EP2315854A1

EP2315854A1 - Process for the quantitative determination of the copier number of a predetermined sequence in a sample

Info

Publication number: EP2315854A1
Application number: EP09733308A
Authority: EP
Inventors: Wolfgang Mann
Original assignee: Beckman Coulter Inc
Current assignee: Beckman Coulter Inc
Priority date: 2008-04-16
Filing date: 2009-04-15
Publication date: 2011-05-04
Also published as: US20110262923A1; WO2009127408A1; DE102008019132A1

Abstract

A process for the quantitative determination of the number of at least one predetermined sequence in a biological sample comprises the steps: a) providing a biological sample which contains a nucleic acid, b) fragmenting the nucleic acid present in the biological sample, c) dividing the sample obtained in step b) into at least two subsamples, d) adding in each case at least two primer pairs to each of the at least two subsamples, where in each case the same primer pairs are added to each of the subsamples and where the individual primer pairs are adapted to amplify, in one amplification reaction, in each case different part-sequences of the at least one predetermined sequence, e) carrying out an amplification reaction with each of the at least two subsamples obtained in step d), f) determining the number of different amplificates obtained by the amplification reactions in step e) for the individual subsamples, and determining the number of subsamples with which in each case identical amplificates have been obtained.

Description

A method for quantitatively determining the copy number of a predetermined sequence in a sample

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.

In molecular diagnostics, methods for quantifying sequences, in particular for quantifying the copy number of nucleic acid sequences per cell, are gaining an increasingly important role. Since a large number of serious illnesses are caused by deviations from the normal copy number of nucleic acid sequences in the genome, by determining the copy number of specific chromosomes or certain gene segments, corresponding diseases can be reliably diagnosed at an early stage of development.

Examples of sometimes severe anomalies, which are due to an increased copy number of whole chromosomes, are trisomy 18 (Edward's syndrome), trisomy 13 (Patau syndrome) and trisomy 21 (Down's syndrome). In each of these diseases, 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. In all three cases, 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.

In addition to diseases which are attributable to an increased copy number of whole chromosomes, a large number of diseases are known which are based on an altered copy number of genes or gene segments.

The cause of 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. Further examples of unstable trinucleotide sequences in humans are Kennedy syndrome and spinocerebral ataxia-1.

In addition, it is known that certain proto-oncogenes can multiply by gene amplification in the genome. Such amplifications are often recognized in the chromosome set as so-called "double minutes" (DM) or as "homogeneously staining regions" (HSR). Due to the enormous increase in gene copy number, the associated protein can be produced in the cells in very large amounts, which allows an increased activation of cell proliferation - without changing the individual gene itself. In particular, the myc proto-oncogene is said to be particularly often affected by the amplification. Due to the need for methods for quantifying sequence copies in a biological sample, a variety of methods have been proposed in the past.

One of the basic quantification methods, which allows at least a statement about the presence or absence of nucleic acid sequences and depending on the procedure also a conditional conclusion on the copy number of the respective nucleic acid sequences per cell, is the so-called FISH method (fluorescence in situ hybridization ). In this method, the biological sample to be tested, after appropriate pretreatment, i. Denaturing with formamide and prehybridization, with one or more different probes, which were previously labeled with different fluorescent color substances, respectively, incubated under conditions which a

Allow hybridization of the probes with homologous sequences in the biological sample. After hybridization, the samples are washed, eliminating nonspecific hybridization signals. Finally, the fluorescence signals of the preparation are evaluated with a fluorescence microscope. 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. However, the absence of a corresponding fluorescence signal can also be due to the fact that in the corresponding tion site of the sequence to be detected, a mutation and / or microdeletion has taken place, which is why the probe no longer binds to the predetermined sequence under the selected hybridization conditions. A further disadvantage of the aforementioned method is that unwanted cross-hybridization leading to incorrect results can never be completely ruled out. In addition, this method is relatively expensive, on the one hand because it is imperative to use fluorescent dyes and, on the other hand, because it requires expensive equipment, such as fluorescence microscopes. Finally, the significance of this method depends to a very considerable degree on the quality of the probes used; reliable results are only obtained if the probes hybridize with an effectiveness of more than 90% to the corresponding binding sites, so that only 10% of the target sequences are unhybridized and therefore no longer be amplified. It follows that a wrong choice of probes, but also inadequate hybridization conditions lead to a false result. Another disadvantage of this method is that a minimum amount of biological sample must be used in order to obtain an evaluable fluorescence signal at all. In addition, the sequence must not fall below a minimum length. Furthermore, for a valid result, it is necessary to analyze a variety of cells that were susceptible to hybridization. For this reason, FISH analysis is not adequate for single cell diagnostics. In addition, an automated evaluation by the pathologist is hardly possible.

