WO1993003167A1 - Procede d'isolation de l'adn - Google Patents

Procede d'isolation de l'adn Download PDF

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Publication number
WO1993003167A1
WO1993003167A1 PCT/US1992/006477 US9206477W WO9303167A1 WO 1993003167 A1 WO1993003167 A1 WO 1993003167A1 US 9206477 W US9206477 W US 9206477W WO 9303167 A1 WO9303167 A1 WO 9303167A1
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dna
biological sample
lysis
kinetoplast
mixture
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PCT/US1992/006477
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English (en)
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David S. Sigman
Larry Simpson
Herbert Avila
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The World Health Organisation
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Priority to JP5503805A priority Critical patent/JPH07504801A/ja
Priority to BR9206341A priority patent/BR9206341A/pt
Publication of WO1993003167A1 publication Critical patent/WO1993003167A1/fr

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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage

Definitions

  • DNA-based diagnostic methods are coming into use for the diagnosis of infectious and parasitic diseases.
  • Many of these techniques involve DNA amplification procedures, such as the polymerase chain reaction procedure (PCR) , as described in United States Patents Nos. 4,683,195, 4,683,202, 4,800,159, and 4,965,188 to Mullis et al.
  • PCR polymerase chain reaction procedure
  • DNA-based amplification procedures require that DNA be isolated in a stable, biologically active form from blood or other biological fluids; optimal application of such amplification procedures further requires that the DNA to be amplified be present in the form of linear fragments of less than about 2 kb in length.
  • Disadvantages of this procedure include the necessity to lyse the erythrocytes first, the requirement for use of a detergent, and the expense of the cesium salt.
  • this system may inhibit enzymes such as exonucleases added to the lysate to cleave DNA.
  • the present invention provides a rapid and efficient method of DNA isolation, storage, and cleavage that meets these needs and provides DNA suitable for amplification by a sequence-specific method, such as PCR.
  • the method of the present invention for isolating biologically active DNA from a biological sample having DNA-containing structures comprises the steps of:
  • a biological sample containing DNA-containing structures with a lysis and storage buffer comprising a non-amphipathic chaotropic salt sufficient to lyse DNA-containing structures in the sample and a chelating agent to preserve the DNA from degradation to form a mixture of the biological sample and the lysis and storage buffer;
  • step (a) incubating the mixture formed in step (a) with a metal-containing chemical nuclease that cleaves the DNA to form DNA fragments;
  • the DNA in the DNA-containing structures can be present in the form of catenated closed circles. It can be isolated from a parasite. In particular, the DNA can be isolated from a kinetoplastid parasite such as Trypanosoma cruzi. Alternatively, the DNA can be associated with a virus such as herpes simplex virus (HSV) . RNA can also be isolated from RNA-containing structures, and DNA can be produced from the RNA using reverse transcriptase for amplification.
  • the biological sample can be a fluid sample such as mammalian blood, including human blood.
  • the non-amphipathic chaotropic salt is preferably a guanidinium salt, such as guanidinium chloride or guanidinium thiocyanate. More preferably, the guanidinium salt is guanidinium chloride. Most preferably, the guanidinium chloride is present in at least about 3 molar concentration in the mixture of the biological sample and lysis and storage buffer.
  • the chelating agent is preferably ethylenediamine- tetraacetic acid (EDTA) .
  • EDTA ethylenediamine- tetraacetic acid
  • the EDTA is present in at least about 0.1 molar concentration in the mixture of the biological sample and lysis and storage buffer.
  • the general method can further comprise the steps of:
  • the primers employed in the sequence-specific method can comprise at least two primers that hybridize to a genetic marker of the organism from which the biological sample was isolated for identification of the genetic marker.
  • the metal-containing chemical nuclease can be selected from 1,10-phenanthroline-copper complex, derivatives of ferrous EDTA, metalloporphyrins, or octahedral metal complexes of 4,7-diphenyl-l,10- phenanthroline.
  • the metal-containing chemical nuclease is the 1,10-phenanthroline-copper complex.
  • An application of the general method is a method for detecting a disease caused by a kinetoplastid parasite. This method comprises the steps of:
  • a biological sample from a patient suspected of having a disease caused by a kinetoplastid parasite with a lysis and storage buffer comprising a non-amphipathic chaotropic salt sufficient to lyse cells containing catenated closed circular kinetoplast DNA in the sample and a chelating agent to preserve the DNA in the cells from degradation to form a mixture of the biological sample and the lysis and storage buffer;
  • step (2) incubating the mixture obtained in step (1) with a metal-containing chemical nuclease to linearize catenated closed circular kinetoplast DNA to form kinetoplast DNA fragments; (3) purifying the DNA fragments to form purified kinetoplast DNA fragments suitable for amplification; (4) amplifying the purified kinetoplast DNA fragments by a sequence-specific method employing at least two primers capable of hybridizing to the linearized DNA to form amplified kinetoplast DNA; and (5) detecting disease by identifying the presence of the amplified kinetoplast DNA corresponding to the primers and thus having sequences specific for kinetoplast DNA of the parasite.
