JP2020000011A - Method for detecting copy number of target region in genomic dna and reagent kit - Google Patents

Abstract

To provide methods for detecting the copy number of a target region more accurately, in a method of detecting the copy number of a target region in genomic DNA based on the result of nucleic acid amplification of the target region and the result of nucleic acid amplification of a reference region.SOLUTION: Provided is a method for detecting the copy number of a target region in a genomic DNA, the method comprising: an amplification step of respectively amplifying multiple detection regions per target region, and a reference region in a region other than the target region in the genomic DNA; and a detection step of detecting the copy number of the target region in the genomic DNA based on the amplification result obtained in the amplification step.SELECTED DRAWING: None

Description

  The present invention relates to a method for analyzing the copy number of a target region in genomic DNA.

  The copy number is the number of a specific base sequence in the genomic DNA, and one copy number means that only one region consisting of the specific base sequence exists in the genomic DNA. . The chromosome of human somatic cells is diploid, and the copy number of many genes is 2. However, the copy number may change due to genomic mutation, and copy number variation (CNV) may occur. being called. Among CNVs, those that occur in somatic cells such as cancer cells are called CNA (copy number somatic aberrations / alterations).

  In CNV analysis, generally, the amount of a nucleic acid molecule containing the same base sequence as the target region for copy number analysis, which is present in a sample containing the genomic DNA of interest, and the amount of a nucleic acid containing the same base sequence as the reference region Based on the amount of the molecule, the ratio of the copy number between the target region and the reference region is calculated. The analysis sample is often a very small amount.For this reason, in many cases, a nucleic acid amplification reaction such as PCR is performed using the target genomic DNA as a template to amplify the entire region or a part of the target region and the reference region, From the amplification result, the ratio of the copy number between the target region and the reference region is calculated.

  Genetic abnormalities in the target region can be detected by CNV analysis. For example, in normal cells, the copy number ratio between the target region and the reference region is 1: 1. However, when a chromosomal abnormality such as deletion or duplication occurs, the copy number ratio changes. Thus, CNV analysis makes it possible to detect a copy number abnormality occurring in the target region. However, in the case of CNA, the genomic sample to be analyzed is usually a mixture of genomic DNA derived from a large amount of normal cells and genomic DNA derived from a very small amount of abnormal cells. For this reason, the difference between the copy number of the target region and the copy number of the reference region is very small, and it may be difficult to accurately identify both.

  Various methods have been reported for improving the accuracy of CNV analysis. For example, Patent Document 1 discloses a method of performing a pre-amplification step in which a target region and a reference region are amplified by PCR or the like prior to digital PCR in CNV analysis in which a target region and a reference region are amplified by digital PCR. Is disclosed. Non-Patent Documents 1 and 2 report that the accuracy of CNV analysis can be improved by setting two or more reference regions. It is described that by increasing the number of reference areas, errors in the number of copies in the reference area can be reduced and the detection of the number of copies can be stabilized.

International Publication No. 2009/033178

Long, et al., “Multicopy reference assay (MRef)-a superior normalizer of sample input in DNA copy number analysis”, QIAGEN, 2013, [online], [February 5, 2018 search], Internet <https: //www.qiagen.com/jp/resources/resourcedetail?id=a0c54902-07a6-493b-9352-885f9063689b&lang=en> Hughesman, et al., PLOS ONE, 2016, DOI: 10.1371 / journal.pone.0161274

  An object of the present invention is to provide a method for accurately detecting the copy number of a target region in genomic DNA.

  The present inventors have conducted intensive studies to solve the above problems, and as a result, provided two or more detection regions in a target region, and amplified each of the plurality of detection regions by a nucleic acid amplification reaction. The present inventors have found that by detecting the copy number based on the amplification result of the detection region, the accuracy of the copy number detection of the target region can be improved, and completed the present invention.