Another fluorescence-based method is the CGH analysis (com- parative genomic hybήdizatioή). In this method, 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. In the case of a deletion in the sample to be examined, only dye 2 will be detected at this hybridization site. The reference measurement allows a relative statement about the frequency of sequences in the sample to be analyzed. However, even this method, since absolute fluorescence intensities must be measured, consuming and expensive. In addition, this also requires the use of a certain, relatively high starting amount.

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, however, 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. If, for example, three peaks are obtained in the reaction with a chromosome-specific STR system in capillary electrophoresis, the peak heights being 1: 1: 1, the individual examined contains three different alleles of the corresponding chromosome (triallelic trisomy). If, on the other hand, two peaks are obtained in the method, the ratio between the peaks being 2: 1, then the individual examined has two identical alleles of the chromosome per cell and another allele of the chromosome (diallelic trisomy). In the case that only two peaks with identical peak height are obtained, the individual has two alleles, so that there is no trisomy (heterozygous case). However, in the case where only one peak is 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. In order to be able to differentiate between a monoallelic disomy and a monoallelic trisomy, it is proposed in this method to amplify three different chromosome 13-specific STR DNA regions by PCR. However, this method also has the disadvantage that fluorescently labeled primers must be used. In addition, it requires the use of a minimum amount of DNA, otherwise the stochastic error due to the exponential amplification is very large and no quantitative statement for the diallelic trisomy is possible. Another disadvantage of the aforementioned method lies in the fact that this only in a narrow PCR window with some Reliability works because only in this window the peak heights are proportional to the ratio of the starting material. Furthermore, this method also has the disadvantage that the absolute fluorescence intensity must be determined.

All of the above methods are based on the use of fluorescently labeled primers and require expensive equipment for determining the fluorescence intensity. In addition, these require the use of a minimum amount of starting material in order to obtain at least reasonably reliable results.

It has been proposed in DE 10 2005 045 560 A1, for the quantitative determination of the number of a predetermined sequence and possibly of sequences which are homologous to the predetermined sequence, in a biological sample with a defined amount of a biological sample, at least one amplification reaction which occurs thereon is adapted to amplify a plurality of mutually non-homologous sequences, which are encompassed by the predetermined sequence to perform, before the number of different amplification products obtained in the at least one amplification reaction is determined and the number thus determined is compared with at least one frequency distribution. In this case, 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. This procedure provides good results However, it requires a large number of PCR reactions to be carried out.

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.

According to the invention, 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:

a) providing a biological sample containing nucleic acid, b) fragmenting the nucleic acid contained in the biological sample, c) dividing the sample obtained in step b) into at least two subsets, d) adding at least two primer pairs to each of the at least two subsets in which the same primer pairs are added to each of the subsets, and wherein the individual primer pairs are adapted to amplify in each case different subsequences of the at least one predetermined sequence in an amplification reaction, e) carrying out an amplification reaction with each of the at least two subsets obtained in step d), f) determining the number of different amplification products obtained for the individual subsets with the amplification reactions in step e) and determining with each of them the same number of subsets Amplification products have been obtained.

For the purposes of the present invention, the term 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.

Furthermore, the term 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. In particular, the predetermined sequence may be a chromosome, a chromatid, a gene or a gene segment.

According to a preferred embodiment of the present invention, 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.

In the method according to the invention, 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.