  • a preferred method according to the present invention for the detection of a disease caused by T. cruzi comprises:
  • step (1) incubating the mixture obtained in step (1) with the metal-containing chemical nuclease 1,10- phenanthroline-copper complex to cleave the kinetoplast DNA to form linearized kinetoplast DNA; (3) purifying the linearized kinetoplast DNA to form purified linearized kinetoplast DNA by:
  • the linearized DNA comprises fragments of from about 1 kb to about 1.5 kb in length.
  • a method of isolating and storing DNA from a cell-containing biological sample comprises the steps of: (1) contacting a biological sample containing DNA present in cells with a lysis and storage buffer in order to liberate the DNA from the cells, the lysis and storage buffer comprising: (a) a concentration of a non-amphipathic chaotropic salt sufficient to lyse cells in the biological sample when the lysis buffer is contacted with the biological sample; and
  • the storage temperature is below about 4°C in order to preserve the DNA sample for at least about one year.
  • Figure 1 shows the results of testing the stability of plasmid DNA as described in Example 1, infra.
  • Figure 2 shows the results of cleavage of isolated Trypanosoma cruzi kinetoplast DNA by the 1,10- phenanthroline-copper chemical nuclease (OP-Cu 2+ ) as described in Example 2, infra.
  • Figure 3 shows the frequency of single strand nicks in OP-Cu 2+ linearized minicircle DNA as described in Example 2, infra.
  • Figure 4 shows results of a sensitivity titration of OP-Cu 2+ -cleaved DNA, as described in Example 4, infra.
  • Figure 5 shows results from a blood sample of a chronic chagasic patient analyzed for the presence of T. cruzi minicircle sequences, as described in Example 5, infra.
  • Figure 6 shows the results of PCR amplification of T. cruzi DNA isolated from triatomid bug feces as described in Example 5, infra.
  • the present invention is a rapid and efficient method of storing, isolating, and purifying biologically active DNA from a biological sample having DNA-containing structures.
  • the DNA-containing structures include cells and viral particles.
  • the method is particularly useful for isolating parasitic DNA that is present in the form of catenated closed circles, but the method is not limited to use on such DNA.
  • the method allows for storage of the DNA in a state suitable for subsequent controlled degradation by a chemical nuclease in order to cleave the DNA to fragments of a size suitable for subsequent amplification.
  • the DNA can then be rapidly purified to render it suitable for use in an amplification method such as PCR.
  • the method comprises:
  • a biological sample containing DNA-containing structures with a lysis and storage buffer comprising a non-amphipathic chaotropic salt sufficient to lyse DNA-containing structures in the sample and a chelating agent to preserve the DNA from degradation and prevent coagulation of the blood to form a mixture of the biological sample and the lysis and storage buffer; (2) incubating the mixture formed in step (1) with a metal-containing chemical nuclease that cleaves the DNA to DNA fragments; and
  • the method is particularly adapted for, but is not limited to, detection of parasitic diseases caused by parasites having catenated closed circular kinetoplast DNA, such as Trypanosoma cruzi. the protozoan causing Chagas' disease; (Sturm et al., Mol. Biochem. Parasitol. 33:205 (1989); Avila et al., Mol. Biochem. Parasitol.
  • HIV human immunodeficiency virus
  • CMV cytomegalovirus
  • hepatitis B virus herpesvirus
  • Epstein-Barr virus Epstein-Barr virus
  • Toxoplasma qondii Toxoplasma qondii.
  • the method is suitable for use on any biological sample containing DNA.
  • the method is particularly adapted for storage and isolation of DNA from mammalian blood, including human blood, but the method is suitable for use on other biological samples, such as insect feces, biopsy tissue, urine, sputum, and lymphatic fluid.
  • the method can be used on any volume of fluid sample, from microliters to liters, as needed.
  • the biological sample is mixed with the lysis and storage buffer of the invention.
  • the lysis and storage buffer comprises: (1) a concentration of a non- amphipathic chaotropic salt sufficient to lyse cells in the sample and (2) a concentration of a chelating agent sufficient to preserve the DNA from degradation.
  • a non- amphipathic chaotropic salt is a salt, other than a detergent having distinct polar and non-polar moieties, that disrupts non-covalent bonds, such as hydrogen bonds, salt links, hydrophobic interactions, and van der Waals interactions, that are primarily responsible for the maintenance of secondary, tertiary, and quaternary structure in proteins and nucleic acids.
  • the non-amphipathic chaotropic salt is preferably guanidinium chloride or a chemically related salt such as guanidinium thiocyanate; other chaotropic salts such as lithium bromide, potassium thiocyanate, or potassium iodine are also usable.
  • the guanidinium chloride is present in at least 3 molar concentration in the mixture of the biological sample and the lysis and storage buffer.
  • the chelating agent is preferably EDTA, although other chelating agents, such as sodium citrate, can also be used. Most preferably, the EDTA is present in at least 0.1 molar concentration in the mixture of the biological sample and the lysis and storage buffer.
  • the biological sample is mixed with about an equal volume of the lysis and storage buffer containing about 6 molar guanidinium chloride and about 0.2 molar EDTA, pH 8.0.
