WO2012097849A2 - Séquences d'adn restreintes par le génome mitochondrial - Google Patents

Séquences d'adn restreintes par le génome mitochondrial Download PDF

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WO2012097849A2
WO2012097849A2 PCT/EP2011/006461 EP2011006461W WO2012097849A2 WO 2012097849 A2 WO2012097849 A2 WO 2012097849A2 EP 2011006461 W EP2011006461 W EP 2011006461W WO 2012097849 A2 WO2012097849 A2 WO 2012097849A2
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nucleic acid
gene product
cells
cellular organelle
endosymbiont cellular
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PCT/EP2011/006461
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WO2012097849A3 (fr
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Jason Poole
Adrian Vilalta
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Merck Patent Gmbh
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    • 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
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the invention relates to the design and use of primers based on DNA sequences found exclusively in endosymblont cellular organelles such as mitochondria for the development of quantitative assays and for endosymbiont DNA purification schemes.
  • the invention provides an enabling approach for determining the functioning of a cellular organism by determining the relative ratio of a first endosymbiont cellular organelle nucleic acid and/or gene product thereof in a sample obtained from said organism in relation to the amount of a second nucleic acid and/or gene product thereof.
  • the invention provides mitochondrial DNA (mtDNA) sequences and primers that are unique and show no homology with nuclear DNA (nucDNA) for preventing misleading results in the diagnosis method.
  • a diagnostician of disease studying (mal)functioning of cellular organisms can employ a broad range of inroads into the organism to obtain relevant information as to the various aspects of the malfunctioning.
  • inroads vary widely and may for example include detecting relative ratios of kidney stones by studying urinary samples obtained from various patients, probing for the presence or absence of intestinal ulcers via endoscopy, scanning for detectable tumors by nuclear magnetic resonance ("N R"), detecting diabetes by testing for insulin levels and/or glucose concentration in blood plasma, determining cancer proneness by determining transcriptional levels of oncogenes, and so on.
  • the invention provides an enabling approach for determining the functioning of a cellular organism by determining the relative ratio of a first endosymbiont cellular organelle nucleic acid and/or gene product thereof in a sample obtained from said organism in relation to the amount of a second nucleic acid and/or gene product thereof.
  • the invention provides mitochondrial DNA (mtDNA) sequences that are unique and show no homology with nuclear DNA (nucDNA) for preventing misleading results in the diagnosis method.
  • relative ratio includes the amount of the first endosymbiont cellular organelle nucleic acid and/or gene product thereof in relation to the amount of the second nucleic acid and/or gene product thereof.
  • the relative ratio may, for instance, be determined by (among other) dividing the amount of the first nucleic acid or gene product thereof by the amount of the second nucleic acid or gene product thereof, or vice versa.
  • the amount of one or both nucleic acids may also be divided by, or subtracted from, a reference value.
  • the disease and/or (toxic) compound may affect the organism to such extent that clinical symptoms are present.
  • the disease or (toxic) compound may have an influence upon the organism while clinical symptoms are not (yet) manifested.
  • Endosymbiont cellular organelles include those organelles of a eukaryotic cell that are thought to have been derived from prokaryotic bacteria very early on in the evolution of eukaryotic cells. These bacteria (as it is thought) have engaged in a symbiosis with early eukaryotic cells, and at present, eukaryotic cells comprising these endosymbiont organelles in general cannot live without them.
  • the invention provides a method whereby said relative ratio of an endosymbiont cellular organelle nucleic acid and/or gene product thereof is determined in relation to the amount of nuclear nucleic acid detectable in said sample (be it DNA or RNA), or in relation to gene products (derivable by transcription and/or translation, such as mRNA or (poly)peptides) of said nuclear nucleic acid (nuclear nucleic acid herein comprises chromosomal DNA and the RNA transcribed therefrom), for example present in nuclear or cytoplasmic fractions or parts of said sample.
  • DNA or corresponding mRNA encoding components of small nuclear ribonucleoprotein (snRNP), or other essentially common nucleic acid derived from chromosomal DNA is particularly useful to test, because of its ubiquitous presence.