In contrast to the methods according to the prior art, in the method according to the invention, such as in quantitative PCR, QF-PCR, FISH and CGH, 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. In this respect, fluorescence-marked primers do not have to be used in the method according to the invention. If these are nevertheless used for detecting the number of different PCR products obtained, 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. For example, 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. Preferably, 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

Amplification products have been obtained. From this comparison, the copy number of the predetermined sequence can then be determined with the required security. This is explained in the following thought experiment. A cell may contain zero, one or two copies of a predetermined sequence, for example, 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. On the other hand, in the case of disomy, ie if the cell contains two chromosomes 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

Copies of chromosome 21 included. If the sample does not contain a copy of chromosome 21, it will naturally not be in either of the two subsets.

If the sequence to be determined, ie chromosome 21, 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. In the case of nullisomy, naturally, no partial sequence of chromosome 21 will be found in either of the two subsets. If one then carries out the method steps d) to f) of the method according to the invention, in the case of monosomy 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. Thus, dividing the fragmented sample into two subsets and then Steps d) to f), if the process is carried out under non-denaturing conditions during the process steps a) to c), ie the DNA is double-stranded during these process steps, with a sufficiently large number of primer pairs added during process step d) are decided whether the sample contains zero, one or at least two copies of chromosome 21. By increasing the number of subsets into which the fragmented sample is subdivided in method step c), the cases of nullisomy, monosomy, disomy, trisomy, etc. can also be distinguished from one another, even in the case where the DNA is present before the Division into the subsets is denatured, that is converted into single strands.

The above thought experiment is shown schematically in FIGS. 1 and 2 for the cases of monosomy and disomy.

In 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. Subsequently, 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. In the case illustrated in FIG. 1, 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. In the case illustrated in FIG. 1, 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. Then with the two subsets as well as with a control sample containing a chromosome 21, in each case a PCR is carried out under the same conditions and then a subset of all three PCR approaches is applied to a polyacrylamide gel and separated by electrophoresis. As can be seen from the gel shown schematically in FIG. 1, bottom right, 5 different bands are obtained in the control sample (K), only 2 being obtained for subset 1 (1) and only 3 bands for subset 2 (2) where none of the bands obtained for subset 1 have the same length as the bands obtained for subset 2. From this it can be concluded with high probability that exactly one copy of chromosome 21 was contained in the sample. By increasing the number of primer pairs used in the PCR reactions from 5 to 10, for example, the statistical reliability of the result obtained can be correspondingly increased.

In FIG. 2, the same procedure is schematically reproduced for the case of a biological sample containing two chromosomes 21 (disomy). In this case, 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%. In the case illustrated in FIG. 2, of the two copies of four of the subsequences, one copy each is contained in subset 1 and one copy in subset 2, whereas the two copies of the fifth subsequence are contained exclusively in subset 2. Accordingly, after carrying out the PCR reactions, in the gel electrophoretic analysis, four bands are obtained for the subset 1 (1), whereas five bands are obtained for the subset 2 (2) and the control (K). Thus, subsets 1 and 2 each have four identical PCR products, indicating that the sample contained at least two chromosomes 21. However, the cases of monosomy and nullisomy can be excluded due to the result. By increasing the number of subsets into which the biological sample is divided in method step c), it would also be possible to determine the absolute copy number of chromosome 21 in the sample, ie to determine whether chromosome 21 in the sample has 2, 3 , 4, 5 etc. Copies.

In principle, 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. Alternatively, however, it is also possible to denature the nucleic acid in the biological sample prior to the division of the fragmented sample into at least two subsets in method step c) in order to detect any double-stranded nucleic acid present. 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). In this case, therefore, in 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. Even in the case where the predetermined sequence already exists in the starting sample in single-stranded form, for example as RNA, denaturation can take place before the carrying out of process step c), for example in order to break up secondary structures of the single-stranded nucleic acids prior to the enzymatic fragmentation.

With regard to the type of primer pairs used, the method according to the invention is not limited, so that in principle both specific and unspecific primer pairs can be used. However, 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.

In a further development of the inventive idea, it 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. According to another preferred embodiment of the present invention, it is provided to carry out the fragmentation in method step b) in such a way and / or to select the primer pairs added in method step d) such that the average length of the nucleic acid fragments obtained in method step b) is greater than the length of the partial sequences of the at least one predetermined sequence which can be amplified with the primer pairs added in step d).