  • the DNA-containing structures such as cells or viral particles
  • the DNA is stable in the mixture of the biological sample and lysis and storage buffer and can be stored at a storage temperature below about 65°C.
  • a storage temperature of 37°C DNA stored in a mixture of lysis and storage buffer and sample remains intact for at least a month.
  • the DNA is stable for at least one year.
  • the DNA can be stored at this stage for subsequent cleavage with the chemical nuclease and purification according to the remainder of the process of the present invention, or may be used in other techniques, such as nick translation to produce hybridization probes.
  • the biological sample is blood
  • blood is drawn into tubes containing anticoagulants such as citrate and/or heparin and then used after a period of storage of about 24 hours.
  • the DNA in the lysis and storage buffer is fragmented by incubation of the biological sample-lysis and storage buffer mixture with a metal-containing chemical nuclease capable of nicking DNA by oxidative attack on the deoxyribose moiety (Sigman & Chen, Annu. Rev. Biochem. 59:207 (1990)).
  • the chemical nuclease can be a 1,10-phenanthroline-copper complex (Sigman, Biochemistry 29:9097 (1990)); a derivative of ferrous EDTA such as methidiumpropyl-EDTA-iron (Hertzberg &
  • the chemical nuclease is a 1,10- phenanthroline-copper complex, because it is not sensitive to inhibition by buffer components.
  • the 1,10-phenanthroline-copper complex is inexpensive and its reaction can be efficiently quenched by chelating agents in order to control the extent of cleavage.
  • the phenanthroline-copper chemical nuclease reagent introduces random single strand nicks into duplex DNA in the presence of peroxide (Sigman & Chen, Annu. Rev. Biochem. 59:207 (1990)). Because, on the average, one double strand cleavage occurs after ten random single strand breaks are introduced into a DNA molecule, this nuclease can be used to digest catenated kinetoplast DNA to linearize minicircles.
  • the chemical nuclease reaction using the 1,10- phenanthroline-copper complex is typically performed as follows: to one volume of the mixture of the biological sample and lysis and storage buffer containing 3 molar guanidinium chloride and 0.1 molar EDTA are added 0.1 volume each of 1 molar MgCl 2 , 200 millimolar CuS0 4 , 20 illimolar 1,10-phenanthroline and 7.5% H 2 0 2 (freshly diluted from 30% stock solution) . The reaction is initiated by addition of 0.1 volume of 3- mercaptopropionic acid and digestion of DNA is allowed to proceed for 30 minutes at 37° C.
  • the reaction is stopped by addition of 0.1 volume of 1.5 molar 2,9-dimethyl-l,10- phena throline. Reactions with other chemical nucleases are carried out as specified in the literature describing them.
  • the cleavage process cleaves DNA to fragments that can be used in an amplification process. These fragments are linearized and are typically of about 1 to about 1.5 kb length.
  • the 1,10-phenanthroline-copper complex is believed to generate a highly reactive oxidative species and to react with the DNA through the formation of a reversible complex between the phenanthroline-copper reagent and the DNA.
  • the reaction produces the following stable products: 5'-phosphorylated-termini, 3'- phosphorylated-termini, free bases and 5- methylenefuranone, as well as minor amounts of 3'- phosphoglycolate termini.
  • the predominant reaction involves initial oxidative attack at the C-l hydrogen of the deoxyribose by the DNA-bound coordination complex.
  • Oxidative reaction is initiated within the minor groove of the DNA and the reagent exhibits preferential reactivity for DNA in the B form of the helix relative to DNA in the A form of the helix.
  • the reaction is not specific for the nucleotide at the site of scission, but its rate does depend on local sequence. The most important influence on the intensity of cutting by the phenanthroline-copper complex at any sequence position is the neighboring 5'-nucleotide.
  • the reaction can be terminated at any time by the addition of a chelating agent with high affinity for copper, such as 2,9- dimethyl-1,10-phenanthroline, 2,9-dimethyl-4,7- phenanthroline, or EDTA, compensations can be made for any variations in reactivity due to local sequence.
  • the chelating agent used to quench the reaction is 2,9-dimethyl-l,10-phenanthroline.
  • T. cruzi kinetoplast DNA which is originally present in the form of closed circular catenated minicircles, results in individual linear DNA molecules of about 1.4 kb in length.
  • the DNA is preferably purified to remove substances that can interfere with subsequent primer- based DNA amplification.
  • Purification typically includes: (1) deproteinization; (2) precipitation of the DNA; and (3) filtration.
  • the product of purification is suitable for amplification by PCR or another primer-based amplification step.
  • the deproteinization of the sample is typically performed by extraction with phenol or a 1:1 mixture of phenol and chloroform, which denatures proteins and separates proteins from nucleic acid.
  • the deproteinization is performed with extraction with a 1:1 mixture of phenol and chloroform.
  • Precipitation of the DNA is typically conducted using ethanol in the presence of a glycogen carrier and is preferably carried out in the presence of about 80 ⁇ g/ml glycogen and 0.3 molar sodium acetate at room temperature.
  • the final purification step can be performed by filtration through a microconcentrator.
