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

• US 20050282202A1
• Filed: 05/20/2005
• Published: 12/22/2005
• 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.
• Granted Patent: US 7,459,548 B2
• Time in Patent Office: Days
• US Class Current: 435/6.18
• 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 and compositions, e.g., kits, for removing contaminants from nucleic acids in a sample, e.g., environmental or biological samples such as soil, food, plant, animal, microorganism or water samples. The invention provides methods and compositions for isolating nucleic acids from environmental and biological samples in a scaleable process free of contaminating substances that inhibit PCR and other downstream applications. Exemplary sample types include soil, water, plant and food. The methods and compositions of the invention can be used for isolating and/or detecting nucleic acids from prokaryotic and eukaryotic organisms and for detecting multiple types of organisms in a sample. Thus, the methods and compositions of the invention are useful for detecting organisms pertaining to agriculture, forensics biology and/or combating bioterrorism.

Claims

1. A method for removing at least one contaminant or selectively removing at least one compound from a nucleic acid-comprising sample, wherein the contaminant or compound (i) inhibits amplification or hybridization of nucleic acids in the sample, or, (ii) inhibits an enzymatic reaction, the method comprising the steps of:

(A) (a) contacting the nucleic acid-comprising sample with at least one flocculant to form a flocculant precipitate; and

(b) separating the nucleic acid from the flocculant precipitate,

wherein optionally the method further comprises purifying or isolating the nucleic acid after step (b), and optionally the sample is an unprocessed, preserved, freshly isolated, crude or unrefined sample, or, the sample is broken up, denatured or disrupted before contacting with the flocculant;

or

(B) (a) contacting the nucleic acid-comprising sample with at least a first flocculant to form a first flocculant precipitate, wherein optionally the sample is an unprocessed, preserved, freshly isolated, crude or unrefined sample, or, the sample is broken up, denatured or disrupted before contacting with the flocculant;

(b) separating the nucleic acid from the first flocculant precipitate;

(c) contacting the nucleic acid with a second flocculant to form a flocculant precipitate; and

(d) separating the nucleic acid from the second flocculant precipitate,

wherein optionally the method further comprises purifying the nucleic acid after step (d);

or,

(C) (a) processing the sample to break up, denature or disrupt the sample before contacting it with a flocculent, wherein the processing treatment comprises mixing or contacting the sample with a solution comprising a chaotropic agent, a detergent a buffer, a homogenizing agent or a combination thereof;

(b) contacting the nucleic acid-comprising sample with at least a first flocculant to form a flocculant precipitate, wherein optionally the contacting comprises mixing or vortexing the flocculant and the sample;

(c) separating a nucleic acid-comprising solution from the first flocculant precipitate, wherein optionally the separating comprises centrifuging the flocculant and the sample and harvesting a nucleic acid-comprising supernatant;

(d) contacting the nucleic acid-comprising solution with a second flocculant to form a second flocculant precipitate; and

(e) separating the nucleic acid from the second flocculant precipitate, wherein optionally the separating comprises centrifuging the flocculant and the sample and harvesting a nucleic acid-comprising supernatant;

wherein optionally the method further comprises purifying the nucleic acid after step (e),

and optionally the method further comprises detecting or characterizing a purified, isolated, amplified or hybridized nucleic acid.

10. The method of claim 1, wherein the flocculant comprises

(a) a cationic chemical substance, an anionic chemical substance, a zwitterionic chemical substance, a non-charged chemical substance or a combination thereof, and optionally the cationic, anionic, zwitterionic or non-charged substance comprises a quaternary ammonium or tertiary amine containing polymer, or,

(b) ammonium acetate, magnesium chloride (MgCl₂), ferric chloride (FeCl₃), a salt of iron, a salt of aluminum, calcium chloride (CaCl₂), a polyacrylamide, aluminum ammonium sulfate, derivatives thereof, or a combination thereof.

