+

WO1991015599A1 - Procede de detection de l'adn - Google Patents

Procede de detection de l'adn Download PDF

Info

Publication number
WO1991015599A1
WO1991015599A1 PCT/AU1991/000131 AU9100131W WO9115599A1 WO 1991015599 A1 WO1991015599 A1 WO 1991015599A1 AU 9100131 W AU9100131 W AU 9100131W WO 9115599 A1 WO9115599 A1 WO 9115599A1
Authority
WO
WIPO (PCT)
Prior art keywords
dna
antibody
target
target dna
binding protein
Prior art date
Application number
PCT/AU1991/000131
Other languages
English (en)
Inventor
David Kemp
Andrew Lew
Simon Carroll
Nicholas Martin Gough
Janice Kelly
Original Assignee
Amrad Corporation Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Amrad Corporation Limited filed Critical Amrad Corporation Limited
Publication of WO1991015599A1 publication Critical patent/WO1991015599A1/fr

Links

Classifications

    • 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
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates generally to a rapid method for the detection of target DNA in a sample comprising capturing a first DNA containing a target DNA followed by amplifying target DNA by the polymerase chain reaction and then detecting the amplified target DNA.
  • PCR polymerase chain reaction
  • in vitro has had widespread applications including the detection of pathogens, the diagnosis of genetic disorders, forensic analysis and many genetic manipulations for research purposes 1-2 .
  • Various sources of DNA have been used for PCR.
  • Phage plaques or bacterial colonies carrying plasmid DNA have multiple copies of the DNA of interest and, hence, PCR can be performed directly without the need for DNA purification 3 .
  • Some purification step is, however, generally required for PCR amplification of single copy genes of genomic DNA, especially if there is a mixed population of cells 4 .
  • one aspect of the present invention contemplates a method for detecting target DNA in a sample which method comprises capturing a first DNA containing said target DNA, amplifying said target DNA DNA using a PCR and then detecting said amplified target DNA.
  • the first DNA is captured using an antibody to an antigen associated with said first DNA such as an anti-histone antibody.
  • the first DNA is captured using a DNA binding protein such as GCN4 or GST-GCN4.
  • target DNA refers to that region of said first DNA which is subject to amplification by the PCR and is determined by the appropriate primers used.
  • the target DNA and first DNA may also be co-terminus, i.e. be the same molecule.
  • Target DNA may comprise an entire gene or a portion thereof or may represent a particular region in, for example, prokaryotic, eukaryotic or proviral DNA.
  • the immuno-PCR aspect of the subject invention is exemplified using anti-histone antibodies
  • its ambit extends to antibodies to any antigenic material, such as protein, that is tightly complexed to the first DNA.
  • the present invention extends to the use of antibodies directed to regulatory proteins capable of binding to specific (regulatory) regions of a first DNA.
  • the protein may bind to a first DNA in a particular organism from a mixture of organisms wherein the particular organism carries the specific regulatory protein associated with its genome.
  • the present invention extends to the enrichment of DNA from specific cells (eg. cancer cells and pathogens and non- pathogens such as viruses, bacteria, parasites, mycoplasma, fungi and yeast) via antibodies to one or more antigenic molecules, eg. proteins, exclusive to said specific cells.
  • specific cells eg. cancer cells and pathogens and non- pathogens such as viruses, bacteria, parasites, mycoplasma, fungi and yeast
  • Target DNAs contemplated by the present invention include chromosomal and genomic DNA, proviral and other pathogenic DNA whether or not associated with chromosomal or genomic DNA of the host, oncogenic and normal and abnormal eukaryotic genes or DNA (such as those involved in cancers and genetic disorders).
  • the target DNA contained in said first DNA is isolated from a sample.
  • sample is meant to include a source of chromosomal or genomic DNA.
  • the sample would include a source of histone-complexed or associated DNA or, as referred to above, antigenic material associated DNA, such as would be obtainable from eukaryotic cells and/or their extracts.
  • a sample would include biological fluids, extracts, suspensions or samples, such as blood, lymph, respiratory fluid or extract, excreta, tissue, tissue-extracts and the like in animals, for example, mammals, humans, avian and reptilian species.
  • the source may also be non-biological such as environmental and/or industrial effluent or solid waste.
  • fluid includes particular suspension as well as solution.
  • the antibodies used in the immuno-PCR contemplated herein may be polyclonal or monoclonal and naturally occurring or synthetic (eg. recombinant) and include parts of said antibodies.
  • the source of such antibodies may be from mice, rat, goat, horse, rabbit, human or other animal; the choice dependent upon cost, ease of manipulation and quantity of antibody required. Techniques for generating polyclonal and monoclonal antibodies to histones are well known in the art.
