Kits and processes for removing contaminants from nucleic acids in environmental and biological samples

• US 7,459,548 B2
• Filed: 05/20/2005
• Issued: 12/02/2008
• Est. Priority Date: 05/21/2004
• Status: Active Grant

• Current Assignee: QIAGEN Sciences, LLC (Qiagen NV)
• Original Assignee: MO BIO Laboratories, Inc. (Qiagen NV)
• Inventors: Stolow, David, Venugopal, Raveendran J., Brolaski, Mark N.
• Primary Examiner(s): Jiang; Shaojia Anna
• Assistant Examiner(s): CRANE, LAWRENCE E
• Application Number:
  US11/134,849
• Publication Number:
  US 20050282202A1
• Time in Patent Office: 1,292 Days
• Field of Search: None
• US Class Current: 536/25.41
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2565/113 based on agglutination/precipitation

Abstract

The invention provides methods for removing a contaminant or inhibitor from a nucleic acid-comprising sample, wherein the contaminant or inhibitor inhibits the amplification or hybridization of the nucleic acid in the sample, or inhibits an enzymatic reaction utilizing the nucleic acid in the sample, the method comprising the steps of: (a) providing a reaction mixture comprising the sample, a chaotropic agent, ammonium acetate or an equivalent, and a detergent, (b) isolating the nucleic acid and remaining contaminants and inhibitors from the reaction mixture in a supernatant; and (c) contacting the nucleic acid supernantant with a flocculant resulting in the further removal of the contaminant or the inhibitor from the supernatant. The invention also provides kits that comprise the components necessary to carry out the method.

Claims

1. A method for removing a contaminant or inhibitor from a nucleic acid-comprising sample, wherein the contaminant or inhibitor inhibits the amplification or hybridization of the nucleic acid in the sample, or inhibits an enzymatic reaction utilizing the nucleic acid in the sample, the method comprising the steps of:

(a) providing a reaction mixture comprising the sample, a chaotropic agent, ammonium acetate or an equivalent, and a detergent;

(b) isolating the nucleic acid and remaining contaminants and inhibitors from the reaction mixture in a supernatant; and

(c) contacting the nucleic acid supernantant with a flocculant resulting in the further removal of the contaminant or the inhibitor from the supernatant.

2. The method of claim 1, wherein the contaminant or inhibitor is selected from the group consisting of a polyphenol, a polysaccharide, a humic substance, an enzymatic inhibitor from soil, a humic polymer, an organic compound from compost, a decomposing plant material, a plant pigment, a plant cell wall, a chitin, a photosynthetic pigment, a humic acid, a fulvic acid, a phenolic polymer and/or phenolic oligomer, a tannin, a humin, and a phenolic.

3. The method of claim 1, wherein the equivalent is sodium chloride, ammonium sulfate, potassium acetate, or sodium acetate.

4. The method of claim 1, wherein the flocculant is aluminum ammonium sulfate, ammonium sulfate dodecahydrate;

aluminum ammonium sulfate dodecahydrate;

aluminum potassium sulfate, aluminum chlorohydrate, aluminum sulfate, calcium oxide, iron (III) chloride, iron (II) sulfate, sodium aluminate, or sodium silicate.

5. The method of claim 1, wherein the sample comprises an environmental or a biological sample, and optionally the environmental or biological sample comprises a sample derived from an animal, animal remains, a food, a microorganism, a plant or its components, soil, sediment, rock, reef, sludge, compost, decomposing biological matter, a biopsy, a histological sample, a semen sample, a blood or saliva sample, any body fluid sample, a hair sample, a skin sample, a fecal sample, archaeological remains, a peat bog, compost, oil, water, terrestrial water or subterranean water, atmospheric and industrial water, dust, urban dust, commercial potting mixtures or soil amendments, deep sea vents, or air.

6. The method of claim 1, wherein the nucleic acid comprises an RNA (mRNA, tRNA, rRNA, iRNA) or a DNA or a combination thereof.

7. The method of claim 1, wherein the detergent is selected from the group consisting of sodium dodecyl sulfate (SDS), sarkosyl, sodium lauryl sarcosinate, cetyltrimethyl ammonium bromide (CTAB), cholic acid, deoxycholic acid, benzamidotaurocholate (BATC), octyl phenol polyethoxylate, polyoxyethylene sorbitan monolaurate, tert-octylphenoxy poly(oxyethylene)ethanol, 1,4-piperazinebis-(ethanesulfonic acid), N-(2-acetamido)-2-aminoethanesulfonic acid, polyethylene glycoltert-octylphenyl ether (Triton®

X-100), (1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol (Triton®

X-114), and a combination thereof.