20. The method of claim 1,

(a) wherein the sample comprises an environmental or a biological sample, and optionally the 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,

wherein optionally the sample is processed by mechanical filtering, sedimentation or centrifugation;

or

(b) wherein the nucleic acid comprises an RNA or a DNA or a combination thereof;

or,

(c) wherein the step of extracting a nucleic acid from the sample comprises a step of homogenizing a processed, an unprocessed, freshly isolated, preserved, crude or unrefined sample;

(d) wherein the sample is homogenized by contacting the sample with a mechanical force, shear force, sound vibration, mechanical vibration, a vortex or a Vortex Adapter, wherein optionally the mechanical or shear force comprises use of a glass, a ceramic, a metal, a mineral or a plastic material or a combination thereof, and optionally the material is in the form of a bead;

or,

(e) further comprising adding a homogenizing material to the sample for the homogenizing step, wherein optionally the homogenizing material comprises a glass, a ceramic, a metal, a mineral, a plastic or a combination thereof;

or,

(f) wherein the nucleic acid is extracted from the sample by a step comprising contacting the sample with a liquid or a composition comprising a detergent or a surfactant or a combination thereof, 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, 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;

or,

(g) the method further comprising identifying an organism or nucleic acid component in the sample, wherein optionally the organism is identified by identifying or characterizing the purified, isolated, amplified or hybridized nucleic acid, and optionally the detected organism or nucleic acid component is derived from a microorganism, an animal, a plant, an insect, a yeast, a virus, a phage, a nematode, a bacteria or a fungi, and optionally the bacteria detected comprises a gram positive or a gram negative bacteria;

or,

(h) wherein the nucleic acid is contacted with the flocculant after separating a substantial amount of the detergent from the nucleic acid;

or,

(i) wherein the flocculant does not substantially precipitate the nucleic acid, or, the flocculant precipitates some but not all of the nucleic acid, or, the flocculant substantially precipitates the nucleic acid, or, the flocculant precipitates one or more substances selected from the group consisting of a humic acid, a fulvic acid and humin;

or,

(i) wherein the flocculant is separated from the nucleic acid by contacting the flocculant and nucleic acid with a solid support under conditions in which the nucleic acid selectively binds to the solid support, and optionally the solid support comprises or consists of a glass, an agarose, a plastic, a silica, a polyacrylamide, a hydrogel or a gel;

or,

(k) the method further comprising amplifying the nucleic acid or a portion thereof after the step of separating the flocculant or the flocculant precipitate from the nucleic acid, wherein optionally the nucleic acid is amplified using a polymerase chain reaction (PCR) procedure, rolling circle replication, ligase-chain reaction or derivative methods thereof, and optionally the nucleic acid separated from the flocculant or the flocculant precipitate is substantially free of a substance that inhibits a polymerase chain reaction procedure;

or

(l) wherein the nucleic acid comprises an RNA, and the RNA is reverse transcribed after the flocculant or the flocculant precipitate is separated from the nucleic acid;

or,

(m) wherein the nucleic acid is contacted with a restriction enzyme after the flocculant or the flocculant precipitate is separated from the nucleic acid;

or,

(n) wherein the nucleic acid is analyzed by mass spectrometry;

agarose, capillary or polyacrylamide electrophoresis;

hybridization;

an array;

a microarray;

an enzymatic reaction;

a fluorescent assay;

a radioactive assay;

a chromatographic assay;

or, a combination thereof, after the flocculant or the flocculant precipitate is separated from the nucleic acid;

or,

(o) wherein the nucleic acid is contacted with one or more oligonucleotides after the flocculant or the flocculant precipitate is separated from the nucleic acid, and optionally one or more of the oligonucleotides hybridizes to the nucleic acid;

or,

(p) wherein the nucleic acid is immobilized to a solid surface or is hybridized to a nucleic acid immobilized on a solid surface after the flocculant or the flocculant precipitate is separated from the nucleic acid.