  • the target DNA is detected in a sample by capturing a first DNA containing said target DNA by contacting said first DNA with a binding effective amount of an antibody specific to an antigen associated with said first DNA for a time and under conditions sufficient for an antibody-first DNA complex to form, subjecting the captured first DNA to PCR to amplify said target DNA and then detecting said target DNA.
  • the antibodies are first immobilised to a solid surface.
  • the solid surface could be glass or a polymer, for example cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • the solid supports may be in the form of tubes, beads, discs or microplates or any other surface for conducting immuno-PCR.
  • Binding processes for immobilising antibodies to solid surfaces are well known in the art and include cross-linking, covalent binding or physically adsorbing the molecule to the insoluble carrier.
  • the antibodies may also be immobilised by binding to a second set of antibodies specific to the first antibodies, said second set bound to the solid support.
  • the capturing of the first DNA may occur after the formation of an antibody - first DNA complex by, for example, using antibodies specific to the first antibodies referred to above.
  • a first DNA containing a target DNA is contacted with a binding effective amount of a first antibody specific to an antigen associated with said first DNA for a time and under conditions sufficient for a first antibody-first DNA complex to form, contacting said first antibody-first DNA complex to a antibody- binding effective amount of a second antibody, said second antibody immobilised to a solid support, amplifying the target DNA by PCR and then detecting said amplified target DNA.
  • the second antibody may be monoclonal and/or polyclonal or parts or combinations thereof.
  • the antibody specific to the antigen associated with the first DNA is an anti-histone antibody and the antigen is histone.
  • variants extend to the use of DNA binding proteins to capture a first DNA containing target DNA in place of anti-histone antibodies.
  • Suitable DNA binding proteins include the double-stranded DNA binding specific protein - GCN4.
  • the present invention therefore extends to capturing and detecting target DNA in a sample using DNA binding proteins, amplifying said target DNA in said captured DNA using PCR and then detecting amplified target DNA.
  • Other variations embodied within the scope of the present invention relate to the use of alternate cell capture methods.
  • the DNA can be prepared and submitted for amplification without precipitation and centrifugation.
  • antibodies specific to the cells most likely to contain the target DNA.
  • cells expressing the determinants CD4 and CD8 could be captured by coating tubes with specific antibodies to these markers.
  • a first antibody specific to a cell surface antigen of a cell or organism containing the target DNA is used to capture said cell or organism. Depending on the sample, unwanted or unbound cells or organisms may then be washed away. The cell is then lysed or otherwise disrupted to release a first DNA (e.g. genomic DNA) which contains a target DNA. The target DNA in the first DNA is then amplified using PCR and the amplified product detected.
  • This method can be used to identify specific cell or organism types in a sample using antibodies to a cell surface antigen followed by amplification and detection of target DNA within the genomic DNA and/or as a rapid and convenient source of target DNA in a particular cell type in a sample.
  • the present invention extends to a method for the selective enrichment and/or isolation of target DNA and/or the identification of certain cell types in a sample which method comprises contacting the cells in said sample with a first antibody specific to an antigen on a desired cell type for a time and under conditions to capture said cells, disrupting said cells to release a first DNA containing a target DNA, amplifying the target DNA therein and then detecting the amplified target DNA.
  • This method may also contain the additional step after the release of the first DNA of capturing same using either an antibody specific to an antigen associated with said first DNA (e.g. an anti-histone antibody) or using a DNA binding protein (e.g. GCN4 or GST-GCN4).
  • the capture of the first DNA may be by either a second antibody specific to an antigen associated with said first DNA or by using a DNA binding protein as discussed above.
  • the first and second antibodies or the first antibody and the DNA binding protein are immobilised on a single solid support such as on the inside surface of a container adapted to receive and contain said sample.
  • one or both antibodies are monoclonal antibodies.
  • the first DNA will be the same as the target DNA or the target DNA will reside in the same strand as a first DNA strand or may be on a different strand but still associated, eg. by hydrogen bonds, to the first DNA strand.
  • the present invention further extends, therefore, to capturing and detecting target DNA directly or in addition to when the target DNA is associated in any form with said first DNA.
  • any number of means exist for detecting the amplified target DNA One convenient means is the use of the ELISA.
  • Other examples include, spectrophotometric and radioimmunoassay procedures or the amplified DNA assay (ADA) as disclosed in International Patent Application No. PCT/AU89/00526 and which is incorporated herein by reference. All such means are within the scope of the present invention.