The method for detecting the copy number of the target region in the genomic DNA according to the present invention is as follows.
[1] A method for detecting the copy number of a target region in genomic DNA,
An amplification step of amplifying each of a plurality of detection regions per target region and a reference region in a region other than the target region in the genomic DNA,
A detection step of detecting the copy number of the target region in the genomic DNA based on the amplification result obtained in the amplification step,
A method for detecting the copy number of a target region in genomic DNA, comprising:
[2] The method for detecting the copy number of a target region in genomic DNA according to [1], wherein each of the plurality of reference regions is amplified in the amplification step.
[3] The method for detecting a copy number of a target region in genomic DNA according to [2], wherein the plurality of reference regions are present on two or more chromosomes.
[4] The method for detecting a copy number of a target region in genomic DNA according to [3], wherein the plurality of reference regions are present on different chromosomes.
[5] The method for detecting the copy number of a target region in genomic DNA according to any one of [1] to [4], wherein the detection region and the reference region are present on different chromosomes.
[6] A method for detecting the copy number of a target region in genomic DNA,
An amplification step of amplifying the first detection region and the second detection region in the target region, and a reference region in a region other than the target region in the genomic DNA,
A detection step of detecting a copy number of the target region in the genomic DNA based on the amplification result;
Including
A method for detecting a copy number of a target region in genomic DNA, wherein the first detection region and the second detection region are present on the same chromosome.
[7] The method for detecting a copy number of a target region in genomic DNA according to [6], wherein the reference region includes a first reference region and a second reference region.
[8] The method for detecting a copy number of a target region in genomic DNA according to [7], wherein the first reference region and the second reference region are present on different chromosomes.
[9] a fragmentation step of fragmenting the genomic DNA before the amplification step;
The method for detecting a copy number of a target region in genomic DNA according to any one of the above [1] to [8], wherein in the amplification step, an amplification reaction is performed using the fragment obtained in the fragmentation step as a template.
[10] In the fragmentation step, the genomic DNA is converted into a nucleic acid fragment containing two or more detection regions, a nucleic acid fragment containing two or more reference regions, and a nucleic acid fragment containing both the detection region and the reference region. The method for detecting the copy number of a target region in genomic DNA according to the above [9], wherein a treatment with a restriction enzyme is carried out so that neither occurs.
[11] The method for detecting a copy number of a target region in genomic DNA according to the above [9], wherein the detection region is cleaved by a restriction enzyme in the fragmentation step.
[12] The method for detecting a copy number of a target region in genomic DNA according to any one of [1] to [11], wherein the detection region is 3 or more.
[13] The method for detecting a copy number of a target region in genomic DNA according to any one of [1] to [12], wherein the amplification is performed by digital PCR or real-time PCR.
[14] The method for detecting a copy number of a target region in genomic DNA according to any one of [1] to [13], wherein the target region is in a HER2 gene region or an AKT1 gene region.
[15] amplifying a plurality of detection regions in a target region in genomic DNA using a plurality of detection primer sets;
Amplify a reference region in a region other than the target region in the genomic DNA using a reference primer set,
A copy number detection method for a target region in genomic DNA, wherein the copy number of the target region in the genomic DNA is detected based on the amplification result of the detection region and the amplification result of the reference region.
[16] A reagent kit including a plurality of primer sets for amplifying the plurality of detection regions, respectively, and being used in the copy number detection method for a target region in genomic DNA according to any one of [1] to [15]. .
[17] The reagent kit of [16], further comprising a primer set for amplifying the reference region.

  According to the present invention, the copy number of a target region in genomic DNA can be accurately detected.

FIG. 3 is a diagram schematically showing a target region existing on both of a pair of homologous chromosomes and a detection region set in the region. FIG. 2 is a diagram schematically showing one embodiment of a reagent kit according to the present invention. FIG. 5 is a diagram showing the results of the copy number of a target region (HER2 gene region) determined by performing ddPCR using NA18943 as a template genomic DNA in Example 1. FIG. 5 is a diagram showing the results of the copy number of a target region (HER2 gene region) determined by performing ddPCR using NA19078 as a template genomic DNA in Example 1. FIG. 10 is a view showing the results of the copy number of a target region (AKT1 gene region) obtained by performing ddPCR using NA18943 as a template genomic DNA in Example 2. FIG. 7 is a diagram showing the results of the copy number of a target region (AKT1 gene region) determined by performing ddPCR using NA19089 as a template genomic DNA in Example 2. FIG. 11 is a diagram showing the results of the copy number of a target region (AKT1 gene region) obtained by performing ddPCR using NA18943 as a template genomic DNA in Example 3. FIG. 10 is a view showing the results of the copy number of a target region (AKT1 gene region) determined by performing ddPCR using NA19089 as a template genomic DNA in Example 3. In Example 4, it is the figure which showed the result of the copy number of the target area | region calculated | required by performing ddPCR using the human genomic DNA which adjusted the copy number of the target area | region (AKT1 gene area) to 2.0-3.0 as a template. .