If the fragmentation is carried out by restriction hydrolysis, 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. This is preferably done by electrophoresis, for example by gel electrophoresis, such as polyacrylamide gel electrophoresis or agarose gel electrophoresis, or by capillary electrophoresis. Alternatively, a fluorometric determination of the amplification products is possible.

In order to facilitate the workload in electrophoresis in this embodiment of the present invention, in particular when polyacrylamide gel electrophoresis or agarose gel electrophoresis is used, and in order to reduce the duration of the electrophoresis, it is proposed in development of the inventive idea that in the process step d ) are selected so that all of the different partial sequences amplifiable with the individual primer pairs differ in length by at least 10 base pairs, preferably by at least 25 base pairs, more preferably by at least 50 base pairs and most preferably by at least 100 base pairs. In this way, no special requirements are placed on the separation efficiency in gel electrophoresis.

In order to increase the reliability of the result of the method according to the invention, it is provided according to a further preferred embodiment of the present invention, 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. In this way, from the comparison of the band pattern obtained with the control sample with the band pattern expected for the control sample, any errors in the performance of the amplification can be determined. fikationsreaktion, such as a temporary failure of the thermocycler during the amplification reaction can be detected. For example, 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.

For this reason, the amplification reaction with the at least one control sample is preferably carried out in parallel with step e).

As an alternative to the aforementioned embodiment, instead of the method step gi), 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).

In principle, 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. However, the experimental effort increases as the number of primer pairs used increases. As a compromise between these two opposing tendencies, it is preferred in process step d) of each of the subsets, per predetermined sequence to be determined, in each case 2 to 50, preferably 5 to 25, particularly preferably 8 to 15, very particularly preferably 10 to 15 and most preferably 12 primer pairs added. For the number of subsets produced in method step c), too, 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). As a compromise between these two opposing tendencies, it is preferable to divide the fragmented sample in process step c) into at least 3 aliquots, preferably at least 4 aliquots and more preferably 5 to 20 aliquots. Preferably, 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.

To keep the apparatus and manual effort as low as possible, it is proposed in a further development of the inventive idea to perform the amplification reactions in step e) for all subsets in parallel on an existing example of glass substrate, the individual subsets or subsamples of these subsets on one Reaction point of the substrate are positioned. In order to ensure a firm adhesion of the individual partial quantities or partial samples on the substrate, in particular also after the overlaying with the subsequent oil drop, it is provided according to a further preferred embodiment of the present invention that 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.

In this embodiment, good results are obtained, in particular, when 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.

Of course, prior to carrying out the amplification reactions according to method step e), 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.

With the method according to the invention, 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. Preferably, 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. According to a further preferred embodiment of the present invention, 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:

a) providing a biological sample containing DNA, b) fragmenting the DNA contained in the biological sample, c) dividing the sample obtained in step b) into y subsets, d) adding each z primer pairs to each of the y subsets, each the subsets are each given the same primer pairs, and wherein the individual primer pairs are adapted to amplify different partial sequences of the x predetermined sequence (s) in a PCR, e) performing a PCR with each of the y obtained in step d)

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.

Preferably, x is an integer between 1 and 23, y is an integer greater than or equal to 4, preferably from 5 to 20, and 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.

As detailed in particular in the above thought experiment, from the number of how many of the z subsets identical amplification products have been obtained, the number n of the predetermined sequence (s) can be determined. For the simple case of four subsets (y = 4) and 8 primer pairs used (z = 8) means, for example, if no denaturation of the DNA was carried out in single-stranded DNA prior to process step c), the result that none of the subsets of the same amplification products were obtained but the individual amplification products each occur in only a subset that the copy number of the predetermined sequence in the biological sample 1 (monosomy) is. On the other hand, 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.

Preferably, 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:

By means of the method described below, for example, it should be possible to make a statement as to how many copies of a particular chromosome are present in a sample.

