EP2004848A1 - Procede de caracterisation d'acides nucleiques dans un echantillon mixte - Google Patents

Procede de caracterisation d'acides nucleiques dans un echantillon mixte

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Publication number
EP2004848A1
EP2004848A1 EP07711550A EP07711550A EP2004848A1 EP 2004848 A1 EP2004848 A1 EP 2004848A1 EP 07711550 A EP07711550 A EP 07711550A EP 07711550 A EP07711550 A EP 07711550A EP 2004848 A1 EP2004848 A1 EP 2004848A1
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EP
European Patent Office
Prior art keywords
particles
reaction
mixed sample
individual
substrate
Prior art date
Legal status (The legal status 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 status listed.)
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Application number
EP07711550A
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German (de)
English (en)
Inventor
Christoph Gauer
Mann Wolfgang
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Beckman Coulter Inc
Original Assignee
Advalytix AG
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Filing date
Publication date
Application filed by Advalytix AG filed Critical Advalytix AG
Publication of EP2004848A1 publication Critical patent/EP2004848A1/fr
Withdrawn legal-status Critical Current

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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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • the present invention relates to a method for characterizing a mixed sample comprising at least two particles with nucleic acids of different individuals, each particle comprising nucleic acid of one or more individuals, in particular for the quantitative determination of the absolute and / or relative number of particles present in a mixed sample with nucleic acid of an individual and / or for determining the genotype of one or more individuals from a mixed sample, in particular for the quantitative determination of the absolute and / or relative copy number of a predetermined sequence of an individual of which nucleic acid is contained in the mixed sample.
  • the present invention relates to a kit for determining the genotype of one or more individuals from a mixed sample, which contains particles with nucleic acids of different individuals, which in particular for the quantitative determination of the absolute and / or relative copy number of a predetermined sequence of an individual, of the Nucleic acid contained in the mixed sample is suitable.
  • mixed samples ie biological samples containing nucleic acids from different individuals, in order to draw conclusions about the identity and / or one or more specific genotypic features of one or more of the individuals whose nucleic acid (s) ) is / are included in the mixed sample.
  • applications in forensics in genetic engineering, eg in the context of cloning, or in medical diagnostics are mentioned.
  • forensics for example, often only samples are available from a crime scene, which contain the nucleic acids of two or more different people in order to gain inferences from this mixed sample on the identity of the offender permitting insights.
  • it is unknown how the mixed sample is composed in particular of how many different individuals nucleic acid is contained in the mixed sample and what is the ratio of the nucleic acids of the individual individuals in the mixed sample with each other.
  • the copy number of the corresponding chromosome is 18, 13 and 21 per cell, respectively, whereas healthy individuals have only two copies of the aforementioned chromosomes per cell.
  • the increase in the number of copies of the the chromosome to severe developmental disorders.
  • a variety of diseases is also known, which are based on an altered copy number of genes or gene segments.
  • Huntington's disease a progressive neurodegenerative disease characterized by abnormal, involuntary movements with increasing decay of mental and physical abilities, may be mentioned in this context.
  • determining the number of copies of the corresponding chromosomes, genes or gene segments in the fetal cells it is thus possible to prenatally diagnose a possible corresponding disease.
  • the determination of the genotype of a fetus from maternal blood is problematic because fetal cells in maternal blood occur only at a frequency of about 1: 1,000,000 (fetal cells / maternal cells) and the exact relative relationship between maternal and fetal cells initially not is known and can not be determined without further, complex investigations.
  • a similar problem also arises in cancer diagnostics, for example when examining a body sample for the presence of cancer cells. If the body sample actually contains cancer cells, it is a mixed sample containing healthy cells and cancer cells (which are considered in the present invention as cells of two different individuals), wherein the quantitative ratio of the individual cell types with each other is unknown and with elaborate studies must be determined to determine at what advanced stage the cancer is located.