  • a suitable microconcentrator is a Centricon-100 microconcentrator manufactured by Amicon (Beverly, Massachusetts) .
  • the isolated and purified DNA can be amplified in a system, as described below, or can be used for other purposes for which highly purified DNA fragments are used, such as the generation of hybridization probes or incorporation into cloning vectors.
  • the purified and fragmented DNA can be amplified by a sequence-specific method employing at least two oligonucleotide primers that hybridize to the DNA fragments.
  • a sequence-specific method employing at least two oligonucleotide primers that hybridize to the DNA fragments.
  • Such methods include, but are not limited to, the polymerase chain reaction (PCR method) of Mullis et al. as described in United States Patents 4,683,195, 4,683,202, 4,800,159, and 4,965,188 to Mullis et al. , all of which are herein incorporated in their entirety by this reference.
  • the PCR method is performed using a thermostable DNA polymerase such as the Thermus aquaticus polymerase Taq I described in U.S. Patent Number 4,889,818 to Gelfand et al., and incorporated herein by this reference. Further details on the PCR method are given in PCR Protocols (M.A. Innis et al. , e
  • sequence-specific nucleic acid amplification systems employing primers are also known, such as the transcription-based amplification system of Gingeras et al. described in European Patent Application No. 368906, and the similar system of Davey and Malek described in European Patent Application No. 329822, both incorporated herein by this reference. These systems make use of an alternating cycle of amplification as DNA and RNA employing a primer incorporating a promoter, a DNA-dependent RNA polymerase such as bacteriophage T7 RNA polymerase, and a RNA-dependent DNA polymerase or reverse transcriptase.
  • the product of the amplification is a discrete linear DNA fragment or series of discrete DNA fragments that can be separated by electrophoresis on an agarose gel and identified by hybridization methods such as "Southern blot” hybridization following transfer of the DNA to nitrocellulose filters or other filters.
  • hybridization methods such as "Southern blot” hybridization following transfer of the DNA to nitrocellulose filters or other filters.
  • Useful hybridization procedures are described in E.M. Sutter, "Detection of Specific Sequences Among DNA Fragments Separated by Gel Electrophoresis," J. Mol. Biol. 98: 503 (1975) , incorporated herein by this reference.
  • the hybridization is typically carried out with radioactively labeled DNA oligonucleotide probes; this serves to identify specific sequences originally present in the DNA used for amplification.
  • the hybridization procedure can be used to identify DNA sequences associated with the disease- causing agent in the original sample and thus the presence of the disease.
  • the DNA storage, cleavage, purification, and amplification method of the present invention is useful for the detection of parasitic diseases, particularly diseases caused by kinetoplastid trypanosomes.
  • Chagas' disease a major health problem in Latin America caused by Trvpanosoma cruzi.
  • Current serological and xenodiagnostic methods for the diagnosis of Chagas*' disease are unreliable, especially in the case of chronic Chagas' disease in which the parasites are detectable within the blood only with difficulty, if at all, because of the low concentration of parasites within the blood.
  • T. cruzi kinetoplasts which are DNA-containing structures in the mitochondria of the cells, contain DNA in the form of catenated minicircles, that, when cleaved by the chemical nuclease and purified according to the method of the present invention, can be efficiently amplified by the PCR process or another sequence-specific primer-based amplification process.
  • Oligonucleotide primers specific to conserved regions in T. cruzi kinetoplast minicircles (kDNA) are used (Degrave et al., Mol. Biochem. Parasitol. 27:63-70 (1988); Sturm et al. , Mol. Biochem. Parasitol. 33:205 (1989); Avila et al. , Mol. Biol. Parasitol. 42:175 (1990)).
  • These primers may be synthesized and are also commercially available from AMAC, Inc. (a Division of Genset, located in Westbrook, ME) .
  • DNA isolated and amplified as described herein may be used to detect disease associated with the presence of the DNA using standard methods, such as by DNA hybridization using radioactively labeled probes to bind to specific sequences in the DNA (Sturm et al., Mol. Biochem. Parasitol. 33:205 (1989) and Avila et al., Mol. Biochem. Parasitol. 42:175 (1990)).
  • the method of the present invention is not limited to storage, purification or detection of parasitic DNA or to minicircular DNA. Because the chemical nuclease that is employed typically cleaves DNA into linear fragments of from about 1 kb to about 1.5 kb in length, an optimal size for amplification with primers of less than 1000 bases as required for PCR, the lysis, storage, cleavage, and purification process of the present invention can be used to detect DNA specific for any biological entity of interest from any biological sample, including blood, urine, sputum, or lymphatic fluid.
  • the DNA detected can be from any etiological agent, including the DNA of parasitic protozoans, bacteria, or viruses such as herpes viruses, cytomegalovirus (CMV) , hepatitis B virus, herpes virus, Epstein-Barr virus, or Toxoplas a gondii.
  • CMV cytomegalovirus
  • hepatitis B virus herpes virus
  • Epstein-Barr virus Epstein-Barr virus
  • Toxoplas a gondii a gondii.
  • Cellular DNA can also be detected, including mammalian or human DNA, and retroviral DNA such as the DNA of human immunodeficiency virus (HIV) associated with AIDS.
