échantillons biologiques Download PDF

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WO2021205470A1
WO2021205470A1 PCT/IN2021/050335 IN2021050335W WO2021205470A1 WO 2021205470 A1 WO2021205470 A1 WO 2021205470A1 IN 2021050335 W IN2021050335 W IN 2021050335W WO 2021205470 A1 WO2021205470 A1 WO 2021205470A1
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buffer
nucleic acid
buffer composition
extracted
compositions
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Benet Bosco DHAS D
Sasikanta Naga Pavani PAVULURI
Yaswanth Reddy K
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30m Genomics Private Ltd
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30m Genomics Private Ltd
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    • 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

Definitions

  • Buffer composition for one step nucleic acid sample preparation and storage from biological samples
  • the present invention pertains to the field of buffers for extraction and storage of nucleic acids. More particularly, the invention relates to optimized functional single-buffer compositions capable of extraction of nucleic acid from micro-volumes of various biological samples at ultra-rapid speed, without need of any equipment.
  • the nucleic acids thus extracted can be directly used for downstream processes, and can be stored for at least 7 to 14 days and transported without any further processing steps.
  • Nucleic acids play central role in routine procedures in molecular biology, forensic analysis and diagnostics. Nucleic acid extraction and purification are important steps that determine efficiency and accuracy of nucleic acid based diagnostic tests. Each of the steps in nucleic acid extraction, further require several other processes, buffers and equipment, that needs to be skillfully operated in order to obtain good quality nucleic acids for further down-streaming processes.
  • nucleic acid extraction from biological samples is the first step in any molecular diagnostics.
  • Basic procedure of nucleic acid extraction and purification involves the steps of cell lysis, precipitation and purification.
  • nucleic acid extraction Several methods have been proposed so far for isolating nucleic acids.
  • biological samples like blood, sputum, saliva contains inhibitors that restrict direct amplification of nucleic acid present in them and hence it has to be purified for further analysis.
  • blood sample can be used directly in a PCR (Polymerase Chain Reaction) mixture with an additional preheating step or by adding PCR enhancing chemicals like gelatine, betaine, etc. It is also shown that the same can be performed with high fidelity polymerases like HotStart Polymerases or GoldTaq Polymerases.
  • Zou et al. (PMCID: PMC5697807), teaches an ultra-rapid DNA isolation using a cellulose-paper-based dipstick that can bind, wash, and elute purified nucleic acids in under 30 seconds without requiring any pipetting or electrical equipment.
  • This method uses cellulose-paper-based dipstick for DNA isolation, requiring additionally use of lysis buffer, washing buffer and extraction buffer for the complete process.
  • the over-all extraction process involves more than one step in its processing.
  • Patent publication CN104404030A teaches a rapid extracting plant genome kit comprises different combinations of reagents and DNA adsorption column for separating and purifying DNA with different combination of silicon matrix, which is capable of DNA extraction within 15 minutes.
  • This publication is limited to plant genome extraction, and uses toxic chemicals such as CTAB. This method involves several steps and is limited to plant genome, taking several minutes to carry out the DNA extraction process.
  • the present invention addresses the problems in the existing prior art of nucleic acid extraction technology. It provides for ultra-rapid nucleic acid extraction functional single buffer compositions.
  • the present invention is an optimized cocktail of reagents which extracts nucleic acids from micro volume of various biological plant and animal samples including but not limited to yeast, blood, cell culture, saliva, forensic samples, bacterial cell, cheek cells, sperm cells, plant leaves, root, seeds, pollen grains, insect tissues in less than five (05) seconds without need of any equipment and zero processing steps.
  • the present invention also has added advantage of storing and transporting the extracted nucleic acid samples at room temperature, 4°C for a period of 7 to 14 days and at -20°C for long-term storage, without any additional processing. Extracted nucleic acid samples can be used directly for downstream applications like PCR and qPCR.
  • the present invention is extremely useful in the development of point-of-care genetic diagnostics among other scientific, clinical and industrial usage.
  • the main object of this invention is to provide functional single-buffer compositions for ultra-rapid and single-step nucleic acid extraction.
  • Another object of the invention is to provide a single-buffer technology, wherein an optimized cocktail of chemicals is used for the entire process of nucleic acids extraction, storage and transportation.
  • Yet another object of the invention is to provide a method of nucleic acid extraction in a single-step using the single-buffer technology.
  • Yet another object of the invention is to provide a nucleic acid extraction technology wherein the nucleic acid can be either RNA or DNA.