  • nucleic acid (s) of other individuals contained in addition to the nucleic acid of the individual to be characterized disturb the characterization of the nucleic acid of the individual to be characterized. This is especially true when the amount of nucleic acid of the individual to be characterized in the mixed sample is significantly less than the amount of adjacent nucleic acid of another individual, as in the case of maternal fetal cells.
  • the mixed sample is enriched with respect to the individual to be characterized, for example by enrichment of the fetal cells by means of fluorescence-labeled antibodies, it is generally not possible to obtain a pure sample with regard to the individual to be characterized. False positive results (a maternal cell is falsely typed as a fetal cell) and / or false negative results (a fetal cell is overlooked) occur in the known methods.
  • a cancer cell overexpresses a particular gene, which is why the cancer cell to be detected has a double copy number of mRNA of the aforementioned gene than the healthy cell.
  • the gene expression quantifiable by the mRNA content is measured in comparison with the healthy cell.
  • the corresponding gene expression of a healthy cell is 100%, of 2 healthy cells 200% and of 3 healthy cells 300%, whereas the gene expression of a cancer cell is 200%.
  • the mixed sample containing nucleic acids from different individuals their qualitative and / or quantitative composition, i. of which the number of different individuals of which the mixed sample contains nucleic acid and / or the quantitative ratio of the individual, different nucleic acids among each other is unknown, can be reliably and quickly analyzed for the genotype of an individual contained in the mixed sample.
  • the methods known for this purpose lead to false or at least unreliable results.
  • these methods are usually time consuming and costly.
  • the object of the present invention is therefore to provide a method for characterizing a mixed sample containing at least two particles with nucleic acids of different individuals, with which the absolute and / or relative number of particles present in a mixed sample with nucleic acid of an individual and / or the genotype of a nes or more individuals from the mixed sample can be determined simply, quickly and in particular reliably.
  • this object is achieved by a method according to claim 1 and in particular by a method for characterizing a mixed sample comprising at least two particles with nucleic acids of different individuals, each particle comprising nucleic acid of one or more individuals, in particular for the quantitative determination of the absolute and / or relative number to particles present in a mixed sample with nucleic acid of an individual and / or to determine the genotype of one or more individuals from a mixed sample, in particular for the quantitative determination of the absolute and / or relative copy number of a predetermined sequence of an individual of which nucleic acid is present in the mixed sample comprising the steps:
  • a mixed sample is understood as meaning a sample, in particular a biological sample, which comprises at least two particles each containing nucleic acid, wherein in the sample, either per particle or in the entirety of the particles, nucleic acids of at least two different individuals are included.
  • the term particles in this context means a small particle.
  • the nucleic acid may be contained in the particle or bound to the particle. Examples of corresponding particles are cells, in particular unlysized cells with nucleic acid contained therein, and magnetic particles having nucleic acid bound thereto, for example via hybridization to a primer.
  • the term individual comprises not only a person different from another in the case of humans, but in particular also different cell types of a person, which differ from one another with regard to their genotype.
  • genetic mosaics or chimeras ie cells of different genotype of a person, which are formed by mixing or exchange of different genotypes (chimera) or arise in an individual (genetic mosaic).
  • An example of a genetic mosaic are cancer cells that have been produced, for example, by LOH ("loss of heterozygosity").
  • a method for characterizing a mixed sample means, in particular, that a mixed sample is qualitatively and / or quantitatively characterized with regard to its composition.
  • a characterization of a mixed sample therefore includes, for example, the quantitative determination of the absolute number of different individuals present in a mixed sample, the quantitative determination of the relative number / frequency of an individual in the mixed sample Mixed sample (for example, the determination of the percentage of a cell type A in a biological sample comprising cell types A and B) and / or the determination of the genotype of one or more individuals represented in a mixed sample.
  • the determination of the genotype of one or more individuals in the context of the present invention is understood in particular to characterize at least one predetermined sequence of an individual with regard to presence or absence, copy number and / or nucleic acid sequence, ie in particular the determination of the absolute or relative number of a predetermined sequence, for example a genome, a gene or a gene segment.
