EP2315854A1 - Procédé pour déterminer quantitativement le nombre de copies d une séquence prédéterminée dans un échantillon - Google Patents

Procédé pour déterminer quantitativement le nombre de copies d une séquence prédéterminée dans un échantillon

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Publication number
EP2315854A1
EP2315854A1 EP09733308A EP09733308A EP2315854A1 EP 2315854 A1 EP2315854 A1 EP 2315854A1 EP 09733308 A EP09733308 A EP 09733308A EP 09733308 A EP09733308 A EP 09733308A EP 2315854 A1 EP2315854 A1 EP 2315854A1
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EP
European Patent Office
Prior art keywords
subsets
primer pairs
sample
predetermined sequence
biological sample
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.)
Withdrawn
Application number
EP09733308A
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German (de)
English (en)
Inventor
Wolfgang Mann
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Beckman Coulter Inc
Original Assignee
Beckman Coulter Inc
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Filing date
Publication date
Application filed by Beckman Coulter Inc filed Critical Beckman Coulter Inc
Publication of EP2315854A1 publication Critical patent/EP2315854A1/fr
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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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification

Definitions

  • the present invention relates to a method for the quantitative determination of the number of at least one predetermined sequence in a biological sample, preferably in a single cell, and more particularly to a method for determining the absolute copy number of alleles per cell.
  • trisomy 18 Eras syndrome
  • trisomy 13 Patau syndrome
  • trisomy 21 Down's syndrome
  • 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 copy number of the affected chromosome to severe developmental disorders. While carriers of trisomy 21 are drastically inhibited in their development and sometimes have severe malformations, carriers of trisomy 18 and trisomy 13 usually die within the first year of life.
  • Huntington's disease a progressive neurodegenerative disease characterized by abnormal, involuntary movements with increasing decay of mental and physical abilities, is said to be the cascading of more than 37 copies of a particular subject (CAG), with the predisposition to disease education increases with the number of repetitions of this motif in the genome.
  • CAG a particular subject
  • Further examples of unstable trinucleotide sequences in humans are Kennedy syndrome and spinocerebral ataxia-1.
  • FISH fluorescence in situ hybridization
  • any fluorescence signal present indicates the presence of the sequence corresponding to the probe provided with the corresponding fluorescent label.
  • the intensity of the fluorescence may allow a limited inference to the number of sequence copies in the biological sample. If, on the other hand, no signal or only a signal lying below a defined threshold is obtained at the wavelength of one of the fluorescence-labeled probes used, it is possible to deduce the absence of the sequence corresponding to the corresponding probe in the biological sample.
  • Another fluorescence-based method is the CGH analysis (com- parative genomic hyb ⁇ dizatio ⁇ ).
  • CGH analysis CGH analysis
  • the nucleic acid of the sample to be analyzed is completely labeled with a dye 1.
  • the same amount of nucleic acid of a reference sample is with a dye 2 marked.
  • Both reaction mixtures are hybridized together on a sprouted metaphase chromosome set, wherein the sequences contained in both reaction mixtures compete for the binding sites on the spread chromosomes. Essentially, a ratio of dye 1 to dye 2 of 1: 1 will be established at all hybridization sites. If the sample to be analyzed contains amplified regions (more than the usual copy number of the reference), then dye 1 will predominate at this hybridization site.
  • a special variant is the array CGH, which hybridizes not to chromosomes but to immobilized sequences whose physical address is known in the genome.
  • Another known method for quantifying nucleic acid sequences is the real-time PCR method, in which a PCR (polymerase chain reaction or polymerase chain reaction) is performed with fluorescently labeled primers and the increase of the fluorescence signal is observed as a function of the number of cycles.
  • the threshold PCR cycle (also known as the threshold cycle) is assigned to the reaction time at which the fluorescence signal stands out significantly from the background fluorescence and the PCR product formation proceeds exponentially. This correlates with the initial copy number of the DNA sequence to be amplified. In this way, DNA samples can be analyzed according to the same with a DNA dilution series relatively quantify.
  • a disadvantage of this method is that the amount of starting material can not be arbitrarily reduced because with a few starting molecules, for example 10 to 100 copies, as starting material, the stochastic error due to the exponential amplification is very large, which no longer quantitative statements allows. Furthermore, this method also requires expensive and expensive equipment for measuring the fluorescence intensity.