  In CNV analysis, a target region and a reference region are detected by using a nucleic acid amplification method, and the ratio of the copy number between the target region and the reference region is determined. Conventionally, the copy number ratio is determined by nucleic acid amplification of the entire target region or only a part of the target region, and comparing the obtained amplification product amount with the amplification product amount of the reference region similarly amplified. Was. However, in a general nucleic acid amplification reaction including PCR, amplification efficiency is affected by the sequence and specificity of primers and probes used in the amplification reaction. This difference in amplification efficiency is directly linked to the amplification product detection efficiency and affects the detection result of the copy number of the target region.

  In the method for detecting the copy number of a target region in genomic DNA according to the present invention (hereinafter, sometimes referred to as the "copy number detecting method of the present invention"), two or more detection regions are set in one target region. Is done. The plurality of detection regions and reference regions are each subjected to nucleic acid amplification, and the copy number of the target region is detected based on the obtained amplification results. As a result, the error in the amount of amplification product in the target region is reduced as compared with the conventional CNV analysis in which only one detection region is used, and the copy number detection of the target region can be stabilized.

  Here, the genomic DNA includes a pair of homologous chromosomes. The plurality of detection regions are set in the target region so that all of them are on the same chromosome. When the target region is present on both of a pair of homologous chromosomes, as shown in FIG. 1, there are two copies of the target region in the genomic DNA. In this case, for example, if two detection areas (a first detection area and a second detection area) are set in one target area, the two first detection areas and the two detection areas are used in the copy number detection method of the present invention. Each of the second detection regions is subjected to nucleic acid amplification. On the other hand, when the target region exists only in one of the pair of homologous chromosomes, one copy of the target region exists in the genomic DNA. In this case, if the first detection region and the second detection region are similarly set, in the copy number detection method of the present invention, one first detection region and one second detection region are nucleic acid-amplified, respectively. This is the same for the reference area.

  Primers and probes used for nucleic acid amplification and detection described later are designed based on the wild-type sequence. Therefore, when the region to which the primer or probe hybridizes contains a polymorphism or mutation, the amplification efficiency is reduced, which may affect the detection result of the copy number of the target region. By using a plurality of detection regions, it is possible to minimize the influence of polymorphisms and mutations in the detection region on amplification efficiency and reduce errors in the detection of amplification products due to the detection region. The detection accuracy of numbers is increased.

  In addition, when the amount of the test sample containing the target genomic DNA is very small, when only one detection region is used, the amount of the amplification product obtained is small. By using a plurality of detection regions, the amount of amplification products in the target region can be doubled, and the copy number of the target region can be detected with high accuracy even from a small amount of sample.

That is, the copy number detection method of the present invention is a method for detecting the copy number of a target region in genomic DNA, and has the following amplification step and detection step.
An amplification step of amplifying a plurality of detection regions per one target region and a reference region in a region other than the target region in the genomic DNA.
A detection step of detecting a copy number of the target region in the genomic DNA based on an amplification result obtained in the amplification step.

  In the copy number detection method of the present invention, the target region for which the copy number is to be detected is not particularly limited as long as it is a region where a copy number polymorphism exists in genomic DNA, and may be a gene region. , A non-gene region. In the case of a gene region, it may be an exon or an intron. In addition, the target region may include a part of an exon and a part of an intron.

  Examples of the target region for which the copy number is detected in the copy number detection method of the present invention include, for example, a region encoding a gene whose copy number variation is related to the onset of any disease or drug sensitivity. . Examples of the gene include HER2 gene, AKT1 gene, ALK gene, EGFR gene, MET gene, RET gene, ROS1 gene, PTEN gene, FGFR2 gene, FGFR3 gene, NTRK1 gene, PIK3CA gene, c-myc gene and the like. Can be

  The detection region amplified in the amplification step may be a region in the target region. The detection region is preferably set so that a primer that can be specifically amplified by a nucleic acid amplification reaction or a probe that can specifically detect an amplification product can be designed. The primers and probes used for the amplification reaction of each detection region can be appropriately designed using known primer design software or the like based on the base sequence information of the genomic DNA including the target region.