  • relative quantitative determination of the number of a predetermined sequence in an individual within the meaning of the present invention means determining whether the genome of an individual contains fewer, equal to or more copies of a predetermined sequence than that of a reference sample and absolute quantitative determination of the number of predetermined sequence in an individual, the determination of which specific number of copies of the predetermined sequence is contained in the genome of the individual.
  • homologous sequence in the sense of the present invention denotes sequences which have a similarity with respect to their nucleotide sequence of at least 70%, preferably at least 80%, more preferably at least 90% and most preferably at least 95%, whereas non-homologous sequences are those which have a correspondingly lower sequence similarity with each other.
  • An essential feature of the method according to the invention is that the particles, for example cells, of a mixed sample in step a) are first separated in such a way that a subsequent deposit of exactly one particle or one cell, which in contrast to a large number of those from the prior art known to the art is free of any other cells or components of other cells bound to it, per reaction site of the substrate. This ensures that this cell or particle can be analyzed without background on non-individual nucleic acid.
  • depositing one cell each on the hydrophilic reaction site of a substrate surrounded by a hydrophobic region enables the formation of a liquid drop formed from the liquid contained in the cell isolate or added to the reaction site after being deposited on the reaction site Substrate adheres, so that the subsequent steps b) and c) of the method according to the invention can be carried out directly on the reaction site, without the cell must be transferred to a closed reaction vessel or the like. As a result, laborious and time-consuming transfer steps are avoided.
  • the advantage of the inventively provided deposition of particles on such a substrate in contrast to the conventional new microtiter plate is that immediately before the actual analysis, a visual inspection of the material to be analyzed is possible. For example, it can be determined beyond doubt with the microscope that only exactly one single cell was deposited on each reaction site. In a 3-dimensional reaction vessel this is not possible without considerable effort due to the lack of depth of field of the microscope and other reasons.
  • the optimization of the genotyping in step b) which can be achieved, for example, by means of a PCR, at least 80% of the examined particles can be assigned to an individual or assigned to an individual.
  • the process steps b) and c) can be carried out directly at the reaction site without first having to evaporate the sample or transfer it into a closed reaction vessel. Furthermore, due to the minimal volume of liquid remaining after separation in the cell, the storage of larger amounts of potentially subsequent process steps b) and c), especially if these process steps comprise an enzyme reaction, prevents interfering contaminants on the reaction site.
  • the cell is isolated from cell culture medium or body fluid such as blood or the like, the cell being deposited in a substantial volume of cell culture medium or body fluid in a reaction vessel. Due to the significant amounts of contaminants contained in this volume of cell culture medium or body fluid, an enzymatic reaction is impossible in these processes without further time-consuming and labor-intensive purification of the sample.
  • the at least two individual particles are each in a volume of less than 100 nl, more preferably less than 10 nl, most preferably less than 1 nl and most preferably less than 100 pl on the corresponding reaction site to deposit the substrate.
  • Another essential feature of the method according to the invention is the assignment of the at least two cells or particles individually deposited on the reaction sites to an individual contained in the mixed sample by a determination carried out on the reaction site of the substrate, of which the individuals represented in the mixed sample are those deposited Contain particles of nucleic acid, wherein at least 80% of the investigated particles are assigned to an individual or be assigned.
  • the cells deposited on the reaction sites are actually pure, individual cells which are free of components of cells of a different nature.
  • this can be used to verify whether each of the cells stored is a target cell or a false positive cell.
  • the results of subsequent further characterization of the cell under investigation can be clearly assigned to an individual. Due to the As a result of genetic analysis, a definite statement can be made about this and the subsequent assignment of the deposited particles to an individual contained in the mixed sample.
  • At least 85%, more preferably at least 90%, most preferably at least 95%, most preferably at least 98%, and most preferably 100% of the examined particles are to be assigned to an individual in the examination by genotyping in method step b).
  • the further characterization of the investigated particles according to method step c) comprises the determination of the absolute and / or relative number of particles present in the mixed sample with nucleic acid of an individual and / or the determination of the genotype of the substrate on the reaction site deposited particles.