  • a more recent method for the quantitative determination of a nucleic acid sequence is the QF-PCR (quantitative fluorescence PCR), in which several PCRs are carried out in parallel using fluorescently labeled primers in a PCR approach and the fluorescently labeled PCR products are subsequently subjected to an automatic DNA PCR. Scanner laserdensito be metrically analyzed. In order to make a meaningful quantitative comparison between two side-by-side amplified PCR products, the two partial PCR reactions must proceed with equal efficiency and the fluorescence intensities of the reaction products at the time of exponential product amplification must be quantitatively analyzed. Other methods based on optically active-labeled probes, for example those using infrared-labeled probes, do not solve the problem either.
  • a method based on QF-PCR methodology for the detection of possible numerical aberrations of chromosomes 21, 18, 13, X and Y in amniotic fluid samples is described by Lucchini et al. in Scientific Information, September 2004.
  • This method is based on the in vitro PCR amplification of repetitive and polymorphic STR (short tandem repeats) sequences with fluorescently labeled primers. After completion of the PCR, the amplified PCR products using Capillary electrophoresis quantified. If chromosome-specific STR systems are used in these methods, it is possible to draw conclusions about the copy number of the corresponding chromosome from the number of different PCR products obtained.
  • this method does not allow any statement about the presence or absence of a trisomy, since this result is obtained both in the case of a monoallelic trisomy and in the case of a monoallelic disomy.
  • a method based on this technology for the detection of trisomy 13 is also disclosed in DE 101 02 687 A1.
  • this method also has the disadvantage that fluorescently labeled primers must be used.
  • the frequency distribution is determined by separately carrying out the same and under the same reaction conditions as that used for the biological sample to be examined at least one amplification reaction with at least two different reference samples, each having a known, mutually different copy number of the predetermined sequence, and then determining the number of different amplification products obtained per reference sample.
  • the object of the present invention is to provide a method for the quantitative determination of the number of one or more predetermined sequences in a biological sample, which is simple and inexpensive feasible, which also and especially with a small number of predetermined sequences present in the biological sample to be examined provides reliable results and which is feasible in particular with small amounts of starting material.
  • this object is achieved by a method for the quantitative determination of the number of at least one predetermined sequence in a biological sample, in particular for determining the absolute copy number of alleles per cell, which comprises the following steps:
  • fragmentation is understood to mean the division of nucleic acid molecules into at least two nucleic acid molecules.
  • the nucleic acid fragments resulting from the fragmentation of each nucleic acid molecule therefore have a shorter molecule length than the starting molecules.
  • predetermined sequence in the context of the present invention is understood to mean any sequence which is encompassed by the nucleic acid contained in the biological sample.
  • the predetermined sequence may be a chromosome, a chromatid, a gene or a gene segment.
  • the nucleic acid contained in the sample provided in step a) is lysed between process step a) and process step b). This can be accomplished, for example, by adding lysozyme to the sample, heating the sample, or adding denaturing agents, such as urea, dithiothreitol (DTT), and the like, to the sample.
  • denaturing agents such as urea, dithiothreitol (DTT), and the like
  • the nucleic acid molecules contained in the biological sample are preferably used in fragmentation. fragmented into at least five nucleic acid fragments, wherein the fragmentation can be carried out for example by restriction hydrolysis, by shearing, by ultrasound or by DNase digestion.
  • the process parameters in the fragmentation for example the type of restriction enzyme used or the frequency and duration of the ultrasound, can be adjusted so that fragments having a desired length are obtained.
  • the absolute fluorescence intensity of PCR products is not determined and, as in the case of FISH and CGH, with the Fluorescence intensity of a control or reference sample compared, but it is only determined with how many subsets each of the same amplification products have been obtained.
  • fluorescence-marked primers do not have to be used in the method according to the invention.
  • the fluorescence intensity of the fluorescence-labeled PCR products obtained does not have to be determined in a complex manner, but only evaluated if a fluorescence, if present above a defined threshold, is present in one of the fluorescent dyes used corresponding wavelength is present or not. Therefore, the inventive method is simple and inexpensive to perform without costly equipment for the quantitative detection of fluorescence.