  The number of detection areas is not particularly limited as long as it is two or more. The greater the number of detection regions, the higher the accuracy of copy number detection of the target region can be, but the more labor required for experiments such as amplification reactions. In the present invention, the number of detection regions is preferably 2 or more, more preferably 3 or more, still more preferably 3 to 10, since the accuracy of copy number detection of the target region can be sufficiently improved. ~ 6 are even more preferred. Hereinafter, the number of detection regions set in the target region may be described as “Nt” (Nt is an integer of 1 or more).

  In the amplification step, the first detection region and the second detection region in the target region and the reference region in a region other than the target region in the genomic DNA are amplified. The first detection region and the second detection region are on the same chromosome. The detection area in the target area is not limited to only the first detection area and the second detection area. When Nt is 3 or more, detection regions are set in addition to the first detection region and the second detection region, and these are also amplified in the amplification step.

  In the amplification step, one or more reference regions are also amplified. The reference region is not particularly limited as long as it is located in a region other than the target region in the genomic DNA, and may be a gene region or a non-gene region. In the case of a gene region, it may be an exon or an intron. Since the reference region is preferably a stable region in which mutation and the like hardly occur, in the copy number detection method of the present invention, the reference region is preferably in a gene region, and more preferably in an exon. Further, it is preferable that the chromosome is present in a region relatively far from the crossing site rather than near the crossing site in the chromosome.

  The reference region may be in the vicinity of the target region, but is preferably located in a region that is not easily affected by copy number variation of the target region. For this reason, in the copy number detection method of the present invention, the reference region is preferably present on a chromosome different from the target region. When the reference region is set in the same chromosome as the target region, the reference region is preferably located in a region relatively far from the target region.

  In the copy number detection method of the present invention, the number of reference regions may be one, but a plurality of reference regions is preferable. By using a plurality of reference areas, the number of data on which the copy number analysis of the reference area is based increases, so that a random error in the measured value of the copy number of the reference area can be reduced. As a result, the ratio of the copy number between the target area and the reference area can be obtained with a value closer to the true value.

  When setting a plurality of reference regions, each reference region may be present on the same chromosome or may be present on different chromosomes. Since it is expected that an error in the copy number of the reference region due to an unpredictable mutation or the like on the genomic DNA can be reduced, in the copy number detection method of the present invention, multiple reference regions It is preferable to design. For example, when two reference regions are set, the first reference region and the second reference region can be set so as to exist on different chromosomes. Also, for example, one reference region can be set on all chromosomes.

  The greater the number of reference regions, the higher the accuracy of copy number detection of the reference region can be, but the more labor required for experiments such as amplification reactions. In the present invention, the number of reference regions is preferably 2 or more, more preferably 3 or more, still more preferably 3 to 10, since the accuracy of copy number detection of the reference region can be sufficiently improved. ~ 6 are even more preferred. Hereinafter, the number of reference regions amplified in the amplification step may be referred to as “Nr” (Nr is an integer of 1 or more). Nr may be the same as Nt, or may be different.

  It is preferable that the reference region is set so that a primer that can be specifically amplified by a nucleic acid amplification reaction or a probe that can specifically detect an amplification product can be designed for each reference region. The primers and probes used for the amplification reaction of each reference region can be appropriately designed using known primer design software or the like based on the base sequence information of genomic DNA.

  Next, as an amplification step, Nt detection regions and Nr reference regions are amplified using the target genomic DNA as a template. Specifically, a DNA sample containing genomic DNA whose copy number of a target region is to be analyzed and a primer and / or a probe used for the amplification reaction of each region are added to a buffer containing an enzyme or the like necessary for a nucleic acid amplification reaction. Mix to prepare a reaction solution. A nucleic acid amplification reaction is performed in this reaction solution. The amplification reactions of the Nt detection regions and the Nr reference regions may all be performed in one reaction solution. For example, Nt detection primer sets for amplifying Nt detection regions and Nr reference primer sets for amplifying Nr reference regions are put together in a reaction solution together with the template. Thus, the amplification reactions of the Nt detection regions and the Nr reference regions can all be performed in one reaction solution. Further, the amplification reactions of the Nt detection regions may be collectively performed in one reaction solution, and the amplification reactions of the Nr reference regions may be collectively performed in another reaction solution. The regions may be amplified in separate reaction solutions.

