COMPOSITIONS AND METHODS FOR USING ACTIVATED CARBON PARTICLES FOR PURIFICATION OF NUCLEIC ACIDS

Abstract

Compositions and methods to improve the process of nucleic aids purification from bacterial, prokaryotic and eukaryotic cells through the use of activated carbon particles.

Claims

1-4. (canceled)

5. A method of formulating activated carbon for use in nucleic acid extraction, comprising (a) harvesting cells through centrifugation; (b) resuspending the cells in a lysis buffer; (c) adding activated carbon particles either before or after cell lysis; (d) removing cell debris and activated carbon particles by centrifugation; (e) applying DNA or RNA-containing supernatant to a DNA or RNA-binding column; (f) washing the column with washing buffer to remove cellular contaminants; and (g) eluting DNA or RNA with a buffer or with H.sub.2O.

6. The method of claim 5, wherein the buffer is TE buffer.

7. The method of claim 5, wherein the cells are chosen from bacterial cells, bacterial cell cultures, or biological samples containing bacterial cells.

8. The method of claim 7, wherein the method results in the purification of bacterial genomic DNA or bacterial plasmid DNA.

9. The method of claim 5, wherein the cells are chosen from animal cell cultures, animal tissue samples, and animal fluid samples.

10. The method of claim 9, wherein the animal fluid samples are chosen from blood and sperm.

11. The method of claim 10, wherein the method results in the purification of genomic DNA, cytoplasmic DNA or RNA.

12. The method of claim 11, where the cytoplasmic DNA is mitochondrial DNA.

13. The method of claim 9, wherein the method results in the purification of genomic DNA, cytoplasmic DNA or RNA.

14. The method of claim 13, where the cytoplasmic DNA is mitochondrial DNA.

15. A kit comprising activated carbon, and one or more of DNA or RNA extracting buffers, DNA or RNA binding buffers, DNA or RNA-binding columns, column washing buffers, and DNA elution buffers.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1: Activated carbon particles can remove impurities during the purification of plasmid DNA from E. coli. An equal amount of overnight bacterial cell culture (1.5 ml) was purified using a commercial Q miniSpin plasmid purification kit, or using a similar kit from Virong LLC (Virongy high purity miniSpin plasmid purification kit) in which activated charcoal particles were added to the bacterial cell lysates during purification. Following plasmid DNA purification, an equal amount of plasmid DNA, purified with the commercial Q miniSpin kit or with the Virongy miniSpin kit was digested with different amounts of E.coRI that was serially diluted (1:2 dilution). Following digestion, DNA was resolved by agarose gel electrophoresis. As shown, activated charcoal treatment leads to greater plasmid DNA purity that can be readily digested by E.coRI. At low E.coRI concentrations, the presence of impurities in the DNA purified from the Q miniSpin kit inhibits E.coRI activity, resulting in partial digestions.

[0020] FIG. 2: Activated carbon particles can remove impurities during plasmid DNA purification, leading to high purity DNA that can generate longer nucleotide read from DNA sequencing. An equal amount of overnight bacterial cell culture (1.5 ml) was purified using a commercial Q miniSpin plasmid purification kit, or using a similar kit from Virong LLC (Virongy high purity miniSpin plasmid purification kit) in which activated charcoal particles were added to bacterial cell lysates during plasmid purification. Following plasmid DNA purification, an equal amount of plasmid DNA, purified with the commercial Q miniSpin kit or with the Virongy miniSpin kit, was used as the template for DNA sequencing analysis. DNA purified with activated charcoal particles gave a sequence read 20 to 40 nucleotide longer than DNA purified with the commercial Q miniSpin kit (Average sequenced DNA length from activated charcoal/Virongy MiniSpin kit: 91924.77; average sequenced DNA length from Q MiniSpin kit: 89819.45; p=0.00196)

DETAILED DESCRIPTION

[0021] The present disclosure relates to composition and methodology for use activated carbon or charcoal to improve the process of nucleic aids purification from bacterial, prokaryotic and eukaryotic cells. Provided herein are methodology, compositions, and the like for using activated carbon particles to remove impurities in the purification of nucleic acids.

[0022] In one embodiment, activated carbon particles are used to remove impurities present in bacterial cell lysate during the purification of bacterial genomic DNA or bacterial plasmid DNA.

[0023] In another embodiment, activated carbon particles are used to remove impurities present in animal cell lysate during the purification of genomic DNA, cytoplasmic DNA (e.g. mitochondrial DNA) or RNA.

[0024] In another embodiment, activated carbon particles are used to remove impurities present in plant cell lysate during the purification of genomic DNA or cytoplasmic DNA (e.g. chloroplast DNA)

[0025] Activated carbon refers to activated charcoal or any porous and nanoporous carbon materials that can be used to absorb chemicals and biochemical substances.

[0026] In other embodiments, provided is a kit comprising activated charcoal and one or more of DNA/RNA extracting buffers, DNA/RNA binding buffer, DNA/RNA-binding columns, column washing buffers, and DNA elution buffers.