  • Yet another object of this invention is to provide a nucleic acid extraction technology wherein the extraction sample is plant based or animal based.
  • Yet another object of this invention is to provide a nucleic acid extraction technology wherein the extraction samples are yeast, blood, cell culture, saliva, forensic samples, bacterial cell, cheek cells, sperm cells, plant leaves, root, seeds, pollen grains, insect tissues.
  • Yet another object of the invention is to provide a technology wherein the nucleic acids extraction is possible from a micro- volume of sample.
  • Yet another object of the invention is to provide an ultra-rapid or instantaneous method of nucleic acid extraction.
  • Yet another object of the invention is to provide the single-step nucleic acid extraction buffer compositions that does not require additional processing for storing and transportation of the extracted nucleic acid samples.
  • Yet another object of the invention is to provide functional single -buffer compositions having a shelf life of up to 6 months at room temperature, and more than 12 months at 4°C and -20 °C.
  • Yet another object of the invention is to eliminate the use of laboratory infrastructure and equipment for nucleic acid extraction.
  • Yet another object of the invention is to provide ultra-rapid, single-step nucleic acid extraction method with a single buffer technology using functional single-buffer compositions for point-of-care genetic diagnostics.
  • Yet another object of the invention is to provide affordable and efficient diagnostic kits for the point-of-care genetic diagnostics.
  • Yet another object of the invention is to provide ultra-rapid, single-step nucleic acid extraction method with a single buffer technology using functional single-buffer compositions for scientific, clinical and industrial usage.
  • the present invention pertains to the field of buffers for extraction and storage of nucleic acids using micro-volumes of various biological samples at ultra-rapid speed, without need of any equipment in a single-step process.
  • the compositions disclosed herein comprises Tris Hydrochloride, Magnesium chloride hexahydrate, Potassium chloride, Sodium chloride, Ammonium chloride, Sodium bicarbonate, Sodium dodecyl sulphate, and water.
  • the nucleic acids thus extracted can be directly used for downstream processes, and can be stored and transported without any further processing steps.
  • the extracted nucleic acid samples can be stored at 4°C for 7 to 14 days and -20°C for long-term storage.
  • Figure 1 Gel bands of PCR amplified DNA extracted through Buffer A to Buffer G
  • Figure 2A Gel bands of PCR amplified DNA extracted through Buffer H
  • Figure 2B Gel bands of PCR amplified DNA extracted through Buffer J and Buffer K
  • Figure 2C Gel bands of PCR amplified DNA extracted through Buffer L and Buffer M
  • Figure 2D Gel bands of PCR amplified DNA extracted through Buffer N and Buffer O
  • Figure 2E Gel bands of PCR amplified DNA extracted through Buffer P
  • Figure 3A Gel bands for optimized sample volume
  • Figure 3B Gel bands of PCR products amplified from DNA extracted with optimized Buffer 1 at 200 pL buffer and 5pL blood ratio
  • Figure 3C Gel bands of PCR products amplified from DNA extracted with different optimized buffer compositions, Buffer 1, Buffer 2, Buffer 3 and Buffer 4 at 200 pL buffer and 5pL blood ratio
  • Figure 3D Gel bands of PCR products amplified from DNA extracted with different optimized buffer compositions, Buffer 1, Buffer 2, Buffer 3 and Buffer 4 at 300 pL buffer and 5pL blood ratio
  • Figure 5A Gel bands of genomic DNA extracted from animal cells, yeast cells and bacterial plasmid
  • Figure 5B Gel bands of genomic DNA and RNA extracted from insect tissues
  • Figure 5C Gel bands of genomic DNA and RNA extracted from plant seeds
  • FIG. 5D Gel bands of genomic DNA extracted from plant roots, pollen and leaves
  • FIG. 5E Gel bands of genomic DNA extracted from plant leaves with different optimized buffer compositions
  • FIG. 5F Gel bands of genomic DNA extracted from insect tissue with different optimized buffer compositions
  • Figure 7A Gel bands of DNA extracted with Buffer 1 and the corresponding PCR amplified product validated by CIDRF (Central Inter-Disciplinary Research Facility, Puducherry)
  • Figure 7B Gel bands of DNA extracted with Buffer 1 and the corresponding PCR amplified product validated by School of Lifesciences, Manipal University, Manipal
  • Figure 7C Gel bands of DNA extracted with Buffer 1 and the corresponding PCR amplified product validated by Elango Genetics, India
  • Figure 8A Gel bands of PCR amplified DNA extracted from buffers stored for one month at different temperature
  • Figure 8B Gel bands of PCR amplified DNA extracted from buffers stored for three months at different temperature
  • Figure 8C Gel bands of PCR amplified DNA extracted from buffers stored for four months at different temperature
  • Figure 8D Gel bands of PCR amplified DNA extracted from buffers stored for six months at different temperature
  • Figure 8E Gel bands of PCR from buffer extracted DNA stored at 4°C at day 2, day 4, day 7
  • Figure 8F Gel bands of PCR from buffer extracted DNA stored at day 10, day 12, day 14 and day 16
  • buffer compositions mean a cocktail of buffers and chemicals comprising of buffering chemicals, cell lysis chemicals, stabilizing chemicals and protein denaturation chemicals, and particularly referred herein as Buffer 1, Buffer 2, Buffer 3 and/or Buffer 4, and its other optimized variants.