  • a further advantage of the method according to the invention is its quick and easy implementation, because in comparison with the quantification method known from DE 10 2005 045 560 A1, only a comparatively small number of amplification reactions have to be carried out.
  • the method according to the invention is suitable both for the determination the relative number of at least one predetermined sequence in a biological sample as well as for the determination of the absolute number of at least one predetermined sequence in a biological sample.
  • the method according to the invention can be used to determine a trisomy in the context of in vitro fertilization (IVF) or in the context of the analysis of fetal cells from maternal blood.
  • the method according to the invention is also particularly suitable for the quantitative determination of the number of at least one predetermined sequence in a biological sample which contains only a small amount of nucleic acid, for example only a few cells.
  • the method according to the invention is carried out with a biological sample which contains as starting material only one cell, for example a human cell, such as a polar body, an animal cell or a plant cell, or with a suspended single cell.
  • the basic principle of the method according to the invention is based on the determination of the copy number of a predetermined nucleic acid sequence, such as the copy number of a chromosome, by fragmentation of the nucleic acid, dividing the fragmented sample into multiple subsets, performing a multiplex PCR with each of the subsets, wherein in The same primer pairs are used for each subset of multiplex PCR reactions before it is subsequently determined with which number of subsets the same in each case
  • a cell may contain zero, one or two copies of a predetermined sequence, for example, chromosome 21.
  • chromosome 21 When the cell is lysed in the cell suspension and the sample thus divided is divided into two subsets, in the case of monosomy, ie when the cell contains a copy of chromosome 21, chromosome 21 will be distributed to one of the two subsets. Thus, in this case, only one of the two subsets contains chromosome 21.
  • both subsets may each contain one copy of chromosome 21 or one of the subset may not contain a chromosome 21 and the other subset two
  • sequence to be determined ie chromosome 21
  • sequence to be determined is divided into 10 different partial sequences by fragmentation into several partial sequences, for example by restriction hydrolysis, the probability that in the case of disomy at least one pair of identical partial sequences is distributed over both partial amounts is nearly 100 %, whereas in the case of monosomy, each subsequence can be present in at most one of the two subsets.
  • nullisomy naturally, no partial sequence of chromosome 21 will be found in either of the two subsets.
  • each subsequence can be detected only in one of the two subsets, whereas in the case of disomy at least some of the subsequences of the chromosome 21 are present in both subsets and in the case Nullisomie in any of the subsets a partial sequence of chromosome 21 is present.
  • FIGS. 1 and 2 The above thought experiment is shown schematically in FIGS. 1 and 2 for the cases of monosomy and disomy.
  • FIG. 1 in the method according to the invention, a single cell symbolized in the top left by a circle in FIG. 1 is used, which contains a copy of the sequence to be quantified, in this case chromosome 21, which symbolizes the gray bar contained in the circle becomes.
  • This single cell is lysed in suspension (not shown) before the suspension is added a restriction enzyme, the chromosome 21 contained in the sample, as shown above in the middle and top right in Figure 1, in five defined partial sequences with each more distinctive Length fragmented.
  • the sample containing these five partial sequences as shown in the bottom left and in the middle of FIG. 1, is divided into two partial samples, each of the five partial sequences being distributed to one of the two partial quantities with a probability of 50% each.
  • the two short subsequences are contained in subset 1, while the three longer subsequences are contained in subset 1 Subset 2 are present.
  • the two subsets are then each 5 equal primer pairs added, of which each of the five primer pairs for each one of the previously generated by the fragmentation partial sequences of chromosome 21 is specific.
  • the 5 primer pairs are designed in such a way that each of the primer pairs produces a PCR product of different lengths in the subsequent PCR.
  • FIG. 2 the same procedure is schematically reproduced for the case of a biological sample containing two chromosomes 21 (disomy).
  • two copies of the five partial sequences of chromosome 21 are obtained in the fragmentation.
  • Each of these subsequences will have a 50% probability of being split into one of the two subsets so that the probability that the two copies of a subsequence are in a subset is 50% and the likelihood that each one of the subsequences will be 50% two copies of a subsequence are in one subset, whereas the other copy of the same subsequence is in the other subset, also 50%.
  • the probability that at least for one of the five different subsequences the two copies of the subsequence are distributed over two different subsets is therefore more than 95%.