  • the invention provides a method for studying for example endosymbiont cellular organelle related disease, like mitochondrial and/or proplastid related disease.
  • endosymbiont cellular organelle related disease is meant herein a condition wherein the amount and/or at least one property of nucleic acid of said endosymbiont cellular organelle, and/or gene product thereof, is altered as compared to the natural situation. For instance, expression of said nucleic acid may be reduced.
  • Endosymbiont cellular organelle related disease e.g. encoded by defects in said organelle's DNA, manifests in many different syndromes and is often variable in its expression (and thus in general hard to detect by testing for clinical parameters alone) due to heteroplasmy, whereby mutant and wild type nucleic acid can be found in one cell, whereby its distribution can vary.
  • Endosymbiont cellular organelle related disease is often aggravated with increasing age of the affected individual.
  • Endosymbiont cellular organelle related disease can also often be observed after treatment against other disease with various drugs, and then contributes to various side-effects of those drugs that one would like to avoid during treatment. Those side effects can now be better studied by using a method as provided herein.
  • step (iii) Determining the relative ratio of the amplification of the endosymbiont cellular organelle nucleic acid and/or gene product from step (i) in relation to the amplification of the nuclear nucleic acid and/or gene product from step (ii).
  • bioinformatic approaches for the detection of organelle-specific sequences
  • Another preferred method is detection of exclusively endosymbiont cellular organelle nucleic acid and/or gene product using bioinformatic approaches, wherein the method comprises the following steps:
  • the preferred endosymbiont cellular organelle nucleic acid and/or gene product is derived from mitochondria.
  • the preferred cells used in the methods are from human or mouse.
  • the preferred primers used in the methods are derived from Seq. ID 1-4 (human) and 22 - 28 (mouse) Measurement of transcription and/or translation levels
  • the invention provides a method whereby said relative ratio of a first endosymbiont cellular organelle nucleic acid and/or gene product thereof is determined in relation to the amount of a second (distinct) endosymbiont cellular organelle nucleic acid detectable in said sample (be it DNA or RNA), or in relation to gene products (derivable by transcription and/or translation, such as mRNA or (poly) peptides) of said endosymbiont cellular organelle nucleic acid.
  • the method involves determining a ratio between organelle DNA, such as mtDNA, and the corresponding transcriptionally derivable organelle RNA, in the example the related mRNA, or translated gene product.
  • the level of transcription and/or translation can be determined.
  • An alteration of the level of transcription and/or translation, as compared to the natural level of transcription and/or translation, is indicative of the altered functioning of said organelle.
  • Said altered functioning may be malfunctioning of said organelle, because of a disease and/or because of side-effects of a given treatment. Said malfunctioning may for instance comprise a decreased level of transcription.
  • said altered functioning may be an improved functioning of said organelle, for instance during treatment and/or curing of an endosymbiont cellular organelle related disease.
  • a disease, or a treatment of a disease may involve decrease of the amount of endosymbiont organelle DNA. However, said decrease can at least in part be compensated by an increase in transcription of said DNA, at least in the first stage of said disease. That way, the amount of RNA derived from said endosymbiont organelle DNA may not be decreased at all, or may be relatively less decreased as compared to the amount of said endosymbiont organelle DNA. Symptomatic side- effects of said disease or treatment may then not be (fully) sensed yet. However, upon further decrease of the amount of said endosymbiont organelle DNA, the amount of RNA derived from said DNA will eventually also drop significantly. Side- effects can then occur.
  • the ratio between two distinct organelle DNA's or related gene products is determined.
  • a method of the invention is provided wherein said first endosymbiont cellular organelle nucleic acid and said second endosymbiont cellular organelle nucleic acid are obtained from the same kind of organelle.
  • Said organelle for instance comprises a mitochondrion.
  • a method of the invention is particularly suitable for staging of a disease.
  • An organism can already be affected by a disease, while little or no clinical symptoms are present.
  • the relative ratio of a first endosymbiont cellular organelle nucleic acid and/or gene product thereof in relation to the amount of a second nucleic acid and/or gene product thereof can already be altered.