  • DBS dried blood sample
  • Gel bands means gel-electrophoresis bands.
  • the present invention discloses functional single-buffer compositions for extraction and storage of nucleic acids. More particularly, the invention relates to optimized single-buffer compositions capable of extraction of nucleic acid from micro-volumes of various biological samples at ultra-rapid speed in a single-step method, without need of any equipment.
  • the nucleic acids thus extracted can be directly used for downstream processes, and can be stored and transported without any further processing steps.
  • the single-buffer technology using functional single -buffer compositions of the present invention is characterized by the following advantages: a.
  • the ultra-rapid nucleic acid extraction method reduces the turn-around time for the process to less than five (05) seconds, which is an instantaneous method. b.
  • the buffer cocktail contains chemicals that lyse the cells, degrade the proteins present and protect the nucleic acids.
  • a small quantity of biological sample has to be added with the cocktail buffers and mixed properly for 5 seconds.
  • This lysed mixture can be used as the starting sample for any molecular biology research or diagnostics.
  • This mixture can be stored at room temperature, 4°C or -20 °C for any future reference or studies.
  • the invention provides for the said functional single-buffer compositions comprising of buffering chemicals, cell lysis chemicals, stabilizing chemicals and protein denaturation chemicals.
  • compositions comprise of Tris Hydrochloride, Magnesium chloride hexahydrate, Potassium chloride, Sodium chloride, Ammonium chloride, Sodium bicarbonate, Sodium dodecyl sulphate, and water.
  • the invention provides for the said functional single-buffer compositions comprising of buffering chemicals, cell lysis chemicals, stabilizing chemicals and protein denaturation chemicals in water solution.
  • the invention provides for functional single-buffer compositions comprising Tris Hydrochloride and Potassium chloride as buffering agents.
  • the invention provides for functional single-buffer compositions comprising Sodium dodecyl sulphate as cell lysis agents.
  • the invention provides for functional single-buffer compositions comprising Ammonium chloride, Magnesium chloride hexahydrate and Sodium bicarbonate as stabilizing agents.
  • the invention provides for functional single-buffer compositions comprising Sodium chloride as protein denaturation agents.
  • the invention provides for functional single-buffer compositions comprising Tris Hydrochloride at pH 7.0 to 9.0 in concentration range of 10 mM to 50 iTiM or in concentration range of 10 mM to 30 mM, preferably at 20 mM.
  • the invention provides for functional single-buffer compositions comprising Magnesium chloride hexahydrate in the concentration range of 0 mM to 5 mM or in concentration of 1 mM to 3 mM, preferably at 0 mM and 2.5 mM.
  • the invention provides for functional single-buffer compositions comprising Potassium chloride in the concentration range of 10 mM to 100 mM or in concentration range of 15 mM to 40 mM, preferably at 25 mM.
  • the invention provides for functional single-buffer compositions comprising Sodium dodecyl sulphate in the concentration range of 0.01% to 0.1% w/v in the composition, preferably at 0.05% w/v and at 0.02% w/v.
  • the invention provides for functional single-buffer compositions comprising Sodium chloride in the concentration range of 50 mM to 250 mM, preferably at 200 mM and 100 mM.
  • the invention provides for functional single-buffer compositions comprising Ammonium chloride in the concentration range of 2 mM to 20 mM or in concentration range of 2 mM to 10 mM, preferably at 5 mM.
  • the invention provides for functional single-buffer compositions comprising Sodium bicarbonate in the concentration of 1 mM to 5 mM or in concentration range of 1.5 mM to 3.5 mM, preferably at 2.5 mM.