  In the present invention, the genomic DNA for which the copy number of the target region is to be detected is not particularly limited as long as it is expected to contain the target region. The genomic DNA to be used as a template in the amplification reaction includes genomic DNA extracted from cells whose copy number of the target region is to be analyzed. Genomic DNA can be extracted from cells or tissues by a conventional method. Cells or tissues collected from a test animal to obtain genomic DNA are not particularly limited as long as they contain genomic DNA, and may include any cells and tissues. For example, it may be a body fluid sample such as blood, lymph, urine, tears, saliva, or a tissue sample obtained by biopsy.

  The genomic DNA to be used as a template in the amplification reaction may be obtained by previously amplifying a region including a target region and a reference region before the amplification step. The amplification product obtained by such preamplification can be used as a template DNA in the amplification step. The preamplification method can be appropriately selected from amplification methods generally used for amplifying genomic DNA such as PCR, and can be carried out.

  The amplification reaction of the detection region and the like is a method of amplifying a nucleic acid fragment consisting of a specific base sequence, which reflects the abundance ratio of the nucleic acid molecule containing the target region and the nucleic acid molecule containing the reference region that existed at the start of the reaction. The method is not particularly limited as long as it is a method that can obtain the adjusted amplification result. The nucleic acid amplification reaction used in the present invention is preferably an amplification method using a DNA polymerase because it is widely used. For example, traditional PCR, real-time PCR, digital PCR, nucleic acid sequence-based amplification (NASBA) , Helicase-dependent amplification (HDA), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), and the like.

  In digital PCR, a sample is randomly distributed into a number of microcompartments, and then PCR is performed. The PCR product in each microcompartment is detected by a fluorescent label or the like, and the microcompartment where the PCR product is detected and the microcompartment where the PCR product is not detected are detected. And the number of copies of the nucleic acid to be amplified originally contained in the sample per unit volume is calculated from the number of samples using the standard Poisson statistical method. In digital PCR, for example, a droplet (Droplet: an extremely small amount of droplet) is used as a micro compartment so that only one molecule of nucleic acid to be amplified is distributed per micro compartment. Digital PCR is carried out using a commercially available digital PCR device such as the “QX200 Droplet Digital PCR System” (Bio-Rad Laboratories) or the “QuantStudio 3D Digital PCR System” (Thermo Fisher Scientific) in a conventional manner. can do.

  In the present invention, the Nt detection regions and the Nr reference regions are preferably amplified by digital PCR. When the amount of the sample containing the target genomic DNA is very small, the number of nucleic acid molecules containing the target region and the reference region contained in the sample input to each amplification reaction varies greatly, and as a result, the amplification product detection accuracy Becomes lower. On the other hand, in the digital PCR, since the Poisson statistical method is used, the copy number of the target region or the reference region can be obtained with higher accuracy even for a small amount of sample.

  The amplification of the detection region and the reference region in the amplification step can be performed by real-time PCR. The real-time PCR can be performed by a known method such as a TaqMan (registered trademark) probe method or an intercalator method using an apparatus in which a thermal cycler and a spectrofluorometer are integrated.

  Amplification of the detection region and the reference region can also be performed by traditional PCR. A calibration curve showing the relationship between the concentration of the template DNA and the amount of the PCR product is prepared in advance using a sample whose DNA concentration as the template is known. By using this calibration curve, the copy number of the target region and the reference region contained in the reaction solution at the start of PCR can be obtained from the amount of amplification product obtained at the end point of traditional PCR. The PCR and the quantification of the PCR product can be performed by a conventional method.