  • results concerning the quantitative and / or qualitative composition of the mixed sample are obtained.
  • it can be determined from maternal blood containing fetal cells whether the fetus has trisomy 21 or not.
  • the progression of cancer in a patient can be determined by measuring the percentage of cancer cells in a cancerous tissue, i. a mixed sample containing cancer cells and healthy body cells.
  • the particles deposited on the reaction sites of the substrate are cells, particularly preferably unlysed cells.
  • the latter is preferred because unlike a lysed cell in an unlysed cell, optical control ensures that it encompasses the entire genome of an individual.
  • the particles may each be any particle comprising a nucleic acid of a specific individual, such as a DNA or RNA labeled particle having DNA or RNA hybridized to the probe.
  • FACS Fluorescence Activated Cell Sorters
  • the cells are isolated with substantial amounts of fluid. Because of the contaminants contained in the fluid, such as cell culture medium or blood, such as proteases, nucleases, salts, and the like, such isolates require removal of not only the fluid but also the contaminants before the thus isolated cell is used in an enzymatic reaction can be.
  • fluid such as cell culture medium or blood
  • proteases, nucleases, salts, and the like such isolates require removal of not only the fluid but also the contaminants before the thus isolated cell is used in an enzymatic reaction can be.
  • Examples of commercially available devices which use one of the aforementioned techniques are the manual capillary system, for example Eppendorf, Hamburg, the automated system CellCelector from AVISO GmbH, Gera, devices based on laser pressure catapulting technology, for example PALM, Bernried and FACS devices, for example, the companies Becton Dickinson and Dako Cytomation.
  • FACS devices The operation of FACS devices is usually as follows: A liquid suspension containing the particles or cells is passed through a nozzle at which the liquid stream is separated into individual separated liquid droplets, the individual liquid droplets each containing a predetermined number of cells, all or selectively, after separation from the nozzle, individual liquid drops are electrically charged and the individual liquid drops are passed through an electric field, whereby one or more electrically charged liquid drops can be selectively directed onto a substrate.
  • the individual liquid droplets When passing the individual liquid droplets through the electric field, only the electrically charged droplets or only the electrically charged droplets are deflected and applied to the correspondingly positioned substrate.
  • the separation of the liquid suspension at the nozzle is effected by piezoelectric modulation, in which a periodic pressure fluctuation is exerted on the liquid jet flowing through the nozzle, due to which liquid droplets of a defined and reproducible size are formed at the nozzle and torn off from the liquid jet.
  • piezoelectric modulation in which a periodic pressure fluctuation is exerted on the liquid jet flowing through the nozzle, due to which liquid droplets of a defined and reproducible size are formed at the nozzle and torn off from the liquid jet.
  • the substrate used in the method according to the invention is preferably a glass slide, more preferably a glass slide, on the one hand because they are flat and on the other hand because they are outstandingly suitable for applying hydrophilic regions (also referred to as reaction sites) and hydrophobic regions ,
  • the hydrophilic reaction sites on the substrate substantially circular and to surround them with an at least substantially annular hydrophobic region.
  • the annular hydrophobic region should preferably surround the circularly formed hydrophilic regions concentrically.
  • hydrophobic region surrounding the hydrophilic reaction site on the substrate is surrounded on the outside by a hydrophilic region, which is preferably essentially annular and surrounds the hydrophobic region, particularly preferably concentrically.
  • the outer hydrophilic annulus is also surrounded on the outside by a hydrophobic region.
  • a particularly preferred arrangement consists of a circular hydrophilic region concentrically surrounded by two circular rings, wherein the inner of the two circular rings is hydrophobic and the outer of the two circular rings is hydrophilic, wherein the outer hydrophilic annulus is surrounded on the outside by a hydrophobic region.