  • one copy each is contained in subset 1 and one copy in subset 2
  • the two copies of the fifth subsequence are contained exclusively in subset 2.
  • the method steps a) to c) of the method according to the invention can be carried out under non-denaturing conditions, so that the predetermined sequence, if this is double-stranded DNA, is distributed in the form of double-stranded nucleic acid to the individual subsets.
  • To transfer nucleic acid to single-stranded nucleic acid because single-stranded nucleic acid as well as double-stranded nucleic acid can lead to a positive PCR result.
  • This denaturation can be carried out, for example, before or after the fragmentation according to process step b).
  • method step c there are twice as many copies of the fragmented predetermined sequence to be distributed to the partial quantities as in the biological starting sample, namely, in the case of disomy, for example, four single-stranded copies of the fragmented predetermined sequence, whereas in biological Original sample two double-stranded copies of the unfragmented predetermined sequence templates.
  • the method according to the invention is not limited, so that in principle both specific and unspecific primer pairs can be used.
  • the use of specific primer pairs is preferred because it allows the length of the PCR products to be tailored and ensures that only the genetic subsequences are amplified.
  • the at least two primer pairs added in method step d) is proposed to adapt the at least two primer pairs added in method step d) to amplify different non-overlapping partial sequences of the at least one predetermined sequence in an amplification reaction.
  • the restriction enzyme or the restriction enzymes are preferably selected so that they do not intersect in the sequence regions to be amplified. This can be ensured, for example, by first relatively rarely intersecting restriction enzymes being selected in the nucleic acid and then, depending on the specific interfaces, selecting the primers such that the restriction enzyme sites are not in the regions encompassed by the primer binding sites ,
  • An essential process step of the method according to the invention is that after performing the amplification reactions with the individual subsets, it is determined with how many subsets identical amplification products have been obtained in order to determine therefrom the number of predetermined sequences in the biological sample.
  • This determination with how many subsets identical amplification products have been obtained, naturally requires that the different amplification products are distinguished from one another, ie the number of different amplification products obtained per subset is determined.
  • electrophoresis for example by gel electrophoresis, such as polyacrylamide gel electrophoresis or agarose gel electrophoresis, or by capillary electrophoresis.
  • a fluorometric determination of the amplification products is possible.
  • step gi further to perform a method step gi), which comparing the in step f) for each subset determined amplification products with at least amplification products obtained from a control sample in an amplification reaction, wherein the amplification reaction carried out with the at least one control sample is carried out with the same primer pairs as in step d) and the control sample preferably contains a known number of the predetermined sequence.
  • fikationsretress such as a temporary failure of the thermocycler during the amplification reaction
  • the nullisomial case in which no bands are expected for the amplification reactions performed with the fragmented aliquots of the sample can be validated because in the event the expected bands for the control sample are obtained, an error in performing the amplification reaction is excluded can be.
  • the amplification reaction with the at least one control sample is preferably carried out in parallel with step e).
  • the method according to the invention may also comprise the additional method step g2), which comprises comparing the amplification products determined in step f) with a data set for each subset, the data record containing information relating to at least one Control sample in an amplification reaction using the different amplification products available in the primer pairs added in step d).
  • the statistical reliability of the result obtained with the method according to the invention is greater, the more primer pairs are used in the amplification reaction, preferably a multiplex PCR.
  • the experimental effort increases as the number of primer pairs used increases.
  • the statistical reliability of the result obtained with the method according to the invention is greater, in particular for larger copy numbers, the more subsets the fragmented sample is divided in method step c), whereas the experimental effort increases with increasing number of subsets prepared in step c).
  • the sample obtained in process step b) is subdivided in process step c) such that the produced at least two subsets have the same volume. Apart from the fact that this facilitates the implementation of method step c), this also facilitates later calculations of the result.
  • each of the reaction sites on the substrate has a central hydrophilic area which is surrounded on the outside by a first hydrophobic region, which in turn outside of a central hydrophilic Surrounded area, which is surrounded on the outside by a second hydrophobic area.
  • the central hydrophilic region is at least essentially circular and is surrounded on the outside by an at least substantially annular annular first hydrophobic region, which in turn is externally surrounded by an at least substantially circular ring-shaped central hydrophilic region is surrounded concentrically, which is surrounded on the outside by the second hydrophobic region.