  After the amplification step, in the detection step, the copy number of the target region of the target genomic DNA is detected based on the amplification product obtained in the amplification step. For example, in an amplification reaction, genomic DNA may be used as a template as it is, or a fragmented product in which the entire target region is present on one nucleic acid molecule due to fragmentation of genomic DNA may be used as a template. In these cases, the sum of the copy numbers obtained from the respective amplification products of the Nt detection regions corresponds to Nt times the copy number of the target region originally contained in the target genomic DNA. On the other hand, in some cases, such as when each reference region is set on a different chromosome, each reference region is present on a different nucleic acid molecule in the genomic DNA used as a template. In this case, the value obtained by dividing the total of the copy numbers obtained from the respective amplification products of the Nr reference regions by Nr is the copy number per reference region originally contained in the target genomic DNA. Equivalent to. Further, as in the case where all the reference regions are set on the same chromosome, all the reference regions may be present on one nucleic acid molecule in the genomic DNA used as a template. In this case, the value obtained by dividing the sum of the copy numbers obtained from the respective amplification products of the Nr reference regions by Nr is the copy number per reference region originally contained in the target genomic DNA. Equivalent to. For this reason, the copy number ratio between the target region and the reference region ([copy number of the target region]: [copy number of the reference region]) is calculated as [total copy number obtained from the amplification products obtained from the Nt detection regions]. Divided by Nt]: [Number obtained by dividing total copy number obtained from amplification products obtained from Nr reference regions by Nr].

  In the copy number detection method of the present invention, it is preferable to include a fragmentation step of fragmenting genomic DNA before the amplification step. Before subjecting the genomic DNA to be used as a template in the amplification reaction to the amplification reaction, it is preferable that the genomic DNA be fragmented by some method, and the detection region and the reference region be present in different nucleic acid fragments. For example, genomic DNA is treated with a restriction enzyme such that one nucleic acid fragment contains only one detection region and one nucleic acid fragment contains only one reference region. When Nt detection regions are present in nucleic acid fragments different from each other, the value obtained by dividing the total number of copies of each detection region obtained from the amplification product of each detection region by Nt is the genomic DNA of the target. Corresponds to the copy number of the target region originally contained in the target region. Similarly, when all Nr reference regions are present in nucleic acid fragments different from each other, the value obtained by dividing the sum of the copy numbers of the respective reference regions obtained from the amplification products of the respective reference regions by Nr is: This corresponds to the number of copies per reference region originally contained in the target genomic DNA. For this reason, the copy number ratio between the target region and the reference region ([copy number of the target region]: [copy number of the reference region]) is calculated as [total copy number obtained from the amplification products obtained from the Nt detection regions]. Divided by Nt]: [Number obtained by dividing total copy number obtained from amplification products obtained from Nr reference regions by Nr].

  Reagents such as primers and probes used for amplifying a detection region and a reference region, and enzymes and buffers used for an amplification reaction, which are used in the copy number detection method of the present invention, can also be made into a kit. By preparing reagents and the like to be used in the copy number detecting method of the present invention in a kit, the copy number detecting method of the present invention can be more easily performed.

  It is preferable to make a kit of a plurality of detection primer sets for amplifying the plurality of detection regions and a reference primer set for amplifying the reference region. FIG. 2 shows an example of a reagent kit used when two detection regions (the first detection region and the second detection region) are set in one target region. In FIG. 2, 50 indicates a reagent kit, 51 indicates a reagent container including a primer set for amplifying the first detection region, and 52 indicates a reagent container including a primer set for amplifying the second detection region. , 53 indicate a reagent container containing a primer set for amplifying a reference region, 54 indicates a package insert, and 55 indicates a packing box. The package insert 54 describes, for example, the configuration of the reagent kit and the detection protocol.

  EXAMPLES Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to the following Examples.

<Droplet Digital PCR (ddPCR)>
Digital PCR in the following experiments was performed as follows unless otherwise specified.
First, a genomic DNA as a template, a forward primer and a reverse primer for amplifying each region (detection region or reference region), a fluorescent label probe for detecting an amplification product, and ddPCR (trademark) Supermix for Probes ( No dUTP) (including dNTP, polymerase, and Buffer; manufactured by Bio-Rad Laboratories) and water (“Water for Molecular Biology”, manufactured by Millipore) were mixed to prepare a PCR mixture. A FAM-labeled probe was used as a fluorescent-labeled probe for detecting an amplification product in the detection region, and a HEX-labeled probe was used as a fluorescent-labeled probe for detecting an amplification product in the reference region. A primer probe set (manufactured by RIKEN GENESIS) was used as a forward primer and a reverse primer for amplifying each region and a fluorescent label probe for detecting an amplification product.