[0027] All technical terms in this description are commonly used in biochemistry, molecular biology and immunology, respectively, and can be understood by those skilled in the field of this invention. Those technical terms can be found in: MOLECULAR CLONING: A LABORATORY MANUAL, 3rd ed., vol. 1-3, ed. Sambrook and Russel, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, ed. Ausubel et al., Greene Publishing Associates and WileyInterscience, New York, 1988 (with periodic updates); SHORT PROTOCOLS IN MOLECULAR BIOLOGY: A COMPENDIUM OF METHODS FROM CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, 5.sup.th ed., vol. 1-2, ed. Ausubel et al., John Wiley & Sons, Inc., 2002; GENOME ANALYSIS: A LABORATORY MANUAL, vol. 1-2, ed. Green et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1997; CELLULAR AND MOLECULAR IMMUNOLOGY, 4.sup.th ed. Abbas et al., WB Saunders, 1994.

EXAMPLES

[0028] Illustrative Examples are presented below. They are exemplary and non-limiting.

Example 1: The Use of Activated Charcoals to Remove Impurity During the Purification of Plasmid DNA

[0029] As exemplified in FIG. 1, activated carbon particles were used to remove impurities during the purification of plasmid DNA from E. coli. An equal amount of overnight bacterial cell culture (1.5 ml) was purified using a commercial Q miniSpin plasmid purification kit, or using a similar kit from Virong LLC (Virongy high purity miniSpin plasmid purification kit) in which activated charcoal particles were added to bacterial cell lysates during purification. Following plasmid DNA purification, an equal amount of plasmid DNA, purified with the commercial Q miniSpin kit or with the Virongy miniSpin kit was digested with different amounts of E.coRI that was serially diluted (1:2 dilution). Following digestion, DNA was resolved by agarose gel electrophoresis. As shown, activated charcoal treatment leads to greater plasmid DNA purity that can be readily digested by E.coRI. At low E.coRI concentrations, the presence of impurities in the DNA purified from the Q miniSpin kit inhibits E.coRI activity, resulting in partial digestions.

Example 2: The Use of Activated Charcoals to Remove Impurity During the Purification of Plasmid DNA

[0030] As exemplified in FIG. 2, activated carbon particles were used to remove impurities during plasmid DNA purification, leading to high purity DNA that can be sequenced with longer nucleotide read. An equal amount of overnight bacterial cell culture (1.5 ml) was purified using a commercial Q miniSpin plasmid purification kit, or using a similar kit from Virong LLC (Virongy high purity miniSpin plasmid purification kit) in which activated charcoal particles were added to bacterial cell lysates during plasmid purification. Following plasmid DNA purification, an equal amount of plasmid DNA, purified with the commercial Q miniSpin kit or the Virongy miniSpin kit, was used as the template for DNA sequencing analysis. DNA purified with activated charcoal particles gave a sequence read of 20 to 40 nucleotide longer than DNA purified with the commercial Q miniSpin kit (Average sequenced DNA length from activated charcoal/Virongy MiniSpin kit: 91924.77; Average sequenced DNA length from Q MiniSpin kit: 89819.45; p=0.00196)

Experimental Procedure for Using Activated Charcoals to Facilitate Plasmid DNA Purification

[0031] 1) In a microcentrifuge tube that holds at least 1.8 ml, add activated charcoal particles 1.5 ml of an overnight bacterial culture, and mix thoroughly by inverting. [0032] 2) Centrifuge the bacterial culture and activated charcoal mixture at maximum speed (13,000-16,000 rpm) for 60 seconds. Carefully discard the supernatant without disturbing the pellet. Resuspend the pellet by adding 150 l of Buffer A (50 mM Tris.HCl, 10 mM EDTA, 100 ug/ml RNase A) into the microcentrifuge tube and pipetting vigorously. [0033] 3) Add 150 l of Buffer B (200 mM NaOH, 1% SDS) to the resuspended pellet mix, and lyse bacterial cells by inverting the tube 8 times. [0034] 4) Neutralize the lysis reaction by adding 300 l of Buffer C (4.2M Gu-HCl, 0.9M Potassium acetate, pH4.8) to the microcentrifuge tube and inverting the tube 8 times. [0035] 5) Centrifuge at maximum speed for 10 minutes. A compact black pellet will form. [0036] 6) Decant the supernatant, which contains the plasmid DNA, into a spin column assembly. Centrifuge for 30 seconds and discard flow-through. The plasmid DNA will bind to the column. [0037] 7) Add 650 l of Buffer D (10 mM Tris.HCl, pH7.5, 80% ethanol) to the spin column to wash away contaminants. Centrifuge for 30 seconds at maximum speed. Discard the flow-through. [0038] 8) Remove any residual buffer by centrifuging the spin column at maximum speed for 30 seconds. Discard the flow-through. [0039] 9) Elute plasmid DNA by placing the spin column into a clean 1.5 ml collection tube, and adding 50 l of Buffer E (TE buffer or H20). Incubate for 60 seconds at room temperature. Then centrifuge at maximum speed for 60 seconds. [0040] 10) Store eluted plasmid DNA at 4 C.