  • said alteration of said relative ratio can be determined before clinical symptoms and/or conventional tests, like determination of the lactate pyruvate ratio and indicate an altered functioning of an organism.
  • said relative ratio is very suitable for determining the stage of a certain disease.
  • the invention therefore provides in one aspect a method for determining the staging of a disease, by determining the relative ratio of an endosymbiont cellular organelle nucleic acid and/or gene product thereof in a sample obtained from an organism suffering from or at risk of suffering from said disease.
  • the method also encompasses cell-based drug screening.
  • a method of the invention for staging of a disease can be used for diagnosis. For instance, individuals can be routinely tested by a method of the invention with certain time intervals. Alternatively, individuals can be tested at the moment that they exhibit clinical symptoms. An alteration in said relative ratio is indicative of a certain degree of disease. The kind of said disease need not be diagnosed by a method of the invention.
  • Other possible uses of the invention relate to candidate drug testing, for beneficial activity and/or possible side-effects of medicaments or pharmaceutical compositions such as candidate anti-parasitic compounds, antibiotic compounds, cytostatic compounds, and so on.
  • the invention provides a method for determining therapeutic activity and/or possible side-effects of a candidate compound, such as determining its usefulness for treatment of malfunctioning of a cellular organism, determining the relative ratio of an endosymbiont cellular organelle nucleic acid and/or gene product thereof in a sample obtained from said organism, preferably said organism or an essentially related organism, such as belonging to the same species or genus, having been provided with said compound. If the relative ratio of an endosymbiont cellular organelle nucleic acid, and/or gene product thereof, of a certain organism is altered after said candidate compound is administered to said organism, this may indicate therapeutic activity and/or side-effects related to said compound when administered to said organism.
  • this may also indicate therapeutic activity and/or side-effects related to said compound in an essentially related organism. Therefore, for determining therapeutic activity and/or side-effects of a candidate compound for treatment of malfunctioning of a cellular organism, it is not necessary to use exactly the same organism in a method of the invention. An essentially related organism can also be used.
  • the invention provides a method for determining therapeutic activity and/or possible side-effects of a medicament by determining the relative ratio of an endosymbiont cellular organelle nucleic acid and/or gene product thereof in a sample obtained from an organism, preferably said organism having been provided with said medicament.
  • therapeutic activity means the capability of at least in part treating a disease.
  • said therapeutic activity comprises a therapeutic activity against an HIV-related disease and/or a tumor-related disease.
  • Said medicament may for instance comprise a cytostaticum, optionally combined with other antiretroviral therapy.
  • a method of the invention is very much desired during such therapies, because said method can detect side-effects before (severe) clinical symptoms are present or be useful in cases where clinical symptoms are not present. Said therapy can then be stopped and/or changed before said clinical effects are present, thus preventing a lot of suffering.
  • a method of the invention is provided wherein said side-effects are not essentially manifested at the moment that said method is performed.
  • by 'not essentially manifested' is meant that said side-effect is not (yet), or is only partly, manifested by clinical symptoms.
  • a method of the invention wherein said compound or medicament comprises a cytostaticum.
  • cytostatica for instance comprise alkylating compounds, antimitotoxic cytostatica, antitumor antibiotica, and topo-isomerase inhibitors.
  • Non-limiting examples thereof comprise chlorambucil, cyclofosfamide, estramustine, ifosamide, melfalanthiotepabusulfan, treosulfancarmustne, lomustinecisplatine, carboplatine, oxaliplatinedacarbazine, procarbazine, temozolomide, vinblastine, vincristine, vindesinedocetaxel, paclitaxel, daunorubicine, doxorubicine, epirubicine, idarubicine, mitoxanthronbleomycine, dactinomycine, mitomycineirinotecan, topotecanetoposide, teniposide, amsacrine, asparaginase, cladribine, hydroxyarbaide, pentostatine, methotrexate and/or raltitrexed.
  • nucleoside and/or nucleotide analogue are often used. These analogues involve a high risk of side-effects, because they interfere with replication and/or transcription processes in an organism. The amount of endosymbiont cellular organelle nucleic acid is then often altered as well. Therefore, a method of the invention is very suitable when an organism is treated with a medicament involving nucleoside and/or nucleotide analogues.