  Next, after preparing droplets from the PCR mixture, PCR was performed. The droplets were prepared using an Automated Droplet Generator (manufactured by Bio-Rad Laboratories), the plate sealing was performed using a PX1 PCR Plate Sealer (manufactured by Bio-Rad Laboratories), and the PCR was performed using a Veriti 96-Well Thermal Cycler (Thermo Fisher Scientific). (Manufactured by Sharp Corporation). The temperature cycling conditions for PCR were 95 ° C. for 10 minutes, followed by 40 cycles of 94 ° C. for 30 seconds, then 58 ° C. for 1 minute, then 98 ° C. for 10 minutes, and a further 4 ° C. hold. Was performed with 50% and a PCR volume of 22 μL.

  When the genomic DNA as a template is fragmented by restriction enzyme treatment before ddPCR, the restriction enzyme was mixed with the PCR mixture prepared as described above, and reacted at 37 ° C. for 1 hour before preparing droplets. .

<Real-time PCR>
Real-time PCR in the following experiments was performed as follows unless otherwise specified.
First, 2.2 μL of a primer probe set (manufactured by RIKEN GENESIS), 11.0 μL of ddPCR ™ Supermix for Probes (No dUTP) (manufactured by Bio-Rad Laboratories) and 3.8 μL of water (Millipore) Was mixed with a 5 μL sample (genomic DNA solution as a template) to prepare a PCR reaction solution. Next, this reaction solution was set in a real-time PCR device (heating / cooling rate: 2 ° C./sec), held at 95 ° C. for 10 minutes, then 94 ° C., 30 seconds, then 58 ° C., 1 minute + plate read The cycle of holding the time (data acquisition time) was repeated 40 times.

  When the genomic DNA as a template is fragmented by restriction enzyme treatment before ddPCR, the restriction enzyme was mixed with the PCR mixture prepared as described above, and reacted at 37 ° C. for 1 hour before preparing droplets. .

[Example 1]
The effect of the amount of genomic DNA input to the reaction system on the accuracy of CNV analysis was examined.

  The HER2 gene region (human chromosome 17) was used as a target region, and six detection regions (HER2-R1 to HER2-R6) were set in the HER2 gene region. For detection of the HER2-R1 region, a primer probe set “HER2 CNV” including a forward primer and a reverse primer for PCR-amplifying the region and a FAM-labeled probe for detecting an amplification product (product number: A172, RIKEN GENESIS (Manufactured by Sharp Corporation). For detection of the HER2-R2 region, a primer probe set “HER2-2 CNV” containing a forward primer and a reverse primer for PCR-amplifying the region and a FAM-labeled probe for detecting an amplification product (product number: A228, RIKEN GENESIS) was used. For detection of the HER2-R3 region, a primer probe set “HER2-3 CNV” including a forward primer and a reverse primer for PCR amplification of the region and a FAM-labeled probe for detecting an amplification product (product number: A229, RIKEN GENESIS) was used. For detection of the HER2-R4 region, a primer probe set “HER2-4 CNV” including a forward primer and a reverse primer for PCR-amplifying the region and a FAM-labeled probe for detecting an amplification product (product number: A230; RIKEN GENESIS) was used. For detection of the HER2-R5 region, a primer probe set “HER2-5 CNV” including a forward primer and a reverse primer for PCR-amplifying the region and a FAM-labeled probe for detecting an amplification product (product number: A231; RIKEN GENESIS) was used. For detection of the HER2-R6 region, a primer probe set “HER2-6 CNV” containing a forward primer and a reverse primer for PCR-amplifying the region and a FAM-labeled probe for detecting an amplification product (product number: A232, RIKEN GENESIS) was used.