  • the present invention is particularly applicable to the ADA.
  • An example of the applicability of these two procedures is shown in Example 6 herein. This example shows the ease by which the improved PCR can be used to capture target DNA from human blood cells and to incorporate the DNA binding sequences for TyrR or GCN4 into amplified DNA as described for ADA.
  • another aspect of the present invention contemplates a method for detecting target DNA in a sample which method comprises capturing on a first solid substrate a first DNA containing said DNA in said sample using an antibody specific to an antigen associated with said target DNA (such as an anti-histone antibody) or a DNA binding protein and then detecting said target DNA by the ADA comprising incorporating a first ligand into said DNA by polymerase chain reaction using a set of primers wherein one of the primers bears the ligand and contacting the so treated DNA with a second solid substrate having a binding reagent for said ligand immobilised thereon and subjecting said captured amplified DNA to a detecting means or alternatively contacting said captured amplified DNA with a detection reagent which is capable of binding to a second ligand previously incorporated into said amplified DNA by the polymerase chain reaction using a set of primers wherein one of the primers bears the ligand capable of binding to a detection reagent.
  • the present invention also extends to a kit for detecting target DNA sequences
  • kit comprises in compartmental form a first container adapted to contain an immobilised antibody specific to an antigen associated with said first DNA (e.g. an anti-histone antibody) or a DNA binding protein; a second container adapted to contain a sample containing a source of a first DNA; a third container adapted to contain the reagents for a PCR; and a fourth container adapted to contain a detection means.
  • the kit may be varied so that the contents of two or more containers may be combined.
  • the reagents for PCR and detecting means may be in one container.
  • the first and second containers may be combined to form a single container.
  • the first container may alternatively contain an immobilised antibody specific to the first antibody and the second container may contain the first antibody and be adapted to receive the sample.
  • the kit may also contain reagents for the ADA as described in PCT/AU89/00526. All such variations are within the scope of the subject invention.
  • the kit will comprise a container having a capturing means for a first DNA containing a target DNA.
  • the capturing means may be an antibody specific to an antigen associated with said first DNA such as an anti-histone antibody or may contain a DNA binding protein such as GCN4 or GST-GCN4.
  • the antibody or DNA bindingp rotein is immobilised to the surface inside the container.
  • This container is adapted to receive a sample containing a first DNA and is used, after suitable incubations and washings, to amplify and detect the target DNA contained in said first DNA.
  • One aspect of the present invention is exemplified by the detection of DNA from Plasmodium falciparu in whole blood.
  • Another aspect of the present invention is exemplified by screening for mutant or wild-type alleles of the cystic fibrosis (CF) gene using whole blood.
  • a third aspect of the invention is exemplified by the detection of the human testis specific form of the pyruvate dehydrogenase El subunit from whole blood or tissue culture.
  • chromosomal DNA can be selectively bound in the presence of a large amount of organic material (eg. blood) and then PCR performed in the same tube.
  • the antibody coated tubes can be stored for at least 1 week, in the cold in PBS containing 0.1% (w/v) azide without significant loss in activity.
  • a minimum of manipulations is required which is an advantage when handling hazardous material.
  • this immuno-PCR procedure would seem to be easily adapted for field use, as there are a low number of manipulations and no centrifugation steps are required.
  • the anti-malaria histone antibody is effective, when the starting material has a parasitaemia as low as 1%.
  • the antibody used to exemplify the present invention was obtained from mice which had received only 2 injections of P. falciparum histones. It is probable that a more avid antibody obtained from hyperimmunization might be more efficacious, or a monoclonal antibody against histone specific for a particular organism could be used.
  • there are ways of increasing the amount of functional antibody bound to the tube eg. by coating the tube with protein A or protein G which are polyvalent for the Fc of immunoglobulin (thus, not only might more antibody be bound but all the antibodies would be orientated in the appropriate way) or by using cross- linking agents, eg. carbodimide.
  • Example 6 The detection of the CF gene is exemplified in Example 6.
  • Figure 1 is a graphical representation showing the optimum concentration of immunoglobulin for coating polypropylene tubes.
  • Protein A purified fraction of human anti-histone serum at 0, 5, 10, 30, 90 ⁇ g/ml in PBS was added to polypropylene tubes, incubated for 2h at room temperature and washed 3X in PBS.
  • ELISA was performed using peroxidase conjugated sheep anti-human ig.
  • Figure 2 is a series of photographs of agarose gel showing the MgCl 2 concentration dependence of the amplification of the 316 base pair fragment of the PDH gene and the 193 base pair fragment of the CF gene. Arrows point to the amplified product.