2-4. -4. (canceled)

5. A method for amplifying, hybridizing, isolating or purifying a nucleic acid from a nucleic acid-comprising sample, the method comprising the steps of:

(A) (a) processing the sample to break up, denature or disrupt the sample before contacting it with a flocculant, wherein the processing treatment comprises mixing or contacting the sample with a solution comprising a chaotropic agent, a detergent, a buffer, a homogenizing agent or a combination thereof;

(b) contacting the nucleic acid-comprising sample with at least a first flocculant to form a flocculant precipitate, wherein the contacting comprises mixing or vortexing the flocculant and the sample,

wherein optionally the first flocculant comprises an ammonium acetate;

(c) separating a nucleic acid-comprising solution from the first flocculant precipitate, wherein the separating comprises centrifuging the flocculant and the sample and harvesting a nucleic acid-comprising supernatant;

(d) contacting the nucleic acid-comprising solution with a second flocculant to form a second flocculant precipitate, wherein optionally the second flocculant comprises an aluminum sulfate dodecahydrate;

(e) separating the nucleic acid from the second flocculant precipitate, wherein the separating comprises centrifuging the flocculant and the sample and harvesting a nucleic acid-comprising supernatant; and

(f) amplifying, hybridizing, isolating or purifying the nucleic acid after step (e);

or,

(B) (a) releasing a nucleic acid into the sample medium;

(b) contacting the sample medium with at least one flocculant after the nucleic acid is released from the sample; and

(c) separating the nucleic acid from the flocculent, wherein optionally the method further comprises purifying, hybridizing isolating or amplifying the nucleic acid after step (c);

or,

(C) (a) extracting a nucleic acid from the sample;

(b) contacting the nucleic acid with at least one flocculant after the nucleic acid is extracted from the sample; and

(c) separating the nucleic acid from the flocculant, wherein optionally the method further comprises purifying the nucleic acid after step (c);

or,

(D) (a) extracting a nucleic acid from the sample comprising a step of adding a first flocculant to;

(i) an unprocessed. preserved, freshly isolated, crude or unrefined sample, or (ii) a processed sample, wherein the processing comprises breaking up, denaturing or disrupting the sample before contacting it with the first flocculant, wherein optionally the processing treatment comprises mixing or contacting the sample with a solution comprising a chaotropic agent, a detergent, a buffer, a homogenizing agent or a combination thereof, such that a flocculant precipitate and a nucleic acid-comprising supernatant is formed;

(b) removing the flocculant precipitate from the nucleic acid-comprising supernatant, wherein optionally the separating comprises centrifuging the sample to form a precipitate-free nucleic acid-comprising supernatant;

(c) contacting the nucleic acid with a second flocculant to form a second flocculant precipitate; and

(d) separating the nucleic acid from the second flocculant and flocculant precipitate, wherein optionally the separating comprises centrifuging the sample to form a precipitate-free nucleic acid-comprising supernatant, and

(e) purifying, isolating, amplifying or hybridizing the nucleic acid after step (d);

or,

(E) (a) extracting a nucleic acid from the sample comprising a step of adding a first flocculant to;

(i) an unprocessed, preserved, freshly isolated, crude or unrefined sample, or (ii) a processed sample, wherein the processing comprises breaking up, denaturing or disrupting the sample before contacting it with the first flocculant, and the processing treatment comprises mixing or contacting the sample with a solution comprising a chaotropic agent, a detergent, a buffer, a homogenizing agent or a combination thereof, such that a flocculant precipitate and a nucleic acid-comprising supernatant is formed, wherein optionally the first flocculant comprises ammonium acetate;

(b) removing the flocculant precipitate from the nucleic acid-comprising supernatant, wherein the separating comprises centrifuging the sample to form a precipitate-free nucleic acid-comprising supernatant;

(c) contacting the nucleic acid with a second flocculant to form a second flocculant precipitate, wherein optionally the second flocculant comprises aluminum ammonium sulfate dodecahydrate;

(d) separating the nucleic acid from the second flocculant and flocculant precipitate, wherein the separating comprises centrifuging the sample to form a precipitate-free nucleic acid-comprising supernatant, and

(e) purifying, isolating, amplifying or hybridizing the nucleic acid after step (d);

and optionally the method further comprises detecting or characterizing a purified, isolated, amplified or hybridized nucleic acid.