  • HIV human immunodeficiency virus
  • genetic markers of the host species can be assayed in the same multiplex PCR assay in addition to genetic markers of parasites or other etiological agents of diseases.
  • the genetic markers assayed can include a family of immune response genes such as the major histocompatibility locus (MHC) genes which have been shown to be correlated with resistance to various diseases and susceptibility to autoimmune diseases.
  • MHC major histocompatibility locus
  • Other appropriate genetic loci for assay in multiplex PCR include the human homologues of those genetic markers found in animal models to confer resistance to parasitic diseases such as those caused by Leishmania or Trypanoso a cruzi. Any desired genetic locus, such as oncogenes and anti-oncogenes or genes involved in genetic diseases can be targeted for selective amplification during this assay, using the appropriate selection of primers and the isolated DNA.
  • RNA can also be isolated from RNA-containing structures such as cells or viruses and stored in a mixture of the sample and the lysis and storage buffer. The RNA can then be transcribed by reverse transcriptase to generate a RNA-DNA hybrid. The RNA in the hybrid is then degraded by the enzyme ribonuclease H specific for the RNA strand of a double-stranded RNA-DNA hybrid, and the resulting DNA strand made double-stranded by hybridization of a suitable primer and elongation of the primer by DNA polymerase.
  • An additional advantage of the method of the invention is that the use of the lysis and storage buffer and chemical nuclease of the invention can contribute to reduction of the infectivity of endogenous infectious organisms such as viruses or bacteria present in the biological sample, because the infectivity is substantially destroyed by lysis of the bacterial cells or virus particles and also by cleavage of the viral nucleic acid.
  • Treatment of membrane enveloped viruses with the l,10-phenanthroline-copper chemical nuclease destroys their infectivity (Lembech et al., Fed. Proc. 44:1072 (1985).
  • plasmid DNA pGEM 7Z (Promega, Madison, Wisconsin) was added to 2 tubes of GEB lysates. Each tube was stored at either 37°C or 65°C. Equivalent aliquots from each tube were taken at different time intervals for up to four weeks. The aliquots were extracted once with phenol/chloroform (1:1, v/v) and ethanol-precipitated. The isolated DNA was electrophoresed in a 1% agarose gel to determine the percentage of nicked or linearized plasmid DNA, which can be detected by its relatively rapid mobility through the gel in contrast to intact catenanes, which remain at the top of the gel.
  • FIG. 1 The results are shown in Figure 1; the electrophoretic pattern resulting from incubation at 37°C is designated (1A) and the pattern resulting from incubation at 65°C is designated (IB) .
  • the control lane is unincubated plasmid DNA in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA (TE) .
  • Figure 1 shows that the DNA remains intact at 37°C for at least a month, with no apparent nicking or degradation.
  • the DNA is nicked after a two-week incubation, as indicated by the disappearance of the closed circular DNA band and the increase in the nicked circular band. Even at 65°C, at least 50% of the DNA remained in the nicked circular or linear form after one week incubation.
  • DNA 0.05 ml of each of the following solutions was added: 1 M MgCl 2 , 200 mM CuS0 4 , 20 mM 1,10-phenanthroline, and 7.5% H 2 0 2 (diluted fresh from 30% stock) .
  • the reaction was initiated by addition of 0.05 ml of 58 mM 3- mercaptopropionic acid. Digestion of DNA was allowed to proceed for 30 minutes or 60 minutes at 37°C. The reaction was stopped by addition of 0.05 ml of 1.5 M 2,9- dimethy1-1,10-phenanthroline.
  • GEB lysates containing T. cruzi kinetoplast DNA digestion was carried out for 60 minutes at 37°C, and aliquots were removed every 10 minutes.
  • the reaction was quenched by addition of 2,9- dimethyl-1,10-phenanthroline. The aliquots were deproteinized with phenol/chloroform (1:1) and precipitated with ethanol.
  • the DNA was denatured in glyoxal/DMSO and loaded onto a 1% agarose gel and electrophoresed as described by McMaster and Carmichael fProc. Natl. Acad. Sci. U.S.A. 74:4835 (1977)). The gel was blotted and the DNA transferred to a Nytran filter (Schuell and Schuster, Keene, New Hampshire) . Total T ⁇ . cruzi kinetoplast DNA was nick-translated with 32 P-ATP and used as a hybridization probe. Nick-translation and hybridization conditions were as described in Simpson et al., Nucl. Acids Res. 13:9577 (1985)), incorporated by reference herein.
  • the DNA will not be an adequate substrate for PCR amplification.
  • the size of the single-stranded fragment would have to be at least equal to the distance between the two PCR primers in order to obtain successful amplification.
  • the DNA was cleaved with the phenanthroline-copper chemical nuclease in GEB lysate for increasing periods of time and the cleaved DNA was electrophoresed in a denaturing glyoxal gel to determine the size distribution of single-stranded fragments. The gel was blotted onto a nylon membrane and hybridized with 32 P-labeled T.
  • cruzi kinetoplast DNA kDNA
  • kDNA cruzi kinetoplast DNA
  • a few decatenated minicircles undergoing replication can be seen as a 1.4- kb band in the gel.