  • the invention provides for functional single-buffer compositions dissolved in nuclease-free water, wherein the water is undistilled, distilled, double distilled, high pure, ultra-high pure or ultra-filtered.
  • the invention provides for functional single-buffer compositions dissolved in nuclease-free water, wherein the water is sterilized by autoclave or UV rays exposure.
  • the invention provides for functional single-buffer compositions dissolved in nuclease free water, wherein the water is either sterilized or un sterili ed.
  • the invention provides for a method of preparing the functional single-buffer compositions, comprising the following steps: a. preparation of stock solutions of all chemicals in respective concentration ranges: 2 M Tris Hydrochloride, 1 M Magnesium chloride, 1 M Potassium chloride, 5 M Sodium chloride, 1 M Ammonium chloride, 0.5 M Sodium bicarbonate and 10% w/v Sodium dodecyl sulphate. b. addition of chemicals in the aforementioned concentration and the final required volume is adjusted with water. c. The cloudy precipitate, if any, can be dissolved by the heating the composition at 37°C for 10 minutes, wherein the final buffer compositions are maintained between pH 7.0 to 9.0, preferably between pH 7.5 to 8.5. In one embodiment, liquid or solid biological sample is added to required volume of buffer and mixed for complete lysis of cells and isolation of the nucleic acids.
  • liquid sample is mixed with the functional single buffer compositions by pipetting or tapping.
  • the solid tissues samples mixed with the functional single-buffer compositions require complete or mild homogenization.
  • the functional single-buffer compositions have a shelf life of up to 6 months at room temperature, and more than 6 months at 4°C and -20 °C.
  • the functional single -buffer compositions do not require further processing steps such that the extracted nucleic acid samples can be directly used for down-stream processing.
  • the nucleic acid samples, extracted with the functional single-buffer compositions have a shelf life of up to 16 days at 4°C and long-term storage at -20°C.
  • the functional single-buffer compositions comprise of buffering chemicals, which maintains the pH of the solution during the nucleic acid extraction.
  • the cell lysis chemicals break the cell wall and cell membranes of the cell and releases the nucleic acids, cell organelles and proteins.
  • the stabilizing chemicals stabilizes the nucleic acid in the solution.
  • the protein denaturation chemicals denature the proteins present in the solution.
  • buffers and chemicals are added together in an optimized concentration to make a cocktail composition that is finally diluted with nuclease-free water to obtain the functional buffer composition for nucleic acid sample extraction, preparation and storage.
  • compositions comprising buffers and chemicals of the present invention may further comprise a pharmaceutically acceptable carrier or excipient.
  • the carriers include, but are not limited to sterile aqueous media, solid diluents or fillers, excipients, and various non-toxic organic solvents.
  • the biological materials used herein this invention were solely for the purposes of testing the effectivity and efficacy of the functional buffer compositions. None of the biological material is part of any of the compositions or the invention, and is merely used as a source of nucleic acids to demonstrate the effectiveness of the functional single-buffer compositions.
  • the blood samples, cheek cells and sperm cells were obtained from the first named inventor of this present invention. Plant leaves, roots and pollen were obtained from plants in and around the School of Life Sciences at University of Hyderabad. Insect tissues were obtained from dead honeybees and ants in and around the School of Life Sciences at University of India.
  • the cultured cells, which were used-up, completely matured and waste to be discarded, were obtained from Oncoseek Private Limited, India. Watermelon seeds and pulses were purchased from local market/store. Yeast and bacterial cells were obtained from a startup from Albus Eco Projects incubated at BioNEST.
  • the functional single-buffer compositions were prepared after optimization of various chemical constituents that are generally present in an isolation/extraction buffer for nucleic acid extraction.
  • Table 1 elucidates a complete extraction buffer, used as control, selected from a range of concentrations of various constituents, for optimal nucleic acid extraction. The complete extraction buffer, so prepared, was then was tested for its significance on the nucleic acid extraction with and without one or more constituent.
  • the complete extraction-buffer was prepared in different compositions used as control, and other buffers were prepared by keeping all the constituents concentration constant and by modifying concentrations and/or removing one constituent at a time.
  • the new buffer compositions were then compared for its nucleic acid extraction effectiveness against that of the complete extraction-buffer, which is presented in Table 2.
  • Table 1 details the complete-buffer composition with respective concentration which was used for the optimization process.
  • Buffer A was prepared by removing EDTA from the complete extraction-buffer and then this Buffer A was used to extract DNA from blood sample, followed by PCR analysis.
  • Buffer B was prepared from complete extraction-buffer without Triton X100 and used to extract DNA from blood sample, followed by PCR analysis.