The probability of correctly determining the copy number depends on all of the following parameters:

- k = actual copies

- N = number of partial reactions

(Multiplex degree = number of different lengths studied

Bands per part reaction)

(Number of cells examined). The following statistical considerations clarify the determination of the probability of a correct copy number determination:

1) Determination of all possible combinations in the distribution of the existing sequence copies on the partial reactions:

Combinations with repetition Aerwünscht = (N + k - l)! / (N - l)! * k!

In principle, the following conditions apply:

a) All elements (= partial reaction, for example 1 to 8) differ from each other. b) Some elements are selected (a partial reaction with sequence copy is considered "selected"). c) One element can be selected multiple times (i.e., multiple sequence copies of a given partial response may be assigned in the initial distribution).

2) Determination of the possible combinations in the distribution of the existing sequence copies on the partial reactions, in which only one copy per partial reaction is present:

Combinations with repetition A total = N! / (N - k)! * k!

In principle, the following conditions apply:

a) and b) as above. c) 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.

Probability P (correct statement) = Aerwünscht / Agesamt = (N - 1)! * N! / (N + k - 1)! * (N - k)!

Example:

- k = 4

- N = 8

- degree of multiplexing = number of investigated bands of different lengths per partial reaction).

Probability for successful detection of all 4 existing sequence copies

= Aenvünscht / A total = ((8 - 1)! * 8!) / ((8 + 4 - 1)! * (8 - 4)!) = (7! * 8!) / (11! * 4!) = 0.2121.

The probability of making an incorrect statement is therefore 1 - 0.2121 = 0.7878. This applies if only one PCR product per subset is analyzed.

However, if one analyzes several PCR products per subset (multiplex PCR), one obtains the total probability by multiplying the individual probabilities. Eg for a 3-Plex: P (proof of all bands) = 0.7878 * 0.7878 * 0.7878. One comes to the following table, depending on the multiplex degree.

With a multiplex degree of 13, one thus achieves an error probability of less than or equal to 5%.

But because For the detection of chromosomes after their denaturation always 2n single strands are present, one can proceed as follows in the statistical statement.

P (successful detection of n chromosomes) = P (successful detection of exactly 2n chromatids) + P (successful detection of exactly (2n - 1) chromatids). Thus, one can conclude from the detection of 1 or 2 amplificates on the presence of one chromosome (= 2 chromatids), from the detection of 3 or 4 amplificates on the presence of 2 chromosomes (= 4 chromatids), from the detection of 5 or 6 amplicons indicate the presence of 3 chromosomes (= 6 chromatids), etc.

P (detection of exactly 2n chromatids) + P (detection of exactly (2n - 1) chromatids) = (N - 1)! * N! / (N + k - 1)! * (N - k)! + (N! / K _o ! * K ₁ ! * K ₂ ! * ... * k _n !) / ((N + k - 1)! / (N- l)! * K!) Where:

Ko = number of reaction vessels in which no sequence copy is present, K ₁ = number of reaction vessels in which exactly one sequence copy is present, K ₂ = number of reaction vessels in which exactly two sequence copies are present, etc.

In the case of the detection of 2n-1 chromatids, two copies of the sequence are present in one of the partial reactions and only in all others, if at all

1 sequence copy: thus applies here:

Ko = N-k + l

K ₁ = k-2

K ₂ = 1

So the formula is:

P (detection of exactly 2n chromatids) + P (detection of exactly (2n - 1) chromatids)

= ((N-1)! * N!) / ((N + k-1)! * (N-k) l) + (N! / (N-k + 1)! * (K-2)!) / ((N + k-1)! / (N-1)! * K!)

Example: - k = 4

- N = 8

Probability for successful detection of all 4 existing sequence copies = ((8 - 1)! * 8!) / ((8 + 4 - 1)! * (8 - 4)!) + (8! / (8-4 + 1)! * (4-2)! ) / ((8+ 4 - 1) 1 / (8-1) 1 * 4!) = 0.212 + 0.509 = 0.721

The probability of making an incorrect statement lies at 1 - 0.721 = 0.278. This applies if only one PCR product per subset is analyzed.

However, if several PCR products per subset are analyzed (multiplex PCR), the overall probability is obtained by multiplying the individual probabilities. So, for example for a 3-Plex: P (detection of all bands) = 0.278 * 0.278 * 0.278 = 0.021.