  • the invention provides a method wherein said compound or medicament comprises a nucleoside and/or nucleotide analogue.
  • analogues are fludarabine, mercaptopurine, thioguanine, cytarabine, fluorouracil, and/or gemeytabine.
  • a method of the invention is provided wherein said compound or medicament comprises AZT, ddl, ddC, d4T, 3TC and/or tenofofir.
  • said organism or an essentially related organism has preferably been provided with said compound or organism.
  • a method of the invention is particularly suitable during treatment of a disease during a long period of time. During said long period, many side-effects can evolve, and a patient can now be monitored regularly even though no clinical symptoms are evident (yet). Therefore, in one aspect a method of the invention is provided wherein said medicament is used during at least 3 months, preferably during at least 6 months, more preferably during at least 12 months. In one aspect, said medicament is used for treatment of a chronic disease.
  • a chronic disease is meant herein a disease which cannot be completely cured. Once an individual has acquired said disease, said disease is always present in said individual, albeit the clinical symptoms may vary widely. Said symptoms may sometimes even be unnoticed by said individual.
  • a chronic disease for instance comprises an HIV-related disease.
  • a side effect of a compound is meant herein another effect than the purpose of said compound.
  • Said side-effect may be an unwanted effect.
  • a therapeutic compound may counteract a disease and simultaneously reduce the metabolism of an organism. Said reduction of said metabolism is then referred to as a (negative) side-effect.
  • a side-effect of a compound may be a beneficial effect, like for instance immunity against yet another disease.
  • the invention provides a method for determining toxic activity of a candidate compound, for example in determining its usefulness for causing malfunctioning of a cellular organism, e.g. by having a cytostatic or even cytotoxic effect, comprising determining the relative ratio of an endosymbiont cellular organelle nucleic acid and/or or gene product thereof in a sample obtained from an organism, preferably said organism or related organism having been provided with said compound.
  • selectivity is also tested, using or applying the method as provided herein (preferably in parallel experiments) on or to a first organism and on or to an essentially unrelated second organism, if desired belonging to a different family or order, but preferably belonging to at least a different class or phylum, most preferably belonging to a different kingdom of organisms.
  • Selectivity aspects are for example tested by testing the compounds in (if desired only in cells of) a first target organism (such as a bacterium or parasite) as well as testing the host or cells thereof, being an essentially unrelated second organism, for example a mammal or plant, or by testing a crop plant or cells thereof as well as testing an essentially unrelated weed plant or cells thereof with said compound, to determine for example selective toxic or selective therapeutic effects. It is also provided to test normal cells derived from an individual in parallel or comparison with aberrant cells, such as tumour cells derived from the same individual, to detect or screen for a tumour- specific or at least selective cytostatic or cytotoxic compound for use in therapy of said individual or others with similar or related disease.
  • a first target organism such as a bacterium or parasite
  • the invention provides a mitochondrial DNA (mtDNA) sequence that is unique and shows no homology with nuclear DNA (nucDNA) for preventing misleading results in the diagnosis method. Furthermore the invention provides primers derived from the mtDNA sequence that is unique and shows no homology with nucDNA for preventing misleading results in the diagnosis method. If the oligonucleotide primers used for the detection of a particular mtDNA PCR- product also hybridizes with nucDNA the relative ratio of a first endosymbiont cellular organelle nucleic acid and/or gene product thereof in a sample obtained from said organism in relation to the amount of a second nucleic acid and/or gene product thereof would be wrong.
  • ID 1 - 4 human and Seq. ID 22 - 28 (mouse) was identified by comparing human or mouse mtDNA sequences with respective nuclear genomes using bioinformatics tools. Unique regions in the human or mouse mtDNA were identified (i.e., without significant homology to nuclear sequences) by this process. These mtDNA sequences can be used, amongst other purposes, to design sequencing primers compatible with Next Generation Sequencing technologies, to design capture oligonucleotide probes useful in purifying mtDNA from total cellular DNA preparations and to develop PCR assays to validate rho zero cells (i.e., cells devoid of mitochondria).