  • a. is a 1% agarose gel showing MgCl 2 concentration dependence.
  • Lane 1 is Drigcst III markers;
  • Lane 2 shows MgCl 2 concentration of O.OmM; Lane 3 shows MgCl 2 concentration of 1.5mM; Lane 4 shows MgCl 2 concentration 2.25mM; Lane 5 shows MgCl 2 concentration of 3.0mM; Lane 6 shows MgCl 2 concentration of 3.75mM; Lane 7 shows MgCl 2 concentration of 4.5mM: Lane 8 shows MgCl 2 concentration of 5.25mM; Lane 9 shows MgCl 2 concentration of 6.0mM; Lane 10 shows MgCl 2 concentration of 7.5mM; Lane 11 shows positive control using Plasmid DNA.
  • b. is a 1% agarose gel showing MgCl 2 concentration dependence.
  • Lane 1 shows a BRL 1 Kb ladder;
  • Lane 2 show MgCl 2 concentration of 3mM;
  • Lane 3 shows MgCl 2 concentration of 3.75mM;
  • Lane 4 shows MgCl 2 concentration of 7.5mM;
  • Lane 5 shows MgCl 2 concentration of 10.5mM;
  • Lane 6 shows positive control using Plasmid DNA.
  • c is a 1.5% agarose gel showing MgCl 2 concentration dependence of the amplification of the 193 base pair fragment of the CF gene.
  • Lane 1 shows a BRL 1Kb Ladder; Lane 2 shows a
  • Lane 3 shows a MgCl 2 concentration of 5.0mM and human anti-histone antibody (HHAb) coating concentration of lO ⁇ g/ml
  • Lane 4 shows a MgCl 2 concentration of 2.5mM and murine anti- histone antibody (MHAb) coating concentration of 1:3000
  • Lane 5 shows MgCl 2 concentration of 5.0mM and MHAb coating concentration of 1:3000
  • Lane 6 shows MgCl 2 concentration of 2.5mM and
  • Lane 7 shows MgCl 2 concentration of 5.0mM and MHAb concentration of 1:25000; Lane 8 shows positive control using genomic DNA.
  • FIG. 3 is a photographic representation showing the specificity of immuno-PCR.
  • Polypropylene tubes were coated with human anti-histone (Ab+) or normal lg (Ab-).
  • Ab+ human anti-histone
  • Ab- normal lg
  • Blood infected with Plasmodium falciparum malaria (Ag+) or uninfected blood (Ag-) was added to the tube containing 0.5% (v/v) Triton X-100 in TE. Additional salt was (10X PBS+) or was not (10X PBS-) added.
  • PCR was performed and the products electrophoresed in 1% (w/v) agarose gel containing 0.1 ⁇ g/ml ethidium bromide for staining DNA. The far right lane shows the DNA size markers (EcoRI cut spp-1).
  • Figure 4 is a photographic representation showing the effect of varying the EDTA concentration to immuno-PCR. As for Fig. 2 except that 1, 10 and 100 mM EDTA was used instead of TE, and in one tube mouse anti-P. falciparum histone (M P. falciparum) was used instead of human anti- histone autoantibody.
  • Figure 5 is a photographic representation showing the sensitivity of immuno-PCR. Using tubes coated with a mouse anti-P. falciparum histone, blood containing 0.04, 0.2 and 1% parasitaemia was used as the source of DNA for immuno-PCR and the products treated as for Fig. 2.
  • Figure 6 is a photographic representation of agarose gels showing the effect of MgCl 2 concentration on the efficiency of amplification of the PDH or CF target sequence using blood and tissue culture cells as the source of DNA, using the DNA binding protein GCN4 as the chromatin capture source.
  • Lane 1 shows a Plasmid DNA (+ve control); Lane 2 shows an MgCl 2 concentration of 3mM; Lane 3 shows an MgCl 2 concentration of 3.75mM; Lane 4 shows a MgCl 2 concentration of 7.5mM; Lane 5 shows a MgCl 2 concentration of 10.5mM
  • Lane 1 shows a BRL 1 Kb ladder
  • Lane 4 shows a MgCl 2 concentration of 1.5mM and tube coating with GST- GCN4
  • Lane 7 shows Plasmid DNA (as a positive control)
  • Lane 1 shows a BRL 1 Kb ladder
  • Lane 2 shows a 2.5mM Mg 2+ 1.5 x 10 6 cells
  • Lane 3 shows a 2.5mM Mg 2+ 0.75 x 10 6 cells
  • Lane 7 Genomic DNA (as a positive control)
  • Figure 7 is a photographic representation showing amplified product of the PDH target gene.
  • Lane 1 shows Genomic DNA as a positive control
  • Lane 2 shows amplification from 1 x 10 4 cells
  • Lane 3 shows amplification from 1 x 10 5 cells
  • Lane 4 shows amplification from 1 x 10 6 cells
  • Lane 5 shows a BRL 1 Kb ladder
  • Figure 8 is a graphical representation showing the combined immuno-PCR/ADA on a sample of human blood to detect the CF gene. Conditions are as described in Example 5.
  • EXAMPLE 1 MATERIALS AND METHODS
  • mice CBA x BALB/c mice were given two intraperitoneal injections (one month apart) of 50 ⁇ g of histone emulsified in Freund's Incomplete Adjuvant. Serum was obtained 3 weeks after the last injection. Im unoglobulin was purified on protein A agarose beads.
  • Antibodies were raised in bcl-2 transgenic mice (Strasser et al, manuscript in preparation) by two intraperitoneal injections (3 weeks apart) of 50 ⁇ g human histones. The serum obtained two weeks after the last injection was shown to react against human histones with a titre of 1/25,000.
  • Human anti-histone antibodies were purified on protein A agarose from serum from a patient treated with hydralazine (such patients often develop autoantibodies to histones).
  • the serum was a gift from Dr S Whittingham, The Walter and Eliza Hall Institute, Melbourne, and was previously shown to react against calf and human histones Western blot analysis.
  • the 5' specific primer (PI) has the sequence 5'-TCGCGGTTTCTGTCACCTGT-3' and the 3' specific primer (P2) has the sequence
  • the 5' specific primer (CFI) has the sequence
  • 5'-GACTTCACTTCTAATGATGATTAT-3' and the 3' specific primer has the sequence