Depending on the degree of multiplexing, the following table is given.

Already at a multiplex grade 3, an error probability of less than or equal to 5% can be achieved here.

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. Thus, 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.

For example, 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.

In the following, the present invention is explained in more detail by means of an example, but not limiting the present invention example.

example

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.

To this end, 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.

Then 1 μl of a PCR mixture containing 0.5 μm 2x Multiplex PCR Master Mix (Qiagen GmbH, Hilden), 0.005 μl bromophenol blue (0.1%), 0.06 μl 5x Q-Solution (Qiagen GmbH, Hilden) and 0.39 μl of water. Finally, the individual reaction mixtures were covered with 5 μl Sealing Solution (Advalytix AG, Munich) and subjected to a PCR with the following temperature profile:

- 10 minutes at 94 ⁰ C, - 35 cycles with

94 ⁰ C for 30 seconds, 63 ⁰ C for 60 seconds and 72 ⁰ C for 60 seconds, as well

- 10 minutes at 72 ⁰ C.

Subsequently, in each case a subset of each of the 8 PCR products was pipetted into a pocket of a polyacrylamide gel, separated by electrophoresis and detected after silver staining.

A photograph of the polyacrylamide gel is shown in FIG. In FIG. 3, 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. Below this, 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.

As can be seen from FIG. 3, for the first sample, whose PCR products for the individual subsets were applied in the left four lanes of the polyacrylamide gel, the same PCR products were obtained in maximum in three of the four subsets, whereas for the second sample, whose PCR products were applied for the individual subsets in the right four lanes of the polyacrylamide gel, the same maximum in two of the four subsets of the same PCR products have been obtained.

As a result, the first sample contained at least three copies of chromosome 21, whereas the second sample contained at least two copies of chromosome 21.

Claims

claims

A method for quantitatively determining the number of at least one predetermined sequence in a biological sample, in particular for determining the absolute copy number of alleles per cell, comprising the steps:

a) providing a biological sample containing nucleic acid, b) fragmenting the nucleic acid contained in the biological sample, c) dividing the sample obtained in step b) into at least two subsets, d) adding at least two primer pairs to each of the at least two subsets in which the same primer pairs are added to each of the subsets, and wherein the individual primer pairs are adapted to amplify different subsequences of the at least one predetermined sequence in an amplification reaction, e) carrying out an amplification reaction with each of the at least two in the F) determining the number of different amplification products obtained with the amplification reactions in step e) for the individual subsets and determining with how many subsets identical amplification products have been obtained.

2. The method according to claim 1, characterized in that the at least one predetermined sequence is a chromosome

Chromatid, a gene or a gene section is.

3. The method according to claim 1 or 2, characterized in that the biological sample is a single cell or a single cell held in suspension, wherein the single cell is preferably a human cell, an animal cell or a Poeanzenzelle, more preferably a polar body.

4. The method according to any one of the preceding claims, characterized in that the fragmentation of the nucleic acid contained in the biological sample in step b) by restriction hydrolysis, by

Shear, by ultrasound or by DNase digestion.

5. The method according to any one of the preceding claims, characterized in that the nucleic acid is double-stranded DNA and this is denatured after the step a) and before the step c) to single strands.

6. The method according to any one of the preceding claims, characterized in that the primer pairs added in step d) are each for partial sequences of the at least one predetermined sequence specific primer pairs.

7. The method according to any one of the preceding claims, characterized in that the at least 2 primer pairs added in step d) are adapted to amplify different non-overlapping partial sequences of the at least one predetermined sequence in an amplification reaction.

8. The method according to any one of the preceding claims, characterized in that the fragmentation in step b) is carried out in such a way and / or the primer pairs added in step d) are selected such that the average length of the nucleic acid fragments obtained in step b) is greater than the length of the partial sequences of the at least one predetermined sequence that can be amplified by the primer pairs added in step d).

9. The method according to any one of the preceding claims, characterized in that the primer pairs added in step d) are selected so that all of the different partial sequences which can be amplified with the individual primer pairs are each at least 10 base pairs in length differ by at least 25 base pairs, more preferably by at least 50 base pairs, and most preferably by at least 100 base pairs.