8. The method of claim 1, further comprising:

(d) purifying or isolating the nucleic acid; and

/or

(e) detecting or characterizing the nucleic acid, wherein the detecting or characterizing results in the determination that the nucleic acid is from an organism that produces a spore or a toxin.

9. The method of claim 8, wherein the toxin is a bacterial toxin.

10. The method of claim 1, further comprising:

(d) purifying or isolating the nucleic acid; and

/or

(e) detecting or characterizing the nucleic acid, wherein the detecting or characterizing results in the determination that the nucleic acid is from an organism that produces a biohazard agent.

11. The method of claim 10, wherein the biohazard agent is a bacterial toxin.

12. The method of claim 1, further comprising purifying or isolating the nucleic acid after step (c).

13. The method of claim 1, wherein the sample is an unprocessed, preserved, freshly isolated, crude or unrefined sample.

14. The method of claim 1, wherein the reaction mixture of step (a) is mixed or vortexed.

15. The method of claim 1, wherein the isolating in step (b) comprises centrifuging the reaction mixture and harvesting a nucleic acid-comprising supernatant.

16. The method of claim 1, further comprising after step (c), detecting or characterizing the nucleic acid.

39. A kit executing the method of for isolating a nucleic acid from a sample as described in claim 1 comprising:

(a) a chaotropic agent;

(b) ammonium acetate or an equivalent;

(c) a flocculant;

(d) a salt solution;

(e) an ethanol based wash solution;

(f) a low salt buffer, wherein optionally the buffer comprises Tris EDTA or water;

(g) a spin filter or spin filters; and

(h) a collection tube or collection tubes.

40. The kit of claim 39, further comprising a detergent or a surfactant, wherein optionally the detergent is selected from the group consisting of sodium dodecyl sulfate (SDS), sarkosyl, sodium lauryl sarcosinate, cetyltrimethyl ammonium bromide (CTAB), cholic acid, deoxycholic acid, benzamidotaurocholate (BATC), octyl phenol polyethoxylate, polyoxyethylene sorbitan monolaurate, tert-octylphenoxy poly(oxyethylene)ethanol, polyethylene glycoltert-octylphenyl ether (Triton®

X-100), (1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol (Triton®

X-114), and a combination thereof.

41. The kit of claim 39, further comprising a homogenizing material.

42. The kit of claim 41, wherein the homogenizing material comprises a bead.

43. The kit of claim 39, further comprising one or more oligonucleotides or free nucleotides.

44. The kit of claim 43, wherein the one or more oligonucleotides hybridize to a nucleic acid from a microorganism, an animal, a plant, an insect, a yeast, a virus, a phage, a nematode, a bacteria, a fungi, a bacterial toxin, or a fungal toxin.

45. The kit of claim 43, wherein the one or more oligonucleotides hybridize to a nucleic acid from:

(a) a Bacillus spp., a Clostridium spp., a Sporolactobacillus spp., a Sporocarcina spp., a Filibacter spp., a Caryophanum spp., a Desulfotomaculum spp., a Corynebacterium spp., a Micrococcus spp., a Mycobacterium spp., a Nocardia spp., a Peptococcus spp., a Peptostreptococcus spp.;

or

(b) a nucleic acid from a Gram negative bacteria selected from the family consisting of Acetobacteriaceae, Alcaligenaceae, Bacteroidaceae, Chromatiaceae, Enterobacteriaceae, Legionellaceae, Neisseriaceae, Nitrobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Rickettsiaceae and Spirochaetaceae;

or

(c) a nucleic acid from B. anthracis, A. globiformis, B. subtilis, C. renale, C. difficile, M. luteus, or R. eryrhropolis;

or

(d) a nucleic acid from a variola, a varicella, a reovirus, a retrovirus, HIV, HIV-1, HIV-2, a viral hemorrhagic fever, Ebola, Marburg, Machupo, Lassa, Variola major, or viral encephalitis.

46. The kit of claim 43, wherein the free nucleotides are sufficient to carry out a PCR reaction, a rolling circle replication, a ligase-chain reaction, or a reverse transcription reaction.

47. The kit of claim 46, wherein the enzyme is a polymerase.

48. The kit of claim 39, further comprising at least one enzyme.

17. A method for removing a contaminant or inhibitor from a nucleic acid-comprising sample, wherein the contaminant or inhibitor inhibits the amplification or hybridization of the nucleic acid in the sample, or inhibits an enzymatic reaction utilizing the nucleic acid in the sample, the method comprising the steps of:

(a) providing a reaction mixture comprising the sample, a chaotropic agent, ammonium acetate, and a detergent;

(b) isolating the nucleic acid and remaining contaminants and inhibitors from the reaction mixture in a supernatant; and

(c) contacting the nucleic acid supernantant with aluminum ammonium sulfate resulting in the further removal of the contaminant or the inhibitor from the supernatant.