  • the minicircles were released from the kDNA networks as double stranded linearized molecules.
  • Figure 3 shows that with increasing incubation time there was a decrease in the size of the single-stranded fragments caused by nicking.
  • 90% of the minicircle fragments were larger than 310 bases.
  • 50% of the fragments were larger than 310 bases, and 80% of the fragments were larger than 118 bases.
  • An incubation time of 30 minutes was selected for routine phenanthroline-copper digestion of blood lysates, at which time approximately 90% of the single-stranded fragments were longer than 310 bases, and would be appropriate amplification target molecules for the three sets of PCR primers which yield products of 83 bp, 122 bp, and 330 bp respectively (Sturm et al., supra) .
  • a control experiment showed the kinetoplast DNA digested with the phenanthroline-copper reagent in GEB lysate under standard conditions for 30 minutes was a suitable template for PCR amplification.
  • the pellet was resuspended in 1 ml of water and transferred to a Centricon-100 microconcentrator (Amicon) containing 1 ml of water.
  • the microconcentrator unit was centrifuged at 1000 x g in a clinical centrifuge for 10 minutes.
  • the retentate was washed a second time with 2 ml of water. After the second 10 minute centrifugation 100 ⁇ l of concentrated retentate was collected as described by the manufacture of the microconcentrator.
  • the retentate was used for PCR amplification as described below.
  • 70 ⁇ l of the retentate material was amplified in a 100 ⁇ l PCR reaction.
  • the reaction conditions were as follows: 10 mM Tris-HCl, pH 8.3, 50 mM KC1, 5 mM MgCl 2 , 0.1 mg/ml BSA, 3 units of Taq DNA polymerase (Perkin- Elmer Cetus) and 100 picomoles of each primer.
  • the primers used were three sets of primers specific to the four conserved regions in T. cruzi minicircles yielding the 330 bp minicircle variable and conserved region DNA fragment and the 83 bp and 122 bp conserved region fragments respectively, as described by Sturm et al.,
  • the cycling profile was as follows: Denaturation, annealing, and elongation were done at 94°C, 60°C, and 72°C, respectively; each step was allowed to proceed for one minute for a total of 30 cycles. A 15 ⁇ l aliquot from each reaction was analyzed on agarose/Nusieve (FMC, Rockland, Maine) gels.
  • Figure 4A shows results from amplification of 83-bp fragments using 35 PCR cycles; the products were electrophoresed on a 2% agarose/3% Nusieve gel. The rapidly migrating band present in all lanes represents PCR primers or primer dimers.
  • the top panel shows the stained gel; the low molecular weight band present in all lanes represents PCR primers.
  • the bottom panel shows the hybridization of the blot with 32 P- labeled S34A oligonucleotide internal probe (Sturm et al., supra) .
  • Figure 4C shows results from amplification of 330 bp variable and conserved region fragments with 30 PCR cycles; the products were electrophoresed on a 1% agarose/3% Nusieve gel.
  • the top panel shows the stained gel.
  • the bottom panel shows results from hybridization of the blot with 32 P-labeled S67 oligonucleotide internal probe (Sturm et al. , supra; Avila et al., Mol. Biochem. Parasitol. 42:175 (1990)).
  • the CI and C2 lanes show 500 ⁇ l of undigested GEB lysate sample containing 10 kDNA networks per 30 ml processed for PCR amplification as described for all other samples.
  • a 10 ml sample of venous blood from the patient was obtained and stored as GEB lysate. Phenanthroline- copper cleavage of the lysate was performed and DNA was isolated from two 500 ⁇ l aliquots and PCR amplified as described in Examples 2 through 4, supra.
  • Figure 5 shows a specific amplification of T. cruzi minicircle sequences from patient blood with two different sets of PCR primers.
  • Figure 5A shows the amplification of an 83 bp fragment; the products are analyzed on a 1% agarose/3% Nusieve gel.
  • the negative control in lane 1 is a GEB sample from a non-chagasic donor.
  • the negative control in lane 2 is a sample lacking kDNA in the PCR reaction.
  • the M lane shows DNA Hae III fragments of 0X174 RF DNA as size markers.
  • Figure 5B shows the amplification of 330 bp variable and conserved region fragments, run on a 2% agarose gel, blotted and hybridized with 32 P-labeled S67 oligonucleotide internal probe (Sturm et al., supra: Avila et al., supra.)
  • a positive control is 100 fg gel- isolated OP-Cu 2+ -cleaved kDNA; the negative controls are the same as (A) .
  • the DNA storage, cleavage, purification, and amplification method was extended to an insect vector and to biopsy material from infected mice.
  • the abdominal contents of two T. dimidiata and two R. prolixus were collected and stored in GE buffer.
  • the samples were processed as described in Examples 1-4.
  • Figure 6 shows a specific amplification of kinetoplast DNA minicircle sequences from the insect abdominal contents. Minicircle variable region fragments of 330 bp were amplified.
  • the PCR reactions were of 100 ⁇ l total volume; 15 ⁇ l was loaded on a 2% agarose gel.