  • Peptide sequences, fragments thereof, variants thereof, polynucleotide sequences, fragments thereof or variants thereof according to the present invention can be used for performing computational searches against public and private sequence collections and thereby for detecting endosymbiont cellular organelle nucleic acid and/or gene product that are not homologues to nuclear nucleic acid and/or gene products.
  • Bioinformatic searches were performed using algorithms such as BLAST (Altschul S. F. et al. 1997, Nucleic Acids Res. 25(17): 3389-3402, default parameters are preferably used).
  • BLAST algorithm were used to compare the selected sequences against a set of other sequences and to report statistically significant hits as unique endosymbiont cellular organelle nucleic acid and/or gene product from, but not limited to, mitochondria. Measurement of relative ratio via amplification of target nucleic acid
  • a relative ratio is for instance determined by measuring the amount of said nucleic acid(s) and/or gene product(s) present in said sample, usually after at least one processing step, like for instance amplification of target nucleic acid. After said amounts have been measured, said relative ratio can be determined by dividing one amount by another.
  • Minute amounts of target nucleic acid can be detected and quantified by using enzymatic amplification.
  • enzymatic amplification techniques include the polymerase chain reaction (PCR) 1 , nucleic acid sequence-based amplification (NASBA) 2 , strand displacement amplification (SDA), transcription mediated amplification (TMA), and others.
  • Specific amplification of a target nucleic acid sequence can be achieved by adding two oligonucleotide primers to a reaction.
  • An amplified region can be detected at the end of an amplification reaction by probes that are specific for said amplified region. Alternatively, an amplified region can be detected during generation of said amplified nucleic acid in said amplification reaction 3 .
  • a signal of a label attached to a probe can become detectable after said probe has hybridised to a complementary nucleic acid.
  • probes that enable real-time homogenous detection in amplification reactions are TaqMan 3 and Molecular Beacon probes 4 ' 5 .
  • sequence-specific probes for detection of amplification product is the use of double-stranded DNA binding dyes such as SYBR Green (Biophys J 66, A159 (1994)
  • Quantification of a target nucleic acid sequence is commonly accomplished by adding a competitor molecule, which is amplified using the same primers and which contains sequences that allow discrimination between competitor and target nucleic acid sequence 2;6 .
  • the ratio between amplified competitor and target nucleic acid sequence can be used to quantify said target nucleic acid sequence.
  • Detection of competitor or target nucleic acid sequence can for instance be achieved at the end of the amplification reaction by probes that are specific for said amplified region of competitor or target nucleic acid sequence or during generation of said amplified nucleic acid in the amplification reaction.
  • a signal of a label attached to a probe can become detectable after said probe has hybridised to a complementary target nucleic acid and when said target has exceeded a threshold level; the time or cycle number to positivity.
  • the time to positivity can be used for quantification without addition of a competitor.
  • a method of the invention is very suitable for, among others, determining (mal) functioning of a cellular organism, candidate drug testing and selective toxin testing. Many reactions have been carried out using a method of the invention, which has proven to be a useful tool (see examples). An even more precise result can be obtained using a method of the invention when double spreading in the result is avoided. Generally, double spreading in the result of a method of the invention is obtained due to varieties in conditions in different reaction mixtures. For instance, to be able to detect and quantify specific nucleic acids present in a sample, an amplification step is often necessary. However, the temperature of the reaction mixture of nucleic acid 1 may be slightly higher than the temperature of the reaction mixture of nucleic acid 2.
  • the determined ratio is not exactly the same as the real ratio of the two nucleic acids present in the initial sample.
  • minute variations in other conditions like for instance the amount of enzyme added can lead to variations in the determined amounts of nucleic acids 1 and 2.
  • the measured amounts of nucleic acids 1 and 2 may vary independently from each other. Independent variations in said determined amounts may result in an even larger variation in the calculated ratio of said measured amounts. This is called the double spreading in the result.
  • double spreading is meant herein at least one variation in an obtained result, due to a variety of at least one reaction condition in at least two reaction mixtures. For instance, also the total amount of volume may differ slightly between two reaction mixtures.