10. The method according to any one of the preceding claims, characterized in that the determined in step f) the number of different amplification products obtained for the individual subsets and / or the determination of how many subsets each same Amplification products have been obtained by gel electrophoresis or by capillary electrophoresis.

11. The method according to any one of the preceding claims, characterized in that it further comprises the step gi) comparing the amplification products determined in step f) for each subset with the amplification products obtained with at least one control sample in an amplification reaction, with the at least one control performed amplification reaction with the same

Primer pairs as performed in step d).

12. The method according to claim 11, characterized in that the amplification reaction with the at least one control sample is carried out in parallel to the step e).

13. The method according to any one of claims 1 to 10, characterized in that it further comprises the step g2) comparing the amplification products determined in step f) for each subset with a data set, wherein the data set contains information on at least one control sample in a Amplification reaction using the used in step d) primer pairs available different amplification products.

14. The method according to any one of the preceding claims, characterized in that in the step d) each of the subsets, per predetermined sequence to be determined, each 2 to 50, preferably 5 to 25, especially preferably 8 to 15, most preferably 10 to 15 and most preferably 12 primer pairs are added.

15. The method according to any one of the preceding claims, characterized in that the sample obtained in step b) is divided in step c) in at least 3 subsets, preferably in at least 4 subsets and more preferably in 5 to 20 subsets.

16. The method according to any one of the preceding claims, characterized in that the amplification reactions are carried out in step e) for all subsets in parallel on a substrate of glass, for example, wherein the individual subsets or subsamples of these subsets are positioned on a respective reaction point of the substrate.

17. The method according to claim 16, characterized in that each of the reaction sites on the substrate comprises a central hydrophilic area, which is surrounded on the outside by a first hydrophobic area, which in turn is surrounded on the outside by a central hydrophilic area, the outside of a second hydrophobic area Area is surrounded.

18. The method according to claim 17, characterized in that the central hydrophilic region is at least substantially circular, which on the outside is concentrated by an at least substantially annular annular first hydrophobic region. is in turn surrounded concentrically surrounded on the outside by an at least substantially circular ring-shaped central hydrophilic region which is surrounded on the outside by the second hydrophobic region.

19. Method according to claim 1, wherein the copy number present in the biological samples is determined to be from 1 to 23, preferably from 1 to 10 and particularly preferably from 1 to 5 chromosomes.

20. A method for the quantitative determination of the number n of x predetermined sequence (s) in a biological sample according to one of the preceding claims comprising the steps:

a) providing a biological sample containing DNA, b) fragmenting the DNA contained in the biological sample, c) dividing the sample obtained in step b) into y partial amounts, d) adding in each case z primer pairs to each of the y partial amounts, wherein the same primer pairs are added to each of the subsets, and wherein the individual primer pairs are adapted to amplify different partial sequences of the x predetermined sequence (s) in a PCR, e) performing a PCR with each of the y in step d f) determining the number of subunits obtained with the PCR reactions in step e) for the individual subsets different amplification products and determining with how many of the z subsets each same amplification products have been obtained.

21. The method according to claim 20, characterized in that x is an integer between 1 and 23, y is an integer greater than or equal to 4, preferably from 5 to 20, and z is an integer which is greater than or equal to 3 ^■ x , preferably 5 to 25 x • • x and particularly preferably 8 to 15 • ^■ x x.

22. The method according to claim 21, characterized in that from the number, with how many of the z subsets in each case the same

Amplification products have been obtained, the number n of the predetermined sequence (s) is determined.

23. The method according to any one of the preceding claims, characterized in that the number n to be determined per predetermined sequences in the biological sample 0 to 100, preferably 0 to 10, more preferably 0 to 5 and most preferably 0, 1, 2, 3 or 4.

24. The method according to any one of the preceding claims, characterized in that this is carried out as part of an in vitro fertilization (IVF) or in the context of the analysis of fetal cells from maternal blood.

25. The method according to any one of the preceding claims, characterized in that the nucleic acid contained in the sample provided in step a) between the process step a) and the process step b) is lysed.