  • Oligonucleotides used as primers corresponded to the first and last 30 nucleotides (94-123 and 859-888) of the translated FC27 MSA-2 gene sequence described in Ref 7 .
  • Polypropylene microcentrifuge tubes were coated with 50 ⁇ l of antihistone antibodies using either human anti-histone antibodies (H «HAb) or normal human IgG at lO ⁇ g/ml in PBS or mouse anti-histone antibodies (M «HAb) at 1/3000 dilution in PBS. Coating was allowed to proceed for 2 hours at room temperature. After three washings in 1 x PBS, 50 ⁇ l of a nuclear lysis buffer (lOmM Tris-HCl/lOmM EDTA pH 8.0 + 0.5% Triton x- 100) was added to the tubes followed by 5 ⁇ l of human heparinized blood.
  • H «HAb human anti-histone antibodies
  • M «HAb mouse anti-histone antibodies
  • Tubes were mixed gently and after 5 minutes incubation at room temperature 2.5 ⁇ l 5MNaCl was added and the tubes left for 2 hours at room temperature. The contents of the tubes were then discarded and the tubes washed 3 times with PBS. PCR was performed directly in the tubes using the captured chromatin as the source of DNA.
  • Microtitre plates (Nunc-Immuno Module, Maxisorp U16) were coated with lOO ⁇ l per well of mouse anti- histone antibodies (antibody production previously described) at 1:1000 dilution in 0.1 M Glycine, 0.13M NaCl. The plates were incubated at 37"C for 1 hour and then blocked with a lOO ⁇ l per well of 0.2% sodium casein in PBS. The plates were incubated for 1 hour at 37 * C and then washed 2x with PBS containing 0.2% Tween 20.
  • Tissue culture cells (SupTl mammalian cell line) were pelleted and washed 2x in PBS. The cells were resuspended in nuclear lysis buffer (lOmM Tris-HCl, 10 mM EDTA pH 8.0 + 0.5% Triton x-100) and the equivalent of 5 x 10 3 , 1 xlO 4 , 1 x 10 5 and 1 x 10 6 cells were added to a coated well in 100 ⁇ l of buffer. After a 5 minute incubation 4.8 ⁇ l of 5 M NaCl was added with mixing. The plate was incubated for 1 hour at 37'C and then washed 6x in PBS containing 0.2% Tween 20.
  • nuclear lysis buffer lOmM Tris-HCl, 10 mM EDTA pH 8.0 + 0.5% Triton x-100
  • the captured chromosomal DNA was then treated with proteinase K by the addition of lOO ⁇ l per well of lysis buffer (50 mM KC1, 10 mM Tris - HC1 pH 8.4, 0.45% Nonidet P40, 0.45% Tween 20 containing 16 ⁇ g per ml proteinase K).
  • the plate was covered with a plate sealer and incubated in a 60 * C water bath for 1 hour.
  • the contents of each well were transfered to micro- centrifuges tubes. Tubes were incubated at 95 * C for 10 minutes to inactivate the proteinase K.
  • the volume of each tube was corrected to lOO ⁇ l with sterile distilled water and a 25 ⁇ l sample was taken for the PCR.
  • Purified GCN4 protein has been purified as a recombinant fusion protein with glutathione S- transferase (GST-GCN4).
  • Polypropylene microcentrifuge tubes were coated with 50 ⁇ l of GST-GCN4 at 5 ⁇ g/ml in PBS. After 2 hour at room temperature the tubes were washed 3x with PBS. The procedure for capture of chromosomal DNA from heparinized blood was as described for the immuno- PCR capture. PCR was performed directly in the tubes as previously described.
  • Tissue culture cells (0.75 x 10 6 and 1.5 x 10 6 cells) were pelleted and washed 2x in PBS. The cells were resuspended in 50 ⁇ l of a lysis buffer containing 50 mM KCl, 10 mM Tris-HCl (pH 8.4), 2.5 mM MgCl 2 , 0.45% Nonidet P40 and 0.45% Tween 20 and added to the coated tubes. After 5 minutes incubation, 6 ⁇ l of 5M NaCl was added with mixing.
  • Amplification was performed in a 50 ⁇ l reaction volume containing lOmM Tris-HCl pH 8.4, 50mM KCl, 1.5-7.5mM MgCl 2 , 200 ⁇ M of each deoxynucleotide triphosphate (dATP, dTTP, dCTP, dGTP), 0.2 ⁇ M of PI & P2 primers or 0.3 ⁇ M of CF1 and CF2 primers, and 2.5 units of Taq polymerase.
  • the PCR was overlayed with mineral oil.
  • Amplification was performed in a DNA Thermal Cycler using a predenaturation step at 95 * C for 5 minutes, after which the Taq polymerase was added followed by a step-cycle programme of 95"C for 1 minute, 55'C for 1 minute, 72'C for 1 minute for the PDH fragment, 95"C for 1 minute, 50'C for 1 minute and 70'C for 1 minute for the P. falciparum fragment and 95"C for 2 minutes, 40'C for 2 minutes, 65'C for 2 minutes for the CF fragment. All step cycles were performed for 35 cycles. Plasmid DNA or genomic DNA was used as a positive control in each PCR run.
  • a DNA fragment of the expected size (approximately 310 bp for the PDH gene and 190 bp for the CF gene) was observed in the samples from tubes coated with H HAb's and M HAb's but no visible band was obtained from tubes coated with normal immunoglobulin.
  • the efficiency of the amplification was dependent on magnesium chloride concentration.
  • the optimum magnesium concentration for amplification of the PDH gene fragment was 7.5mM and for the CF gene fragment it was 2.5 mM. (Figs 2a, 2b and 2c).
  • Fig 2a and 2b are 1% (w/v) agarose gels showing the MgCl 2 concentration dependence of the amplification of the 316 base pair PDH gene fragment. Arrows indicate amplified product.
  • Fig 2c is a 1.5% agarose gel showing the MgCl 2 concentration dependence of the amplification of the 193 base pair fragment CF gene. An arrow indicates amplified product.