  • double spreading in a result may exceed the variations of the relative ratio of an endosymbiont cellular organelle nucleic acid and/or gene product thereof in an organism which is due to a certain disease or treatment.
  • inhibitors of viral polymerase are often used for treatment of HIV.
  • Inhibitors of viral polymerase may also affect mitochondrial polymerase gamma.
  • the amount of mitochondrial polymerase gamma may be reduced during said treatment of HIV, which may result in a decreased amount of mitochondria per cell.
  • a decrease of for instance 50% of the mitochondria may result in side-effects.
  • the ratio of mitochondrial DNA versus nuclear DNA may be diminished by a factor 2.
  • one embodiment of the present invention provides a method for determining functioning of a cellular organism, without double spreading in the result, by determining the relative ratio of a first endosymbiont cellular organelle nucleic acid and/or gene product thereof in a sample obtained from said organism in relation to the amount of a second nucleic acid and/or gene product thereof.
  • Said double spreading can in a preferred embodiment of the present invention be prevented by determination of said ratio in the same assay.
  • a processing step and/or a measurement of the amounts of at least two nucleic acids and/or gene products thereof are performed in the same assay.
  • an assay typically utilises one reaction mixture.
  • all components of an assay of the invention are mixed randomly in said assay.
  • Said reaction mixture may be present in one reaction tube.
  • a person skilled in the art can think of more methods to prevent double spreading in the result. He/she can for instance use a reaction vessel which is divided in different parts by a (semi) permeable membrane. As long as at least one reaction condition varies dependently in said different parts, double spreading is avoided and the obtained result will be more accurate.
  • At least two target sequences are amplified in one assay.
  • Said two target sequences may be said endosymbiont cellular organelle nucleic acid and said second nucleic acid.
  • a method of the invention comprising amplification of said endosymbiont cellular organelle nucleic acid and said second nucleic acid in the same assay.
  • Detection of said two target sequences can be achieved by using two specific probes during the generation of amplified nucleic acids during an amplification reaction.
  • Said two probes may each have a different label allowing discrimination between said two probes and thereby between said two different target sequences.
  • Quantification can be achieved by relating the time to positivity as well as the slope of the relative fluorescence increase of both real time amplification reactions.
  • a reference curve is created beforehand. The quantification of said nucleic acid can then be performed by comparing the obtained value(s) with said reference curve.
  • an internal standard such as for instance a competitor molecule.
  • the invention provides a method, wherein a relative ratio is determined directly by dividing one amount of nucleic acid by another. Preferably, said relative ratio is determined by comparison with a reference curve.
  • determined directly means that an immediate indication of the ratio of the two targets is possible, for instance by comparing the intensity of said two different fluorescent labels of said two specific probes.
  • dividing one amount of nucleic acid by another is performed by dividing the intensity of the corresponding fluorescent label by another. No internal standards are used in a method of the invention wherein said relative ratio is determined directly.
  • a method of the invention wherein said cellular organelle nucleic acid, said gene product thereof, said second nucleic acid and/or said gene product thereof is obtained from a peripheral blood mononuclear cell (PBMC) and/or a fibroblast.
  • PBMC peripheral blood mononuclear cell
  • a method of the invention is provided wherein said sample comprises a blood sample.
  • a method of the invention is especially useful to quantify a target nucleic acid and/or gene product thereof with a variable content in relation to a target nucleic acid and/or gene product thereof with a constant content.
  • An example is the quantification of the variable cellular content of mitochondrial DNA to the constant cellular content of the DNA of a nuclear gene (two per diploid cell).
  • Another example comprises the quantification of variably expressed RNA like mitochondrial RNA to constitutively expressed RNA that is essential for cell survival like the SNRP U1A encoding RNA involved in splicing or other essentially common nucleic acids derived from nuclear DNA with an ubiquitous presence. We found that it is possible to determine a relative ratio of a factor 2.
  • the invention provides a diagnostic kit comprising at least one means for performing a method according to the invention, said kit comprising at least one primer or probe set selective for the amplification and detection of a nucleic acid related to or derived from endosymbiont cellular organelles and, when so desired, necessary amplification reagents, such as can be found exemplified in the detailed description herein or which are otherwise known in the art.