6-9. -9. (canceled)

11-19. -19. (canceled)

21-43. -43. (canceled)

44. A method for post-isolation purification of a nucleic acid isolated by an existing method from an environmental or a biological sample that did not yield a detectable amplification product in a polymerase chain reaction (PCR) process, comprising

(a) contacting the isolated nucleic acid with flocculant;

and optionally contacting the isolated nucleic acid with a second flocculant; and

(b) separating the nucleic acid from the flocculant.

45. A method for post-isolation purification 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

(a) adding a sufficient amount of a first flocculant to the isolated sample to generate a flocculant precipitate and a nucleic acid-comprising supernatant;

(b) removing the flocculant precipitate from the nucleic acid-comprising supernatant; and

,

(c) purifying or amplifying the nucleic acid from the nucleic acid-comprising supernatant.

46. The method of claim 45,

(a) further comprising the steps of contacting the nucleic acid-comprising supernatant generated in step (b) with a second flocculant to generate a second flocculant precipitate and second nucleic acid-comprising supernatant, and the nucleic acid is purified or amplified from the second nucleic acid-comprising supernatant;

or

(b) further comprising contacting the isolated sample or nucleic acid, or the first or second flocculant precipitate, with a detergent;

or,

(c) wherein a substantial amount of the detergent is separated from the nucleic acid before the nucleic acid is contacted with the flocculant, or before the nucleic acid is purified or amplified.

47-50. -50. (canceled)

51. A kit for isolating a nucleic acid from a sample comprising at least one flocculant and instructions describing a method for use

wherein optionally the flocculant comprises an anionic, cationic, zwitterionic or uncharged chemical substance or combination thereof, wherein optionally the cationic substance comprises a quaternary ammonium or tertiary amine containing polymer, or optionally the flocculant is selected from the group consisting of ammonium acetate, magnesium chloride (MgCl₂), ferric chloride (FeCl₃), an iron salt or an aluminum salt, calcium chloride (CaCl₂), a polyacrylamide, aluminum ammonium sulfate and derivatives thereof,

and optionally the kit further comprises 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;

and optionally the kit further comprises a homogenizing material, wherein optionally the homogenizing material comprises a bead;

and optionally the kit further comprises one or more solutions or buffers for performing an instructed method, and optionally the instructions describing a method for obtaining a sample for processing;

and optionally the kit further comprises one or more tube vessels useful for performing the method of use;

and optionally the kit further comprises one or more oligonucleotides, and optionally free nucleotides, and optionally sufficient free nucleotides to carry out a PCR reaction, a rolling circle replication, a ligase-chain reaction, a reverse transcription or derivative methods thereof;

and optionally the kit further comprises at least one enzyme, wherein optionally the enzyme is a polymerase;

and optionally the kit further comprises one or more oligonucleotides, free nucleotides and at least one polymerase or enzyme capable of amplifying a nucleic acid in a PCR reaction, a rolling circle replication, a ligase-chain reaction, a reverse transcription or derivative methods thereof, wherein optionally the one or more oligonucleotides specifically hybridizes to a nucleic acid from a microorganism, an animal, a plant, an insect, a yeast, a virus, a phage, a nematode, a bacteria or a fungi or a bacterial or fungal toxin, or specifically hybridizes to a nucleic acid from 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 a Gram negative bacteria from a family comprising Acetobacteriaceae, Alcaligenaceae, Bacteroidaceae, Chromatiaceae, Enterobacteriaceae, Legionellaceae, Neisseriaceae, Nitrobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Rickettsiaceae or Spirochaetaceae, or from B. anthracis, A. globiformis. B. subtilis, C. renale, C. difficile, M. luteus, or R. ervthropolis, or from variola, varicella, reovirus, retroviruses, HIV, HIV-1, viral hemorrhagic fevers, Ebola, Marburg, Machupo, Lassa, Variola major, viral encephalitis, any of the pathogens listed in Table 1;

and optionally the kit is used to detect organisms that produce the spore or toxin, wherein optionally the toxin is a bacterial toxin;

and optionally the kit is used to detect organisms that produce a biohazard agent, wherein optionally the biohazard agent is a bacterial toxin

52-69. -69. (canceled)