  • the subsets of the fragmented sample must have the necessary DNA polymerase and optionally at least one compound selected from the group consisting of pH buffer substances, salts, water and further conventional PCR additives to set conditions suitable for the performance of the PCR.
  • the copy number of a predetermined sequence contained in the biological sample or preferably the copy number of a plurality of predetermined sequences contained in the biological sample can be determined.
  • the predetermined sequence (s) are chromosomes, such that the method according to the invention determines, in particular, the copy number of 1 to 23, more preferably 1 to 10 and most preferably 1 to 5, chromosomes present in the biological samples may.
  • the above-described method according to the invention for the quantitative determination of the number n of x predetermined sequence (s) in a biological sample comprises the following method steps:
  • Subsets f) determining the number of different amplification products obtained with the PCR reactions in step e) for the individual subsets and determining with how many of the z subsets identical amplification products have been obtained.
  • x is an integer between 1 and 23
  • y is an integer greater than or equal to 4, preferably from 5 to 20
  • z is an integer which is greater than or equal to 3 • x, preferably 5 • x to 25 ⁇ x and particularly preferably 8 • x to 15 • x.
  • the number n of the predetermined sequence (s) can be determined.
  • the number n of the predetermined sequence (s) can be determined.
  • the copy number is 0 (zero isomie) if no amplification product has ever been obtained with any of the subsets. If, on the other hand, at least two subsets of identical amplification products are obtained, the copy number is at least two and so on.
  • the number of primer pairs used in method step e) is selected so that the probability of a false negative result is a maximum of 5%. This can be ensured, for example, by the following statistical approach:
  • the probability of correctly determining the copy number depends on all of the following parameters:
  • An element may not be selected more than once (ie in the initial distribution of the sequence copies only a maximum of one sequence is assigned to a partial reaction). If one then sets the "desired" events (2) in relation to all possible events (1) one obtains the proportion of "desired” events and thus the probability of success of a correct statement of the method using the corresponding parameters.
  • a possible evaluation variant of the amplifications obtained would be, for example, the detection by means of fluorescent dyes, wherein in each case one fluorescent dye is selected for each fragment of different size. Since it is only interesting here whether products were amplified, but the amount of product is not of interest, it would be so easy to measure the fluorescence of the individual wavelengths with an end point determination without having to use a gel.
  • the inventive method is particularly suitable for the quantitative determination of the number n of at least one predetermined sequence in a biological sample, if the number n per predetermined sequence in the biological sample 0 to 100, preferably 0 to 10, particularly preferably 0 to 5 and especially preferably 0, 1, 2, 3 or 4.
  • the method according to the invention can advantageously be carried out in the context of a fitro-fertilization (IVF) or in the context of the analysis of fetal cells from maternal blood.
  • IVF fitro-fertilization
  • the method according to the invention was carried out separately with two biological samples in the form of a suspension containing each cell in order to determine the copy number of the chromosome 21 present in the two samples. While the first sample contained two cells, the second sample contained only one cell.
  • the two samples were lysed and with a mixture of restriction enzymes Xho I, Sac I and Pac at 37 0 C for 5 minutes hydrolyzed (Cek kit Advalytix Products or Olympus Life Science Research Europa GmbH). Subsequently, the obtained fragmented samples were each divided into four aliquots of equal volumes, after which each of the subsamples received 7 primer pairs was added. All seven primer pairs were for different subsequences or subsequences of chromosome 21, with all seven primer pairs selected so that each primer pair gives a different length PCR product. These samples were pipetted onto one reaction site each of a glass substrate.
  • FIG. 3 A photograph of the polyacrylamide gel is shown in FIG.
  • the numbers 1 to 4 designate the tracks for the individual subsets of the PCR reactions for the two samples used, while a length marker was applied in the left unmarked track.
  • the result is summarized in the two tables reproduced to the right of the photograph in FIG.
  • the first Line of the tables represents the individual tracks of the gel, that is, the subsets 1 to 4 corresponding tracks 1 to 4 for the first sample and the subsets 1 to 4 corresponding tracks 1 to 4 for the second sample.
  • the number of bands for the seven possible PCR products which have been obtained for each individual subset ie "O" or "1" is indicated in each column.