  • the invention provides a diagnostic kit wherein said kit comprises more than one primer or probe set for the amplification of nucleic acid sequences related to cellular organelles, preferably supplemented with a primer or probe set for the amplification of nucleic acid related to the chromosomes, such as a snRNP specific primer or probe.
  • the invention provides a kit comprising at least one primer or probe from table 1 for the amplification of nucleic acid sequences related to cellular organelles.
  • said amplification reagents when provided with the kit, comprise an enzyme with DNA polymerase activity, such as required for PCR or NASBA.
  • a kit comprising a means for the detection of a gene product other than nucleic acid, for use in a method according to the invention is herewith also provided.
  • the kit is useful for determining the amount of an endosymbiont cellular organelle in cells by measuring the relative ratio of the amplification of the endosymbiont cellular organelle nucleic acid and/or gene product in relation to the amplification of the nuclear nucleic acid and/or gene product and contains: (i) Primers that are able to bind exclusively the endosymbiont cellular organelle nucleic acid and/or gene product of cells and not the nuclear nucleic acid and/or gene product of cells
  • the preferred endosymbiont cellular organelle nucleic acid and/or gene product in, but not limited to, the kit is derived from mitochondria.
  • the preferred cells in the kit are derived from human or mouse.
  • the preferred primers in the kit are derived from human or mouse DNA.
  • the preferred primers used in the kit are derived from Seq. ID 1-4 (human) and Seq. ID 22 - 28 (mouse).
  • the preferred primers are also useful for sequencing of endosymbiont cellular organelle nucleic acid and/or gene product.
  • the preferred primers are also useful as capture oligonucleotides and/or peptide nucleic acids for the purification of endosymbiont cellular organelle nucleic acid and/or gene product of cells.
  • the preferred amplification method in the kit corresponds, but is not limited to, PCR, real time PCR, nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), or transcription mediated amplification (TMA).
  • NASBA nucleic acid sequence-based amplification
  • SDA strand displacement amplification
  • TMA transcription mediated amplification
  • the human DNA free of mitochondrial DNA is purified from Rho zero cells.
  • the invention furthermore provides the use of a compound obtainable or detectable by a method according to the invention in the preparation of a medicament, a herbicide, insecticide, anti-parasiticum, cytostatic, etc, and a medicament, herbicide, insecticide, anti-parasiticum etc. obtainable or derivable or identifiable by a method according to the invention.
  • Table 1 The complete Human mitochondrial ref sequence NC_012920.1 was blasted against the complete human nuclear genome to search for suitable regions of no or low homology to develop primers. This first blast resulted in multiple pseudogene alignments with 100% coverage of the human mitochondria.
  • the complete human mitochondria ref sequence was then broken into 350bp sections, and each such sections were blasted to look for regions with low pseudogene alignment and low % homology.
  • the output is shown below in tabular format and labelled according to the specific region of mtDNA blasted.
  • the percent homology for the most homologous region is shown in the '% nuclear homology' column, and the number of total hits to the nuclear genome are listed in the '# of pseudo-gene alignments column'.
  • Good sections (white) were determined by having a low % homology and/or ⁇ 3 pseudo-gene alignments.
  • "Possible” sections (light gray) were determined by having a low % homology and/or >3 but ⁇ 6 pseudo- gene alignments. Those sections with high % homology and/or > 6 pseudo-gene alignments were disregarded (dark gray), (see table below)
  • Table 2 Same as table 1 above only with the basepair segments staggered by 140 bases.
  • the advantage of this kind of staggering is that small pseudogenes that may have straddled two of the sections from the initial blast above can also be evaluated for their potential impact on assay performance.
  • mtDNA changes were monitored and compared to signal obtained from nuclear DNA.
  • a human lung epithelial carcinoma cell line (A549) was incubated with a low concentration (50 ng/mL) of ethidium bromide, a compound known to be toxic to mitochondria.