  • Triton X-100 lyses the cell membrane. Divalent cations keep the chromatin insoluble and whole blood contains 2.4mM Ca 2+ and 1.8 mM Mg 2t . Because this would be diluted 1 in 10, ⁇ 1 mM EDTA was used in the buffer. EDTA also has the advantage of inhibiting deoxyribonucleases. Disruption of the nuclear envelope would also have to be achieved to allow the antibody access to the chromatin.
  • Fig 4 Also shown in Fig 4 is that either the human autoantibody, or the mouse anti-malaria histone, was effective in immuno-PCR. Subsequently, 0.5% (v/v) Triton X-100 in 100 mM EDTA was used in experiments.
  • the results described above used cultured P. falciparum with a parasitaemia ranging from 5 to 20%.
  • a P. falciparum culture and a parasitaemia of 20% was diluted in fresh human whole blood to a parasitaemia of 1%, 0.2% and 0.04%.
  • the parasites were mainly in the ring stage of development to mimic the clinical situation.
  • An aliquot of 5 ⁇ l of this was then added to the antibody-coated tube containing 0.5% (v/v) Triton X-100 and 100 mM EDTA and immuno-PCR was performed as above.
  • GCN4 11*12 GCN4 has been shown to bind a 9 base sequence. Protein dimers of GCN4 bind double-stranded DNA and not single-stranded DNA 13 ' 14 . Although GCN4 has a very high specific affinity for the target sequence of GGATGACTC, experiments described here show that GCN4 also possesses a non-specific affinity for double-stranded DNA sequences, sufficient to capture chromosomal DNA from heparinized blood and mammalian tissue culture cells. Two target sequences, the Human testis-specific form of pyruvate dehydrogenase El subunit and the wild-type allele of cystic fibrosis were amplified from captured DNA from blood. Captured DNA from tissue culture cells was used to amplify a target sequence from the cystic fibrosis gene.
  • Fig 6a is a 1.5% (w/v) agarose gel showing the effect of MgCl 2 concentration on the efficiency of amplification of the PDH target sequence using blood as the source of DNA.
  • a modified protocol for the immuno-PCR method involves the capture of chromosomal DNA from tissue culture cells by antihistone antibodies, followed by digestion of nuclear proteins with proteinase K. It can be argued that digestion of the nuclear proteins will breakdown the nucleosome structure and result in the decondensation of chromatin. This may make target DNA sequences more readily accessible to primers during the polymerase chain reaction.
  • the procedure was performed in a 96 well microtitre plate and required no centrifugation steps. The extracted DNA was added to a tube for amplification. The procedure is rapid and may be used to process many samples at one time.
  • Amplification was performed in a 50 ⁇ l reaction volume by mixing a 25 ⁇ l DNA sample with an equal volume of PCR buffer (10 mM Tris-HCl pH 8.4, 50 mM KCl) containing dNTPs, primers and MgCl 2 .
  • the PCR was performed as previously described with 2.5 units Taq polymerase being added after the 5 minute 95'C denaturation step.
  • lO ⁇ l of the PCR product was electrophoresed and DNA fragments identified.
  • a DNA fragment of the expected size was observed for the PDH target gene, using 1 x 10 4 , 1 x 10 5 and 1 x 10 6 cells.
  • Fig 3 shows the amplified product.
  • This example shows the applicability of immuno-PCR to ADA and the combined use of these techniques to identify a target DNA sequence, in this case the wild-type allele of the recently described cystic fibrosis (CF) gene 15 .
  • CF cystic fibrosis
  • the specimen used was whole blood.
  • Mouse anti-human leukocyte histones were purified on protein A agarose beads and used to coat polypropylene microcentrifuge tubes at lOug/ml (Example 1). After washing three times in PBS (3xPBS), 50ul of 0.5% (v/v) Triton X- 100/lOmM EDTA pH8 was added. Five ul of heparinized blood from a healthy adult human whose genotype with respect to the CF gene was unknown was added and mixed. After 5 min, 2.5ul of 5M NaCl was added. After 2 hours, the tubes were washed (3xPBS).
  • PCR was done in an Innovonics robot arm machine using all 3 oligonucleotides in the one tube: a) Biotinylated-CGAAATTAAGCACAGTA b) GGTGTGTAAATATATATTTACACAAAACACCAATGATAT which contains the binding site of tyrR and wild type CF sequence. c) GGATGACTCAAACACCAATGATAT which contains the binding site of GCN4 and mutant CF sequence.
  • oligonucleotides used in this example are different to those used in Example 2 only because they correspond to different regions of the genetic sequence.
  • the blood specimen give a significant signal only when the well of the ADA has TyrR thus binding the PCR product containing the wild type sequence. Only a background signal was seen in the well which has been coated with GST-GCN4 indicating that the oligo primer containing the GCN4 binding site had not been incorporated to any significant extent in the PCR reaction.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Hematology (AREA)
  • General Engineering & Computer Science (AREA)
  • Pathology (AREA)
  • Genetics & Genomics (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Cell Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention se rapporte à un procédé de détection d'un ADN cible dans un échantillon, comprenant le captage d'un premier ADN contenant ledit ADN cible à partir dudit échantillon, l'amplification dudit ADN cible en utilisant une réaction de polymérase en chaîne et la détection dudit ADN cible amplifié.
PCT/AU1991/000131 1990-04-05 1991-04-05 Procede de detection de l'adn WO1991015599A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPJ950690 1990-04-05
AUPJ9506 1990-04-05