  • the hydrophobicity of the hydrophilic reaction site and the hydrophobicity of the surrounding area are set such that, when less than 10 ⁇ l of water is applied to the reaction site, a water droplet having a contact angle of 20 to 70 ° is preferred from 30 to 60 °, and more preferably from 40 to 50 °. This ensures that a stable drop of liquid is formed, which firmly adheres to the reaction site, so that the liquid droplet does not dissolve or even on the glass plate with the slightest vibration of the substrate, such as occur during transport of the substrate, for example within a laboratory the glass plate runs.
  • the diameter of the hydrophilic reaction site is between 0.3 and 3 mm.
  • the method according to the invention is suitable for the characterization of all mixed samples, irrespective of the type of particles used and irrespective of the number of different individuals represented in the mixed sample.
  • good results are obtained when the mixed sample comprises nucleic acid from at least two but less than ten different individuals, more preferably at least two but less than five different individuals, most preferably from two or three different individuals and most preferably from exactly two different individuals Contains individuals.
  • the method according to the invention can be used for all mixed samples, independently of the concentration differences of the individual nucleic acids with one another. Good results are obtained, in particular, when the concentration difference between the nucleic acids contained in the mixed sample of the individual individuals is between 1: 1000 and 1: 1, preferably between 1: 100 and 1: 1 and particularly preferably between 1:10 and 1: 1 ,
  • the proportion of the nucleic acid of the subject to be examined in the mixed sample is less than 1: 1,000, relative to the nucleic acids of the other individuals, as is regularly the case, for example, with maternal blood containing fetal cells, it has proved to be advantageous to enrich the mixed sample prior to isolation and prior to application to the substrate according to step a) with respect to the particles with the nucleic acid of the individual to be examined, otherwise a large number of particles must be examined until statistically a target particle of the individual to be examined on the Substrate is applied.
  • the enrichment can be carried out in any manner known to those skilled in the art, for example by means of fluorescently labeled antibodies which bind specifically to the cell type to be enriched and to it so mark.
  • coated scavenger particles or coated magnetic particles can be used.
  • Another example of a suitable enrichment method is the use of a flow cytometer, in particular a fluorescence activated cell sorter (FACS), which generally works with fluorescently labeled antibodies for the classification of particles and / or their accumulation ,
  • FACS fluorescence activated cell sorter
  • the device has the advantage that the fluorescence detection and the enrichment are combined in one device.
  • the method according to the invention is particularly suitable for characterizing mixed samples comprising maternal blood containing fetal cells, and preferably for characterizing mixed samples consisting of maternal blood containing fetal cells.
  • the inventive method for characterizing a healthy cells as well as mixed sample containing LOH-labeled cancer cells or preferably a mixed sample consisting of healthy cells and LOH-labeled cancer cells is predestined.
  • the method according to the invention has proved to be equally suitable for characterizing a mixed sample containing healthy cells as well as MIN (microsatellite instability) labeled cancer cells or, preferably, a mixed sample consisting of healthy cells and MIN-marked cancer cells.
  • MIN microsatellite instability
  • the assignment of the particles to an individual contained in the mixed sample according to step b) preferably takes place by means of an amplification reaction, wherein in the amplification reaction primer pairs are suitably used for gene sections specific for the target individual.
  • the further characterization of the investigated particles can be, for example, the determination of the absolute number of particles present in the mixed sample containing nucleic acid of an individual or the determination of the relative proportion of the particles containing nucleic acid of an individual based on the entire mixed sample.
  • further characterization of the composite sample may be determination of the relative or absolute copy number of a chromosome, gene or gene segment.
  • the further characterization of the particles according to step c) takes place on the reaction site of the substrate by means of an amplification reaction.
  • the amplification reaction can be any reaction known to the person skilled in the art with which nucleic acids, be it DNA or RNA, can be amplified, preferably multiplied almost exponentially.
  • nucleic acids be it DNA or RNA
  • PCR polymerase chain reaction
  • the amplification reaction carried out is a specific amplification reaction.