18. The method of claim 17, further comprising purifying or isolating the nucleic acid after step (c).

19. The method of claim 17, wherein the sample is an unprocessed, preserved, freshly isolated, crude or unrefined sample.

20. The method of claim 17, wherein the reaction mixture of step (a) is mixed or vortexed.

21. The method of claim 17, wherein the isolating in step (b) comprises centrifuging the reaction mixture and harvesting a nucleic acid-comprising supernatant.

22. The method of claim 17, further comprising after step (c), detecting or characterizing the nucleic acid.

23. A method for efficient separation by flocculation of a contaminant or inhibitor from a nucleic acid-comprising sample, wherein the contaminant or inhibitor inhibits the amplification or hybridization of the nucleic acid in the sample, or inhibits an enzymatic reaction utilizing the nucleic acid in the sample, the method comprising the steps of:

(a) providing a reaction mixture comprising the sample, a chaotropic agent, ammonium acetate or an equivalent, and a detergent, wherein the presence of the chaotropic agent, the ammonium acetate or equivalent, and the detergent in the reaction mixture with the sample, results in the separation of the nucleic acid and the contaminant, the separation of the nucleic acid and the inhibitor, the separation of a protein present in the sample and the nucleic acid, the separation of a protein present in the sample and the inhibitor, the separation of a protein present in the sample and the contaminant, or any combination of the above, present in the sample;

(b) isolating the nucleic acid and remaining contaminants and inhibitors from the reaction mixture in a supernatant; and

(c) contacting the nucleic acid supernantant with a flocculant resulting in the further removal of the contaminant or the inhibitor from the supernatant.

24. The method of claim 23, further comprising purifying or isolating the nucleic acid after step (c).

25. The method of claim 23, wherein the sample is an unprocessed, preserved, freshly isolated, crude or unrefined sample.

26. The method of claim 23, wherein the reaction mixture of step (a) is mixed or vortexed.

27. The method of claim 23, wherein the isolating in step (b) comprises centrifuging the reaction mixture and harvesting a nucleic acid-comprising supernatant.

28. The method of claim 23, further comprising after step (c), detecting or characterizing the nucleic acid.

29. A method for maximum recovery of a nucleic acid from a nucleic acid-comprising sample, the method comprising the steps of:

(a) providing a reaction mixture comprising the sample, a chaotropic agent, ammonium acetate or an equivalent, and a detergent, wherein the presence of the chaotropic agent, the ammonium acetate or equivalent, and the detergent in the reaction mixture with the sample, results in the separation of the nucleic acid and the contaminant, the separation of the nucleic acid and the inhibitor, the separation of a protein present in the sample and the nucleic acid, the separation of a protein present in the sample and the inhibitor, the separation of a protein present in the sample and the contaminant, or any combination of the above, present in the sample;

(b) isolating the nucleic acid and remaining contaminants and inhibitors from the reaction mixture in a supernatant; and

(c) contacting the nucleic acid supernantant with a flocculant resulting in the further removal of the contaminant or the inhibitor from the supernatant; and

(d) recovering the nucleic acid from the nucleic acid-comprising sample.

30. The method of claim 29, further comprising purifying or isolating the nucleic acid after step (d).

31. The method of claim 29, wherein the sample is an unprocessed, preserved, freshly isolated, crude or unrefined sample.

32. The method of claim 29, wherein the reaction mixture of step (a) is mixed or vortexed.

33. The method of claim 29, wherein the isolating in step (b) comprises centrifuging the reaction mixture and harvesting a nucleic acid-comprising supernatant.

34. The method of claim 29, further comprising after step (d), detecting or characterizing the nucleic acid.

35. A method for post-isolation purification and/or amplification of a nucleic acid extracted from an environmental or a biological sample, wherein the isolated nucleic acid does not yield a detectable amplification product in an amplification reaction, and optionally the amplification reaction is a polymerase chain reaction (PCR), comprising the steps of:

(a) providing a reaction mixture comprising the environmental or biological sample, a chaotropic agent, ammonium acetate or an equivalent, and a detergent,

(b) isolating the nucleic acid and remaining contaminants and inhibitors from the reaction mixture in a supernatant;

(c) contacting the nucleic acid supernantant with a flocculant resulting in the further removal of the contaminant or the inhibitor from the supernatant; and

(d) purifying and/or amplifying the nucleic acid.

36. The method of claim 35, wherein the sample is an unprocessed, preserved, freshly isolated, crude or unrefined sample.

37. The method of claim 35, wherein the reaction mixture of step (a) is mixed or vortexed.

38. The method of claim 35, wherein the isolating in step (b) comprises centrifuging the reaction mixture and harvesting a nucleic acid-comprising supernatant.