  • the first sample contained at least three copies of chromosome 21, whereas the second sample contained at least two copies of chromosome 21.

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Abstract

L’invention se rapporte à un procédé destiné à déterminer quantitativement le nombre d’au moins une séquence prédéterminée dans un échantillon biologique et comprenant les étapes consistant a) à fournir un échantillon biologique contenant un acide nucléique, b) à fragmenter l’acide nucléique contenu dans l’échantillon biologique, c) à diviser l’échantillon obtenu à l’étape b) en au moins deux parties, d) à ajouter respectivement au moins deux paires d’amorces à chacune des deux parties ou plus, ces paires d’amorces étant respectivement ajoutées à chacune des parties et les paires d’amorces individuelles étant adaptées pour amplifier respectivement différentes séquences partielles d’au moins une séquence prédéterminée au cours d’une réaction d’amplification, e) à effectuer une réaction d’amplification avec chacune des deux parties ou plus obtenues à l’étape d), f) à déterminer le nombre des différents produits d’amplification obtenus pour les parties individuelles à l’étape e) avec les réactions d’amplification et à déterminer avec combien de parties on a obtenu respectivement des produits d’amplification identiques.
EP09733308A 2008-04-16 2009-04-15 Procédé pour déterminer quantitativement le nombre de copies d une séquence prédéterminée dans un échantillon Withdrawn EP2315854A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102008019132A DE102008019132A1 (de) 2008-04-16 2008-04-16 Verfahren zur quantitativen Bestimmung der Kopienzahl einer vorbestimmten Sequenz in einer Probe
PCT/EP2009/002759 WO2009127408A1 (fr) 2008-04-16 2009-04-15 Procédé pour déterminer quantitativement le nombre de copies d’une séquence prédéterminée dans un échantillon

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EP2315854A1 true EP2315854A1 (fr) 2011-05-04

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EP (1) EP2315854A1 (fr)
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CN104531837A (zh) * 2008-12-22 2015-04-22 赛卢拉有限公司 检测等位基因、基因组和转录物组的方法和基因型分析谱
GB201021499D0 (en) * 2010-12-20 2011-02-02 Loxbridge Res Llp Detection of quantative genetic differnces
CN106033087B (zh) * 2015-03-18 2018-05-18 王峥 内置性标准曲线检测物质分子数之方法系统
DE102015111329B4 (de) * 2015-07-13 2017-02-02 Bernd-Peter Ernst Verfahren zum Bestimmen einer relativen Häufigkeit von verschiedenen Genen oder Chromosomen eines Genoms in einer Probe

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DE10102687A1 (de) 2001-01-22 2002-08-01 Adnagen Ag Trisomie 13-Diagnostik-Kit
DE102004036285A1 (de) * 2004-07-27 2006-02-16 Advalytix Ag Verfahren zum Bestimmen der Häufigkeit von Sequenzen einer Probe
DE102005045560B4 (de) 2005-09-23 2009-02-12 Advalytix Ag Verfahren zur quantitativen Bestimmung der Kopienzahl einer vorbestimmten Sequenz in einer Zelle
DE102005051816A1 (de) * 2005-10-28 2007-05-10 Advalytix Ag Verfahren zur relativen Bestimmung der Kopienzahl einer vorbestimmten Sequenz in einer biologischen Probe
DE102005059227A1 (de) * 2005-12-12 2007-06-14 Advalytix Ag Verfahren zur Bestimmung des Genotyps aus einer biologischen Probe enthaltend Nukleinsäuren unterschiedlicher Individuen
PL3002338T3 (pl) * 2006-02-02 2019-12-31 The Board Of Trustees Of The Leland Stanford Junior University Nieinwazyjne badania przesiewowe płodu poprzez analizę cyfrową
DE102006014000B4 (de) * 2006-03-27 2009-08-06 Advalytix Ag Verfahren zur Charakterisierung einer Mischprobe
EP3770275A1 (fr) * 2007-07-23 2021-01-27 The Chinese University of Hong Kong Détermination d'une aneuploïdie fétale
CA2698545C (fr) * 2007-09-07 2014-07-08 Fluidigm Corporation Determination de la variation du nombre de copies, procedes et systemes

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