Publications (1)

Publication Number Publication Date
WO1991015599A1 true WO1991015599A1 (fr) 1991-10-17

Family

ID=3774592

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU1991/000131 WO1991015599A1 (fr) 1990-04-05 1991-04-05 Procede de detection de l'adn

Country Status (1)

Country Link
WO (1) WO1991015599A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0574345A3 (fr) * 1992-06-12 1995-06-28 Inia Procédé de détection et d'identification des pathogènes virales et subvirales.
EP0629856A3 (fr) * 1993-06-12 1996-07-17 Boehringer Mannheim Gmbh Méthode pour la detection d'ADN métaboliquement marqué.
WO1997044450A1 (fr) * 1996-05-21 1997-11-27 Yale University Regulation de l'expression genetique
WO2008066868A3 (fr) * 2006-11-30 2008-11-06 Canon Us Life Sciences Inc Procédé d'isolement d'un adn cible à partir d'un adn mixte
US7932060B2 (en) 2003-04-18 2011-04-26 Becton, Dickinson And Company Immuno-amplification
WO2020214124A1 (fr) * 2019-04-15 2020-10-22 Istanbul Medipol Universitesi Utilisation d'anticorps anti-histone pour éliminer l'adn eucaryote pendant l'isolement d'adn bactérien et procédé d'immuno-précipitation de chromatine

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU5532386A (en) * 1985-03-28 1986-10-02 F. Hoffmann-La Roche Ag Process for amplifying, detecting, and/or cloning nucleic acid sequences
AU5532286A (en) * 1985-03-28 1986-10-02 F. Hoffmann-La Roche Ag Process for amplifying nucleic acid sequences
AU2735988A (en) * 1987-12-21 1989-07-13 Amoco Corporation Target and background capture methods with amplification for affinity assays
AU3360289A (en) * 1988-03-23 1989-10-16 Cemu Bioteknik Ab Method of sequencing dna
AU4663789A (en) * 1988-12-09 1990-06-26 Chemicon International, Inc. Amplified dna assay

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU5532386A (en) * 1985-03-28 1986-10-02 F. Hoffmann-La Roche Ag Process for amplifying, detecting, and/or cloning nucleic acid sequences
AU5532286A (en) * 1985-03-28 1986-10-02 F. Hoffmann-La Roche Ag Process for amplifying nucleic acid sequences
AU2735988A (en) * 1987-12-21 1989-07-13 Amoco Corporation Target and background capture methods with amplification for affinity assays
AU3360289A (en) * 1988-03-23 1989-10-16 Cemu Bioteknik Ab Method of sequencing dna
AU4663789A (en) * 1988-12-09 1990-06-26 Chemicon International, Inc. Amplified dna assay