  • Mitochondrial DNA was followed by using primers designed to amplify two mitochondrial genes uniquely represented in the mitochondrial genome (ND1 and ND6).
  • Signal representative of the nuclear genome was obtained using primer pairs designed to amplify two nuclear genes, NEB1 and BECN1 .
  • Real Time PCR data were obtained using the double-stranded DNA binding dye SYBR Green.
  • mtDNA was detected using primers specific to two mitochondrial genes, cytb and ND1 ; nuclear DNA was detected using primer pairs specific to single copy nuclear genes NEB1 and BECN1 ; Real Time PCR data were obtained using the double-stranded DNA binding dye SYBR Green.
  • a ratio of 1000 is obtained when signal corresponding to mtDNA in a human osteosarcoma cell line (143B) is compared to that corresponding to nuclear DNA.
  • p° When cells devoid of mitochondria (p°) are used in the analysis, no signal can be detected for mtDNA while corresponding nuclear DNA signal is comparable to that obtained when 143B cells were used in the analysis.
  • the lack of signal in the p° cell line indicates that mitochondria-targeted primers do not amplify nuclear sequences.
  • the mitochondrial genes for mouse and human share the genbank ID for the mitochondrial genome sequence we used since they are part of the same circular molecule containing all of the mitochondrial encoded genes.
  • Human cytochrome B is part of the mtDNA reference sequence NC_012920.1 and is contained within 1 141 bp stretch from 14,747 to 15,887. Its official gene ID is MT- CYB and it's gene ID number is 4519., http://www.ncbi. nlm.nih.gov/gene/4519
  • Human ND6 shares the same genbank number NC_012920.1 but is a 525 bp sequence falling within the range from 14149 and 14673 of the 16569 bp molecule. http://www.ncbi.nlm.nih.gov/gene/4541
  • Human ND6 shares the same genbank number NC_012920.1 but is a 956 bp sequence falling within the range from 3307 and 4262 of the 16569 bp molecule. http://www.ncbi.nlm.nih.gov/gene/4535, 3307-4262
  • Fig. 1 Data shown below indicate a decrease in the intensity of the mitochondrial DNA signal with longer incubation of the cells in the mitochondria-toxic compound, ethidium bromide (light traces). At the same time, corresponding nuclear signal remains unchanged (dark trace). Ethidium bromide treatment is used to deplete the mtDNA in 143B Osteosarcoma and A549 Lung Epithelial Carcinoma cell lines. Higher passage number and longer time in culture leads to a greater depletion of mtDNA as it transitions to a total loss of mtDNA (passages p6 and p7 in 143B cells).
  • Fig. 2 Same as 143B cell line above except the A549 cell is showing resistance to total mtDNA depletion.

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L'invention concerne un procédé de détermination de la quantité d'organites cellulaires de l'endosymbionte dans des cellules.
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WO2016063122A1 (fr) * 2014-10-20 2016-04-28 INSERM (Institut National de la Santé et de la Recherche Médicale) Procédés de criblage d'un sujet pour dépister un cancer

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EP1229130B1 (fr) * 2000-12-04 2013-11-20 Primagen B.V. Tests basés sur les acides nucléiques des organelles cellulaires endosymbiontiques
ES2269690T3 (es) * 2001-05-29 2007-04-01 The University Of British Columbia Aplicaciones farmacologicas de los ensayos de adn mitocondrial.
EP1523578A1 (fr) * 2002-07-05 2005-04-20 The University of British Columbia Diagnostic d'une sepsis faisant appel a des epreuves biologiques d'acides nucleiques mytochondriaux

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Title
ALTSCHUL S. F. ET AL., NUCLEIC ACIDS RES., vol. 25, no. 17, 1997, pages 3389 - 3402
BIOPHYS J, vol. 66, 1994, pages A159

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WO2016063122A1 (fr) * 2014-10-20 2016-04-28 INSERM (Institut National de la Santé et de la Recherche Médicale) Procédés de criblage d'un sujet pour dépister un cancer
US10704104B2 (en) 2014-10-20 2020-07-07 Inserm (Institut National De La Sante Et De La Recherche Medicale) Methods for screening a subject for a cancer

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