  • other iterations of the new buffers i.e. Buffer A to Buffer G, from complete extraction-buffer with modification and/or without individual components were also prepared and checked for DNA extraction efficiency.
  • Table 2 below enlists such various new buffer compositions prepared from the complete extraction-buffer and their corresponding effect on DNA extraction. Thereafter, PCR amplification was carried out and the gel-electrophoresis results corresponding to Table 2 are depicted in Figure 1.
  • Buffer H to Buffer Q as represented in Table 3.
  • Buffer H to Buffer Q were tested in absence of individual constituent, without any change in concentration of those individual constituent, in half the concentration (0.5x) and in double the concentration (2x). The effects of concentration variations in the resultant buffer were identified. Thereafter, PCR amplification was carried out and the results are depicted in Figures 2A to 2E.
  • Figure 2A shows the gel electrophoresis results of PCR amplified DNA extracted through Buffer H, Buffer I. The insignificance role of Triton X-100 was confirmed and Sodium bicarbonate concentration was optimized.
  • Figure 2B shows the gel electrophoresis results of PCR amplified DNA extracted through Buffer J and Buffer K. Fower concentration of Magnesium chloride was found to be efficient and ammonium chloride concentration is optimized.
  • Figure 2C shows the gel electrophoresis results of PCR amplified DNA extracted through Buffer L and Buffer M. Sodium dodecyl sulphate was found to highly significant for the buffer composition.
  • Figure 2D shows the gel electrophoresis results of PCR amplified DNA extracted through Buffer N and Buffer O. Sodium chloride was found to be important for the buffer composition.
  • Figure 2E shows the gel electrophoresis results of PCR amplified DNA extracted through Buffer P.
  • the role of EDTA and Triton X-100 were found to be insignificant. Role of other constituents and their concentrations were measured and are reported in Table 4.
  • the buffer compositions were checked without EDTA and Trito X-100 and by removing other selected ingredients like SDS, NaCl, KC1 and MgCF, to confirm their significance in the absence of EDTA and Triton X-100.
  • the concentration of all ingredients were checked at 2 times and 0.5 times the earlier concentration. The 0.5 times concentrations of all ingredients were found to be good (without EDTA and Triton X-100), which was finally optimized.
  • Example 1 Based on the optimizations carried out in Example 1 and Example 2, funtional single-buffer compositions were prepared which is represented in Table 5, Table 6 Table 7 and Table 8.
  • the process of selection involved various constituents, and thereafter the selected sets of the buffer constituents were further optimized to a particular sub-set of concentration range. Further, the composition constituents so selected were optimized from its sub-set concentration range to a particular set of concentration.
  • Four buffers, namely Buffer 1, Buffer 2, Buffer 3 and Buffer 4 were found to be the most effective in the nucleic acid extraction from various sets of samples. These four functional single-buffer compositions are elaborated in Table 5, Table 6 Table 7 and Table 8.
  • the water used for preparation of all the functional single-buffer compositions can be nuclease-free water, wherein the water is undistilled, distilled, double distilled, high pure, ultra-high pure or ultra-filtered, unsterilized or sterilized by using autoclave or sterilized by using UV rays.
  • Table 5 provides the sub-set of concentration range of the individual concentration of the Buffer 1, along with the particular concentration of the individual constituents in which the Buffer 1 was prepared and tested for nucleic acid extraction.
  • Table 5 Composition of Buffer 1 Table 6 provides the sub-set of concentration range of the individual concentration of the Buffer 2, along with the particular concentration of the individual constituents in which the Buffer 2 was prepared and tested for nucleic acid extraction. In the Buffer 2, all the constituents of Buffer 1 were maintained as it is, except that Sodium chloride was used in the concentration range of 50 mM to 250 mM and preferably at 200 mM concentration.
  • Table 7 provides the sub-set of concentration range of the individual concentration of the Buffer 3, along with the particular concentration of the individual constituents in which the Buffer 3 was prepared and tested for nucleic acid extraction.
  • Buffer 3 all the constituents of Buffer 1 were maintained as it is, except that Sodium dodecyl sulphate was used in the concentration range of 0.01% to 0.1% w/v, and preferably at 0.05% concentration.
  • Table 7 Composition of Buffer 3 Table 8 provides the sub-set of concentration range of the individual concentration of the Buffer 4, along with the particular concentration of the individual constituents in which the Buffer 4 was prepared and tested for nucleic acid extraction.