  • the M lane shows DNA Hae III fragments of 0X174 RF DNA as size markers.
  • Positive and negative controls are 1 pg kDNA and no kDNA, respectively, in the PCR reaction.
  • Animal biopsy material was also dissolved and stored in GE.
  • Heart tissue obtained from infected and uninfected mice was washed with saline solution and stored in GE buffer. The tissue was dissolved by incubation in GE at 37°C for two days with occasional vigorous mixing. The dissolved tissue was processed as described in Examples 1-4. Specific PCR amplification of minicircle sequences was observed from the heart tissue lysates of the infected mouse but not from uninfected controls. These results indicate that the method could be used for autopsy specimens to detect Chagas' disease.
  • the method of the present invention has a wide variety of potential applications in the diagnosis of bacterial, parasitic, and viral diseases. It is particularly useful for the collection of specimens from patients in the field, in clinics and under other conditions in which storage conditions may be less than optimal. It is particularly adapted to the diagnosis of parasitic diseases caused by kinetoplastid trypanosomes, such as . cruzi, the causative agent of Chagas' disease, because the catenated kinetoplast minicircle DNA characteristic of this organism is efficiently cleaved to linear fragments of a size suitable for amplification by a sequence-specific primer-based amplification technique such as PCR.
  • the method of the invention is simple to carry out, rapid, and highly effective in detecting small quantities of DNA specific to infectious agents. In addition, the infectivity of biological samples containing infectious agents is reduced by application of the method of the invention.

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Abstract

L'invention décrit un procédé rapide et efficace d'isolation, de préservation et de coupure de l'ADN pemettant d'obtenir un ADN capable d'amplification au moyen d'un procédé sequentiel spécifique. Le procédé d'isolation d'un ADN biologiquement actif à partir d'un spécimen biologique possédant des structures contenant l'ADN comprend: (1) la mise en contact d'un spécimen biologique possédant des structures contenant de l'ADN avec une lyse et un tampon de réserve contenant un sel chaotropique non amphipathique suffisant pour effectuer la lyse des structures contenant de l'ADN dudit spécimen, ainsi qu'un agent chélatant servant à préserver l'ADN de la dégradation, afin de constituer un mélange du spécimen biologique, de la lyse et du tampon de réserve; (2) l'incubation du mélange constitué dans l'étape 1) avec une nucléase chimique à teneur métallique coupant l'ADN en fragments d'ADN; (3) la purification desdits fragments d'ADN. L'ADN isolé peut être un ADN circulaire fermé caténarisé, tel qu'un ADN kinétoplaste de Trypanosoma cruzi. On peut utiliser les fragments d'ADN purifiés dans le but d'une amplification dans un système d'amplification d'ADN séquentiel utilisant des amorces s'hybridant à l'ADN, afin de déterminer la présence d'une séquence d'ADN spécifique dans les fragments. La combinaison des méthodes d'isolation et d'amplification peut être efficace pour la détection de maladies parasitaires, bactériennes et virales au moyen de l'identification des séquences d'ADN associées aux organismes qui les provoquent.
PCT/US1992/006477 1991-08-06 1992-08-04 Procede d'isolation de l'adn WO1993003167A1 (fr)

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EP0574227A2 (fr) * 1992-06-08 1993-12-15 Gen-Probe Incorporated Préparation d'acide nucléique de cellules mononucléaires
WO1994026899A1 (fr) * 1993-05-13 1994-11-24 Institut Français De Recherche Scientifique Pour Le Developpement En Cooperation (Orstom) Procede de culture in vitro de differents stades de parasites tissulaires
WO1997040839A1 (fr) * 1996-04-26 1997-11-06 Merck & Co., Inc. Formulations de vaccins a adn
EP0819766A1 (fr) * 1995-04-04 1998-01-21 Sumitomo Electric Industries, Limited Polynucleotides permettant de deceler des leishmanies et procede de detection du protozoaire de la leishmanie
EP0851032A1 (fr) * 1996-12-30 1998-07-01 Becton, Dickinson and Company Méthode pour réduire des inhibiteurs de l'hybridisation des acides nucléiques
WO1999029904A2 (fr) * 1997-12-10 1999-06-17 Sierra Diagnostics, Inc. Procedes et reactifs de conservation de l'adn de fluides corporels
US6210881B1 (en) 1996-12-30 2001-04-03 Becton, Dickinson And Company Method for reducing inhibitors of nucleic acid hybridization
EP1207208A2 (fr) * 2000-11-15 2002-05-22 Becton Dickinson and Company Méthode pour la préservation des cellules et des acides nucléiques cibles