  • the particles in the mixed sample extremely low nucleic acid, for example, less than 1 pg, which may occur, for example, in the case that magnetic particles are used with on the surface via probes of hybridized nucleic acid, may occur
  • the amplification reaction for the assignment of the particles to an individual contained in the mixed sample according to step b) and / or before the amplification reaction for the further characterization of the investigated particles according to step c) in / on the particles contained nucleic acids by non-specific PCR to multiply. After the non-specific PCR, a specific PCR can be performed.
  • step b) at least two particles, each deposited one at a reaction site on the substrate, are examined in order to assign them to individuals from the mixed sample by genotyping on the reaction sites of the substrate.
  • the embodiments described below have proven to be particularly suitable.
  • those necessary for carrying out the amplification reaction Reaction components in the case of a PCR preferably the primer, placed on the hydrophilic reaction site before the particle is deposited on the reaction site.
  • the reaction components it is also possible to apply the reaction components to the particle in the form of liquid after the particle has been deposited on the hydrophilic reaction site of the substrate.
  • the inventive concept it is proposed to adapt the amplification reaction to amplify one or at least two mutually homologous and / or non-homologous sequences from the coding DNA region and / or preferably from the non-coding DNA region. It is known that the non-coding DNA region is substantially more polymorphic than the coding DNA region, so that by amplifying sequences from the non-coding DNA region with a relatively high probability individual-specific sequences can be amplified. This is advantageous in both forensic composite samples and in characterizing the genotype of fetal cells from maternal blood containing fetal cells.
  • the amplification reaction has proven to be advantageous to adapt the amplification reaction to amplify one or at least two mutually homologous and / or non-homologous highly polymorphic sequences.
  • the amplification reaction is adapted to amplify one or at least two mutually homologous and / or non-homologous sequences derived from the
  • STR sequences, VNTR sequences, SNP sequences and any combinations thereof are selected, good results are obtained.
  • STR or short tandem repeat sequences are highly polymorphic sequences consisting of only two to four bp repeats and between individuals have a high variability.
  • VNTR or variable number of tandem repeat sequences consist of about 15 to 30 bp in length constructed repetitive DNA sections whose total length is determined by the number of repetitions of this basic unit.
  • VNTR sequences are usually highly polymorphic, that is, the number of respective repeating units is very different between the different individuals.
  • SNPs single nucleotide polymorphism
  • SNPs single nucleotide polymorphism
  • the amplification reaction in particular an amplification reaction used in step c), is adapted to amplify one or at least two mutually homologous and / or non-homologous sequences, which occur only once in the genome of the individual per allele ,
  • conclusions can be drawn on the individual alleles of an individual, so that, for example, the number of individual alleles of an individual in a mixed sample can be determined.
  • the examination according to step b) and the further characterization of the investigated particles according to step c) are carried out simultaneously, ie in one process step.
  • the amplification reaction is adapted to amplify between 1 and 100, preferably between 2 and 20 and more preferably between 5 and 15 mutually homologous and / or non-homologous sequences of the subject of the mixed sample to be examined.
  • the experimental effort is not too big.
  • the number of different amplification products obtained in the amplification reaction can be determined, the determination of the number preferably determining the presence or absence of at least one amplification product and determining a second physically and / or chemically measurable parameter of the amplification products obtained.
  • the presence or absence of amplification products it is possible to use all methods known to the person skilled in the art for this purpose, examples being merely gel electrophoresis, customary hybridization techniques, for example those on a DNA array.
  • the parameters in the amplification reaction are chosen such that the relative frequency for a positive amplification reaction for each of the homologous and / or non-homologous sequences is between 0.2 and less than 1, preferably between 0.4 and 0.6 and more preferably about 0.5, good results are obtained.
  • At least one amplification reaction should be used to characterize the amplification products before, after or preferably in parallel with the amplification reaction for the at least two particles to be examined the same conditions as used for the at least two of the mixed sample deposited on each reaction site particles used with a reference sample, wherein the reference sample preferably has the same amount of nucleic acid as the deposited particles and the reference sample preferably has a known genotype.
  • At least one amplification reaction of the different amplification products obtained with the number of different amplification products obtained with the amplification reaction (s) carried out with the deposited particles can thus be used to determine the relative copy number of the studied, predetermined sequence of the examined individual.