  • Buffer 4 all the constituents of Buffer 1 were maintained as it is, except that Magnesium chloride hexahydrate was used in the concentration range of OmM to 5 mM, and preferably at 0 mM concentration.
  • the sample/buffer ratio was checked with the optimized i.e. Buffer 1, Buffer 2, Buffer 3 and Buffer 4.
  • Commercial kits and manual methods use a minimum of 50 pL of blood sample for DNA extraction.
  • the functional single-buffer compositions of the present invention are optimized to reduces the sample volume requirement to 1 to 10 pL, and most preferably 5 pL for 200 pL of the functional single-buffer compositions.
  • Figure 3A elucidates the optimized sample volume.
  • the optimized functional single-buffer compositions were tested in concentrations of 200 pL, 300 pL, 400 pL and 500 pL. Functional single-buffer compositions of higher volumes were not used, as the higher volumes were diluting the salt concentration present in the buffers, making it unfit for the intended purpose.
  • Figure 3B elucidates gel- electrophoresis bands of PCR products amplified from DNA extracted with optimized Buffer 1 at 200 pL buffer and 5pL blood ratio.
  • Figure 3C elucidates gel-electrophoresis bands of PCR products amplified from DNA extracted with different optimized buffer compositions, Buffer 1, Buffer 2, Buffer 3 and Buffer 4 at 200 pL buffer and 5pL blood ratio.
  • Figure 3D elucidates gel-electrophoresis bands of PCR products amplified from DNA extracted with different optimized buffer compositions, Buffer 1, Buffer 2, Buffer 3 and Buffer 4 at 300 pL buffer and 5pL blood ratio.
  • Qubit assay utilizes target-selective dyes that emit fluorescence when bound to DNA, RNA or protein, unlike UV absorbance which can overestimate sample concentration due to contaminants in the sample. Fluorescence measurements are much more sensitive than UV absorbance, giving accurate measurements with significantly less noise.
  • the template volume of buffer extracted sample was optimized and it was found that the optimal volume of prepared sample for PCR applications is in the volume range of 1 to 5 pL, and more particularly in the volume range of 2 to 3 pL.
  • Figure 4 depicts the results corresponding to optimized template volume of buffer extracted sample for PCR applications/downstream processes.
  • EXAMPLE 6 Method of nucleic acid (DNA/RNA) isolation from various biological samples
  • Different biological samples like dried blood samples (dried in floor, cloth, tissue paper, cotton), cheek cells, sperm cells, culture mammalian cells, old lysed blood, yeast cells, bacterial plasmids, plant leaves, root, seeds, pollen grains, insect tissues were collected from various sources.
  • DBS tissue samples a few drops of blood sample were dropped on commercial tissue paper and dried at room temperature.
  • DBS cotton samples a few drops of blood sample were dropped on cotton and dried at room temperature.
  • DBS cloth samples few drops of blood sample were dropped on a piece of cloth and dried at room temperature.
  • DBS floor samples a few drops of blood sample were dropped on laboratory floor and dried at room temperature.
  • the functional single-buffer compositions were prepared by using a method comprising the following steps: a. preparation of stock solutions of all chemicals in respective concentration ranges of 2 M Tris Hydrochloride, 1 M Magnesium chloride, 1 M Potassium chloride, 5 M Sodium chloride, 1 M Ammonium chloride, 0.5 M Sodium bicarbonate and 10% w/v Sodium dodecyl sulphate, b. addition of chemicals in the aforementioned concentration and the final required volume of 200 pL of the functional single -buffer compositions are prepared by diluting with water, c. The cloudy precipitate, if any, can be dissolved by the heating the composition at
  • Liquid or solid biological sample is added to 200 pL of the functional single-buffer compositions (Buffer 1, Buffer 2, Buffer 3 or Buffer 4) and mixed for complete lysis of cells and isolation of the nucleic acids.
  • the liquid sample is mixed with the functional single-buffer compositions by pipetting or tapping.
  • the solid tissues samples mixed with the functional single-buffer compositions require complete or mild homogenization.
  • the liquid samples were directly mixed with the optimized buffer compositions, while solid tissue samples were homogenized with pestle and added to the buffers. The samples were gently mixed. After lysis, the buffer extracted samples were directly loaded in 0.8% agarose gel and visualized under UV light.
  • Figure 5A shows the gel electrophoresis results of DNA extracted through Buffer 1 using various samples, namely DBS tissue, DBS cotton, DBS cloth, DBS floor, cheek cells, culture mammalian cells, sperm cells, old lysed blood, yeast cells and bacterial cells.