US6914137B2 (en) 1997-12-06 2005-07-05 Dna Research Innovations Limited Isolation of nucleic acids
US7569342B2 (en) * 1997-12-10 2009-08-04 Sierra Molecular Corp. Removal of molecular assay interferences
US7927870B2 (en) 1996-04-26 2011-04-19 Merck Sharp & Dohme Corp. DNA vaccine formulations
US8691969B2 (en) 1994-12-12 2014-04-08 Life Technologies As Isolation of nucleic acid
CN106868121A (zh) * 2017-02-15 2017-06-20 中山大学 一种检测路氏锥虫的引物对和试剂盒
WO2018170186A1 (fr) 2017-03-15 2018-09-20 Ancestry.Com Dna, Llc Dispositif et procédé de prélèvement d'échantillon
US11655495B2 (en) 2017-01-16 2023-05-23 Spectrum Solutions L.L.C. Nucleic acid preservation solution and methods of manufacture and use

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WO2016136324A1 (fr) * 2015-02-24 2016-09-01 東洋紡株式会社 Procédé de stabilisation d'une sonde dans une solution de réaction de détection d'acides nucléiques

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EP0574227A3 (en) * 1992-06-08 1994-08-24 Gen Probe Inc Preparation of nucleic acid from mononuclear cells
US5639599A (en) * 1992-06-08 1997-06-17 Gen-Probe Incorporated Amplification of nucleic acids from mononuclear cells using iron complexing and other agents
EP0574227A2 (fr) * 1992-06-08 1993-12-15 Gen-Probe Incorporated Préparation d'acide nucléique de cellules mononucléaires
US6458581B1 (en) 1993-05-13 2002-10-01 Institut Francais De Recherche Scientifique Pour Le Developpement En Cooperation (Orstom) Process for the in vitro culture of different stages of tissue parasites
WO1994026899A1 (fr) * 1993-05-13 1994-11-24 Institut Français De Recherche Scientifique Pour Le Developpement En Cooperation (Orstom) Procede de culture in vitro de differents stades de parasites tissulaires
FR2705358A1 (fr) * 1993-05-13 1994-11-25 Orstom Procédé de culture in vitro de différents stades de parasites tissulaires, stades parasitaires obtenus et applications biologiques.
US7317094B2 (en) 1993-05-13 2008-01-08 Institut Francais De Recherche Scientifique Pour Le Developpement En Cooperation (Orstom) Process for the in vitro culture of different stages of tissular parasites
US7282210B2 (en) 1993-05-13 2007-10-16 Institut De Recherche Pour De Developpement (Ird) Process for the in vitro culture of different stages of tissular parasites
US8691969B2 (en) 1994-12-12 2014-04-08 Life Technologies As Isolation of nucleic acid
EP0819766A1 (fr) * 1995-04-04 1998-01-21 Sumitomo Electric Industries, Limited Polynucleotides permettant de deceler des leishmanies et procede de detection du protozoaire de la leishmanie
EP0819766A4 (fr) * 1995-04-04 2000-01-12 Sumitomo Electric Industries Polynucleotides permettant de deceler des leishmanies et procede de detection du protozoaire de la leishmanie
US7927870B2 (en) 1996-04-26 2011-04-19 Merck Sharp & Dohme Corp. DNA vaccine formulations
WO1997040839A1 (fr) * 1996-04-26 1997-11-06 Merck & Co., Inc. Formulations de vaccins a adn
AU727306B2 (en) * 1996-04-26 2000-12-07 Merck Sharp & Dohme Corp. DNA vaccine formulations
EP0851032A1 (fr) * 1996-12-30 1998-07-01 Becton, Dickinson and Company Méthode pour réduire des inhibiteurs de l'hybridisation des acides nucléiques
US6210881B1 (en) 1996-12-30 2001-04-03 Becton, Dickinson And Company Method for reducing inhibitors of nucleic acid hybridization
US6914137B2 (en) 1997-12-06 2005-07-05 Dna Research Innovations Limited Isolation of nucleic acids
US6458546B1 (en) * 1997-12-10 2002-10-01 Sierra Diagnostics, Inc. Methods and reagents for preservation of DNA in bodily fluids
WO1999029904A3 (fr) * 1997-12-10 1999-12-23 Sierra Diagnostics Inc Procedes et reactifs de conservation de l'adn de fluides corporels
WO1999029904A2 (fr) * 1997-12-10 1999-06-17 Sierra Diagnostics, Inc. Procedes et reactifs de conservation de l'adn de fluides corporels
US7569342B2 (en) * 1997-12-10 2009-08-04 Sierra Molecular Corp. Removal of molecular assay interferences
EP1207208A3 (fr) * 2000-11-15 2003-12-10 Becton Dickinson and Company Méthode pour la préservation des cellules et des acides nucléiques cibles
EP1207208A2 (fr) * 2000-11-15 2002-05-22 Becton Dickinson and Company Méthode pour la préservation des cellules et des acides nucléiques cibles
US11655495B2 (en) 2017-01-16 2023-05-23 Spectrum Solutions L.L.C. Nucleic acid preservation solution and methods of manufacture and use
CN106868121A (zh) * 2017-02-15 2017-06-20 中山大学 一种检测路氏锥虫的引物对和试剂盒
CN106868121B (zh) * 2017-02-15 2020-11-06 中山大学 一种检测路氏锥虫的引物对和试剂盒
WO2018170186A1 (fr) 2017-03-15 2018-09-20 Ancestry.Com Dna, Llc Dispositif et procédé de prélèvement d'échantillon
US11826027B2 (en) 2017-03-15 2023-11-28 Ancestry.Com Dna, Llc Sample collection device and method

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