  • the absolute copy number of a predetermined sequence of the individual to be examined is to be determined, it is proposed in a refinement of the invention to determine the number of different amplification products having at least one frequency distribution obtained with the at least two deposited particles to compare.
  • a frequency distribution is preferably obtained by separately carrying out the same amplification reaction with the same reaction conditions as those used for the at least two particles deposited on the reaction sites with at least two different reference samples, the same in the amplification reactions as in The amount of nucleic acid contained in the particles is used and the at least two different reference samples each have a known, mutually different copy number of the predetermined sequence.
  • the amplification reactions for the reference samples can be carried out before, after or particularly preferably parallel to the amplification reaction for the particles to be examined.
  • the absolute copy number of a predetermined sequence can be determined. quency of the individual to be examined or of the individuals to be examined.
  • a frequency distribution is used for whose recording the amplification reaction carried out for each of the at least two reference samples was carried out several times, for example ten or one hundred times. Since starting material having a known copy number of the predetermined sequence is used in the amplification reactions for recording the frequency distribution, it is possible to reliably deduce from this comparison the number of copies of the predetermined sequence in the sample of the mixed sample to be examined.
  • the assignment the investigated particles to an individual contained in the mixed sample and / or the further characterization of the examined particles on the reaction site of the substrate also by a gene expression examination at mRNA level done.
  • Another object of the present invention is a method for characterizing a mixed sample containing at least two particles with nucleic acids of different individuals, each particle nucleic acid of one or more individuals, in particular for the quantitative determination of the absolute and / or relative number of particles present in a mixed sample with Nucleic acid of an individual and / or for the determination of the genotype of one or more dividing from a mixed sample, in particular for the quantitative determination of the absolute and / or relative copy number of a predetermined sequence of an individual, of which nucleic acid is contained in the mixed sample, comprising the steps:
  • the present invention further relates to a kit for determining the genotype of one or more individuals from a mixed sample which contains particles with nucleic acids of different individuals, for carrying out the method according to the invention described above, comprising:
  • a polymorphic region is understood as meaning a region of the genome which is between two randomly selected, mutually unrelated individuals with a probability of at least 25%, preferably at least 50%, particularly preferably at least 80% and very preferably at least 90%, for example in the length of the sequence or in the sequence itself, differs.
  • the hydrophilic reaction sites provided on the substrate contained in the kit are substantially circular in shape and are each concentrically surrounded by a substantially annular hydrophobic region, which is surrounded on the outside by a substantially annular hydrophilic region. wherein the diameter of the hydrophilic reaction sites is between 0.3 and 3 mm.
  • the outer hydrophilic annulus is surrounded on the outside by a hydrophobic region.
  • the kit according to the invention may comprise, in addition to constituents a), b), d) and if appropriate c), at least one of the following constituents:
  • a reference sample having a known genotype and preferably having a copy number known with respect to a predetermined sequence and / or ⁇ 2) the result of at least one amplification reaction with a reference sample under the same conditions as prescribed in the protocol according to e), the reaction conditions being such that at least one amplification product has a probability between 20% and less than 100 % and / or at least one frequency distribution which results from separately carrying out the same and under the same reaction conditions as described in protocol e) at least one amplification reaction with at least two different reference samples, the at least two different reference samples each being known , having different copy numbers of a predetermined sequence from each other, and then determining the number of different amplification products obtained per reference sample, and
  • the aim of the following study was the quantitative relative determination of the number of cancer cells contained in a mixed sample comprising healthy cells and cancer cells of a person.
  • a glass slide was used as a substrate, on the 48 spatially separated circular, hydrophilic reaction sites, each of which, viewed from inside to outside, of an annular, hydrophobic area and a thereto subsequent annular, hydrophilic area were concentrically surrounded were arranged.
  • cells were mixed from the mixed sample with a LaserCapture microscope.
  • the cancerous tissue isolated and randomly each 1 cell from the mixed sample in a volume of less than 1 ul deposited on the 48 hydrophilic reaction sites of the substrate.