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DERWENT ABSTRACT, Accession No. 91-03676, Class L (En) A1, D5, C1, KEMP, D.J. et al., Gene (1990), Volume 94(2), "Simplified Colorimetric Analysis of Polymerase Chain Reactions: Detection of HIV Sequences in AIDS Patients", see pages 223-228. *
KEMP, D.J. et al, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE USA, Volume 86, issued April 1989, "Colorimetric Detection of Specific DNA Segments Amplified by Polymerase Chain Reactions", see pages 2423-2427. *
PANACCIO, M. AND A. LEW, NUCLEIC ACIDS RESEARCH, Volume 19(5), issued 1991, (Oxford University Press), "PCR Based Diagnosis in the Presence of 8% (v/v) Blood", see page 1151. *
ZELDIS, J.B. et al., JOURNAL OF CLINICAL INVESTIGATIONS, Volume 84, issued November 1989, (The American Society for Clinical Investigations, Inc.), "Direct Method for Detecting Small Quantities of Hepatitis B Virus DNA in Serum and Plasma Using the Polymerase Chain Reaction", see pages 1503-1508. *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0574345A3 (fr) * 1992-06-12 1995-06-28 Inia Procédé de détection et d'identification des pathogènes virales et subvirales.
EP0629856A3 (fr) * 1993-06-12 1996-07-17 Boehringer Mannheim Gmbh Méthode pour la detection d'ADN métaboliquement marqué.
WO1997044450A1 (fr) * 1996-05-21 1997-11-27 Yale University Regulation de l'expression genetique
US6022863A (en) * 1996-05-21 2000-02-08 Yale University Regulation of gene expression
US7932060B2 (en) 2003-04-18 2011-04-26 Becton, Dickinson And Company Immuno-amplification
US8372605B2 (en) 2003-04-18 2013-02-12 Becton, Dickinson And Company Immuno-amplification
US9499858B2 (en) 2003-04-18 2016-11-22 Becton, Dickinson And Company Immuno-amplification
WO2008066868A3 (fr) * 2006-11-30 2008-11-06 Canon Us Life Sciences Inc Procédé d'isolement d'un adn cible à partir d'un adn mixte
WO2020214124A1 (fr) * 2019-04-15 2020-10-22 Istanbul Medipol Universitesi Utilisation d'anticorps anti-histone pour éliminer l'adn eucaryote pendant l'isolement d'adn bactérien et procédé d'immuno-précipitation de chromatine

Similar Documents

Publication Publication Date Title
US5534406A (en) Method of detecting antigenic nucleic acid-containing macromolecular entities
EP1330553B1 (fr) Trousse destinee a la detection d'un virus grippal de type a et de sous-type h5 non pathogene ou pathogene
EP0439222B1 (fr) Réactif insoluble, sonde d'acide nucléique, kit de test et procédés de diagnostic et purification
EP0726312B1 (fr) Procédés pour la capture et la libération sélective d'acides nucléiques
Vitale et al. Detection of Mycobacterium tuberculosis complex in cattle by PCR using milk, lymph node aspirates, and nasal swabs
JPH06209775A (ja) 温度感受性ポリメラーゼ阻害剤、それを含んでなる熱安定性ｄｎａポリメラーゼ組成物及び標的核酸の増幅方法
AU2002214936A1 (en) A kit for detecting non-pathogenic or pathogenic influenza a subtype H5 virus
JP2001269187A (ja) 遺伝病を検出するための熱安定リガーゼによるｄｎａ増幅系
JPS60262055A (ja) 核酸のハイブリツド形成分析法およびそれに用いる試薬系
JPH0768280B2 (ja) モノクローナル抗体
Nishimura et al. Direct polymerase chain reaction from whole blood without DNA isolation
US7341833B2 (en) Compositions and methods relating to nucleic acid reference standards
US6225053B1 (en) Detection of hepatitis B virus
Brandon et al. Early detection of bovine leukosis virus DNA in infected sheep using the polymerase chain reaction
WO1991015599A1 (fr) Procede de detection de l'adn
EP0635066A1 (fr) Determination quantitative d'un virus
ES2276362T3 (es) Metodos de aislamiento bacteriano de muestras biologicas.
JP4974528B2 (ja) 核酸検出方法およびその利用
US6280962B1 (en) Whole blood/mitogen assay for the early detection of a subject with cancer and kit
RU2163638C1 (ru) Способ обнаружения днк микобактерий туберкулезного комплекса с дифференциальным выявлением днк mycobacterium tuberculosis и набор реагентов для его осуществления
JP2004513651A (ja) インスリン依存糖尿病に対する患者の体質分析方法、装置とプライマの組
Uera et al. Detection of enzootic bovine leukosis in cattle using nested polymerase chain reaction assay
Koren et al. Antibody variable-region sequencing as a method for hybridoma cell-line authentication
EP0322922A2 (fr) Détection des infections du rétrovirus des oiseaux et des mammifères
WO2000004191A1 (fr) Procede et dispositif permettant de detecter la persistance d'une xenogreffe et la presence d'agents infectieux

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP NO US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU NL SE

NENP Non-entry into the national phase

Ref country code: CA

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载