  • Figure 5B shows the gel electrophoresis results of genomic DNA, RNA and degraded RNA extracted through Buffer lusing various samples, namely insect tissues.
  • Figure 5C shows the gel electrophoresis results of genomic DNA and degraded RNA extracted through Buffer lusing seeds.
  • Figure 5D shows the gel electrophoresis results of genomic DNA extracted through Buffer lusing various samples, namely plant roots, pollens and leaves.
  • Figure 5E shows the gel-electrophoresis bands of genomic DNA extracted from plant leaves with different optimized buffer compositions.
  • Figure 5F shows the gel-electrophoresis bands of genomic DNA extracted from insect tissue with different optimized buffer compositions.
  • the buffer compositions of the present invention extract nucleic acid (DNA/RNA) from 1 pL to 10 pL blood.
  • DNA/RNA nucleic acid
  • the DNA extracted with manual method was diluted equalizing the final volume of blood sample and then compared with the corresponding results of the present invention.
  • the undiluted DNA from manual method was 450.2 ng/pL and was diluted 300 times to match with the buffer extracted DNA.
  • concentration multiple of manual method has to be multiplied with that of the buffer.
  • the Equalization Factor comes to 300 times (40 x 7.5).
  • Manual method of DNA extraction uses 300 pL blood sample and DNA eluted to final concentrated volume of 40 pL, while the buffer use only 5 pL blood and mixed with 200 pL of buffer resulting in further dilution. Hence, the final extracted DNA using manual method was diluted to equalize the initial blood volume and then loaded in 0.8% agarose gel. The yield of DNA (diluted) from manual method was lesser compared to DNA extracted using the buffer.
  • Figure 6 elucidates the comparative DNA yields wherein the gel-electrophoresis band obtained from the buffer are much brighter than those obtained from manual method, establishing the superiority of the buffer of present invention.
  • the inventors got third party validations done through three separate independent organizations of repute, including both private and government recognized public organizations.
  • Figure 7A depicts the gel-electrophoresis bands of the DNA extracted from Buffer 1 and the corresponding PCR amplified products, which was validated by Central Inter- Disciplinary Research Facility, Puducherry (CIDRF), which is a scientific and industrial research organization (SIRO) as recognized by the Department of Scientific and Industrial Research (DSIR), Ministry of Science and Technology, Government of India. PCR was performed to amplify product of 692bp size.
  • CDRF Central Inter- Disciplinary Research Facility
  • SIRO scientific and industrial research organization
  • L indicates DNA ladder to identify the product size
  • E is empty lane
  • F is blood sample collected through finger prick
  • V is blood collected from veins
  • 3F & 2V are PCR products amplified from buffer extracted DNA
  • C is PCR product amplified from DNA extracted through commercial kit
  • 3V & 2F are buffer extracted genomic DNA
  • 3 & 2 are random sample numbers. Blood sample of 5pF was mixed with 200 pF of Buffer 1.
  • Figure 7B depicts the gel-electrophoresis bands of the DNA extracted from Buffer land the corresponding PCR amplified products, which was validated by School of Fifesciences, Manipal University, Manipal, India. PCR was performed using Buffer 1 extracted DNA. Blood sample of 5pF was mixed with 200 pF of Buffer 1.
  • Figure 7C depicts the gel-electrophoresis bands of the DNA extracted from Buffer land the corresponding PCR amplified products, which was validated by Elango Genetics, India.
  • the samples were used with and without coagulants in addition to the lysed blood. It was observed that no PCR amplification took place in heparin added blood.
  • PCR was performed using Buffer 1 extracted DNA. Blood sample of 5pF was mixed with 200 pF of Buffer 1.
  • Fane 1 is PCR product of EDTA anticoagulated blood
  • lane 2 and 3 are blood sample collected by finger prick
  • lane 4 is Heparin anticoagulated blood
  • lane 5 & 6 are lysed blood samples
  • lane 7 is DNA ladder.
  • the buffer composition vials were stored at room temperature (RT), 4°C and -20°C separately and checked for its storage parameters and shelf life.
  • the buffers were used to extract DNA and thereafter PCR analysis was performed at different time intervals.
  • Figure 8A elucidates gel-electrophoresis bands of PCR amplified DNA extracted from the buffer compositions, wherein the buffers were stored for one month at room temperature (RT), 4°C and -20°C. No significant difference was observed in the gel- electrophoresis bands of PCR amplified DNA at various temperature conditions for a time period of one months.