  • a reaction solution containing a gene pair D85522-amplifying primer pair, reaction buffer and Taq polymerase was added so that the total volume of the liquid present at each reaction site was 1 ⁇ l.
  • the individual liquid droplets were covered with oil, the substrate was transferred to a PCR thermocycler and a PCR was performed. Finally, a subset of each liquid drop was taken, applied to a gel, electrophoresed by gel electrophoresis in the amplification products containing amplifications and the individual DNA bands visualized.
  • a present biological sample is a mixed sample of female and male cells and, if so, how large the proportion of female cells in the mixed sample is.
  • a random sample of the cell suspension was sorted using a FACS sorter from DAKO and one of the cells was deposited on the 48 reaction sites of the substrate described in Example 1 in each case.
  • reaction solution containing male and female specific primer pairs, reaction buffer and Taq polymerase was added.
  • primer pairs 2 pmol of five primer pairs were used, which were adapted to amplify in a multiplex PCR five different PCR fragments from human male DNA (type XY) or 4 different PCR fragments from human female DNA (type XX). These were the following primers:
  • reaction solution contained the following ingredients:
  • chromosome 21 While healthy body cells are diploid, that is, have 2 copies of chromosome 21, corresponding trisomy cells contain 3 copies of chromosome 21.
  • the obtained values were compared with a frequency table in which the above-mentioned PCR was repeatedly performed under the same conditions with two different reference samples, once a healthy cell having two copies of chromosome 21 and one trisomy 21 cell, and the number of each determination Ampliflkations occur and was prepared in the form of a frequency table.

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Abstract

La présente invention concerne un procédé de caractérisation d'un échantillon mixte contenant au moins deux particules d'acides nucléiques d'individus différents, chaque particule d'acides nucléiques comprenant un ou plusieurs individus, notamment un procédé de détermination quantitative du nombre absolu et/ou relatif de copies d'une séquence donnée d'un individu dont l'acide nucléique est contenu dans l'échantillon mixte, ledit procédé comprenant les étapes suivantes : a) isoler la particule et disposer au moins deux particules isolées sur un substrat, chacune desdites au moins particules étant déposée isolément sur un emplacement de réaction hydrophile entouré d'une zone hydrophobe du substrat dans un volume inférieur à 10 µl, de manière à ce qu'exactement une particule soit présente par emplacement de réaction, b) examiner au moins deux des particules déposées sur le substrat sur l'emplacement de réaction du substrat afin de classifier les individus de la particule de l'échantillon mixte par génotypage, au moins 80 % de la particule examinée étant attribuée à un individu, et c) caractériser de manière supplémentaire la particule examinée. La présente invention concerne également un kit convenant notamment à la réalisation dudit procédé.
EP07711550A 2006-03-27 2007-02-15 Procede de caracterisation d'acides nucleiques dans un echantillon mixte Withdrawn EP2004848A1 (fr)

Applications Claiming Priority (2)

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DE102006014000A DE102006014000B4 (de) 2006-03-27 2006-03-27 Verfahren zur Charakterisierung einer Mischprobe
PCT/EP2007/001328 WO2007110124A1 (fr) 2006-03-27 2007-02-15 Procede de caracterisation d'acides nucleiques dans un echantillon mixte

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EP2004848A1 true EP2004848A1 (fr) 2008-12-24

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US (1) US20100184024A1 (fr)
EP (1) EP2004848A1 (fr)
JP (1) JP2009531037A (fr)
DE (1) DE102006014000B4 (fr)
WO (1) WO2007110124A1 (fr)

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JP5686954B2 (ja) * 2009-01-30 2015-03-18 セイコーエプソン株式会社 デバイス

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JP2009531037A (ja) 2009-09-03
DE102006014000A1 (de) 2007-10-25
US20100184024A1 (en) 2010-07-22
WO2007110124A1 (fr) 2007-10-04
DE102006014000B4 (de) 2009-08-06

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