  • Figure 8B elucidates gel-electrophoresis bands of PCR amplified DNA extracted from the buffer compositions, wherein the buffers were stored for three months at room temperature (RT), 4°C and -20°C. No significant difference was observed in the gel- electrophoresis bands of PCR amplified DNA at various temperature conditions for a time period of three months.
  • Figure 8C elucidates gel-electrophoresis bands of PCR amplified DNA extracted from the buffer compositions, wherein the buffers were stored for four months at room temperature (RT), 4°C and -20°C. No significant difference was observed in the gel- electrophoresis bands of PCR amplified DNA at various temperature conditions for a time period of four months.
  • Figure 8D elucidates gel-electrophoresis bands of PCR amplified DNA extracted from the buffer compositions, wherein the buffers were stored for six months at room temperature (RT), 4°C and -20°C. No significant difference was observed in the gel- electrophoresis bands of PCR amplified DNA at various temperature conditions for a time period of six months. Thereby, the shelf life of buffer compositions is at least six months.
  • the buffer extracted DNA samples were stored at 4°C for period of 7 to 16 days and its integrity were observed with PCR amplification.
  • the DNA was found to be intact which produces good PCR bands.
  • the shelf life of buffer extracted DNA samples is at least 7 to 16 days with high integrity.
  • the shelf life of the buffer extracted DNA samples is higher than 16 days at 4°C, and at -20°C, the shelf-life of the same is for a longer period of time.
  • Figure 8E elucidates gel-electrophoresis bands of PCR of Buffer 1 extracted DNA stored at 4°C after 2 days, 4 days and 7 days. No significant difference was observed in the gel-electrophoresis bands of PCR amplified DNA its indicating high integrity.
  • Figure 8F elucidates gel-electrophoresis bands of PCR of Buffer 1 extracted DNA stored at 4°C after 10 days, 12 days, 14 days and 16 days. No significant difference was observed in the gel-electrophoresis bands of PCR amplified DNA indicating its high integrity.

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Abstract

L'invention concerne des compositions tampons d'extraction d'acides nucléiques de micro-volumes de divers échantillons biologiques notamment de plantes, de cellules animales et microbiennes, de sang, de sperme et d'échantillons médico-légistes à une vitesse ultra-rapide inférieure ou égale à 5 secondes, sans avoir besoin d'un quelconque équipement avec un processus d'extraction en une seule étape. L'invention concerne également le fait que lesdits tampons sont constitués de concentrations optimisées de produits chimiques tampons, de produits chimiques de lyse cellulaire, de produits chimiques stabilisants et de produits chimiques de dénaturation de protéines, et leurs processus de préparation. Les acides nucléiques ainsi extraits peuvent être directement utilisés à des fins de processus aval et peuvent être stockés et transportés sans étape de traitement supplémentaire. La durée de conservation des échantillons d'acides nucléiques extraits est de 7 à 14 jours à 4 °C et plus longue à -20 °C. L'invention concerne des solutions et des kits de diagnostic abordables et efficaces pour des diagnostics génétiques délocalisés, d'autres utilisations scientifiques, cliniques et industrielles.
PCT/IN2021/050335 2020-04-06 2021-04-04 Composition tampon pour préparation d'échantillons d'acides nucléiques en une étape et stockage à partir d'échantillons biologiques Ceased WO2021205470A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007068452A (ja) * 2005-09-06 2007-03-22 Fujifilm Corp 核酸の分離精製方法
CA3042088A1 (fr) * 2017-01-16 2018-07-19 Spectrum Solutions L.L.C. Solution de conservation d'acides nucleiques et ses procedes de fabrication et d'utilisation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007068452A (ja) * 2005-09-06 2007-03-22 Fujifilm Corp 核酸の分離精製方法
CA3042088A1 (fr) * 2017-01-16 2018-07-19 Spectrum Solutions L.L.C. Solution de conservation d'acides nucleiques et ses procedes de fabrication et d'utilisation
US20190062806A1 (en) * 2017-01-16 2019-02-28 Spectrum Solutions L.L.C. Nucleic acid preservation solution and methods of manufacture and use

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HILL, V. R ET AL.: "DEVELOPMENT OF A NUCLEIC ACID EXTRACTION PROCEDURE FOR SIMULTANEOUS RECOVERY OF DNA AND RNA FROM DIVERSE MICROBES IN WATER", PATHOGENS, vol. 4, no. 2, 2015, pages 335 - 354, XP055410495, DOI: 10.3390/pathogens4020335 *

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