  As a reference region, a region (Ref-R1) in the GAMT gene region (human chromosome 19), a region (Ref-R2) in the MFSD8 gene region (human chromosome 4), and an ELL3 gene region (human chromosome 15) Region (Ref-R3), the region (Ref-R4) in the SLC16A14 gene region (human chromosome 2), the region (Ref-R5) in the RPP30 gene region (human chromosome 10), and the EFTUD2 gene region ( A region (Ref-R6) in human chromosome 17) was set. To detect the Ref-R1 region, a primer probe set “GAMT CNV (Reference)” including a forward primer and a reverse primer for PCR-amplifying the region and a HEX-labeled probe for detecting an amplification product (product number: A233) And RIKEN GENESIS). To detect the Ref-R2 region, a primer probe set “MFSD8 CNV (Reference)” including a forward primer and a reverse primer for PCR amplification of the region and a HEX-labeled probe for detecting an amplification product (product number: A234) And RIKEN GENESIS). For detection of the Ref-R3 region, a primer probe set “ELL3 CNV (Reference)” including a forward primer and a reverse primer for PCR amplification of the region and a HEX-labeled probe for detecting an amplification product (product number: A235) And RIKEN GENESIS). For detection of the Ref-R4 region, a primer probe set “SLC16A14 CNV (Reference)” including a forward primer and a reverse primer for PCR amplification of the region and a HEX-labeled probe for detecting an amplification product (product number: A236) And RIKEN GENESIS). For the detection of the Ref-R5 region, a primer probe set “RPP30 CNV (Reference)” including a forward primer and a reverse primer for PCR-amplifying the region and a HEX-labeled probe for detecting an amplification product (product number: A121) And RIKEN GENESIS). For the detection of the Ref-R6 region, a primer probe set “EFTUD2 CNV (Reference)” including a forward primer and a reverse primer for PCR amplification of the region and a HEX-labeled probe for detecting an amplification product (product number: A174) And RIKEN GENESIS).

  As a genomic DNA used as a template, human normal genomic DNAs NA18943 (manufactured by Coriell Institute) and NA19078 (manufactured by Coriell Institute) were used. In each genomic DNA, the copy number (Copy Number: CN) of the HER2 gene is 2, and the copy number of each reference region is 2. The final concentration of the genomic DNA in the PCR mixture was 0.5 ng, 2 ng, or 20 ng. Pst1-HF (manufactured by New England Biolabs) was used as a restriction enzyme for digesting genomic DNA, and 10 units were added to the PCR mixture.

<One detection area and one reference area are used (Nt, Nr = 1, 1)>
The HER2-R1 region was set as a detection region, and the Ref-R1 region was set as a reference region.
The final concentration of each primer for amplifying each detection region and the reference region in the PCR mixture was 900 nM, and the final concentration of each fluorescently labeled probe for detecting an amplification product in each region was 250 nM.

<Six detection areas and six reference areas are used (Nt, Nr = 6, 6)>
The HER2-R1 region to HER2-R6 region were defined as detection regions, and the Ref-R1 region to Ref-R6 region were defined as reference regions.
The final concentration of the primer for amplifying each detection region and the reference region in the PCR mixture was 573 nM, and the final concentration of the fluorescently labeled probe for detecting the amplification product in each region was 159 nM.

  The results of the copy number of the target region (HER2 gene region) calculated by ddPCR are shown in FIGS. FIG. 3 shows the results when NA18943 was used as the template genomic DNA, and FIG. 4 shows the results when NA19078 was used as the template genomic DNA. In the figure, “Target (1), Reference (1)” is the result when one detection area and one reference area are used (Nt, Nr = 1, 1), and “Target (6), Reference (1)” “(6)” is a result when six detection areas and six reference areas are used (Nt, Nr = 6, 6). Numerical values in each figure show the calculated copy number of the target region.

  As shown in FIG. 3, when the input amount of genomic DNA is 20 ng, both the case where one detection region and one reference region are used and the case where six detection regions and six reference regions are used are used. The copy number of the target region almost coincided with the true value of 2, and the error bar (standard error) was very small. On the other hand, when the input amounts of the genomic DNA are 2.0 ng and 0.5 ng, the copy number of the target region is determined using one detection region and one reference region (Nt, Nr = 1, 1). ), The width of deviation from 2 and the error bar were smaller when 6 detection areas and 6 reference areas were used (Nt, Nr = 6, 6). More specifically, when one detection region and one reference region are used (Nt, Nr = 1, 1), if the input amount of genomic DNA is 2.0 ng, the obtained target region The copy number is slightly out of 2, and when the genomic DNA input amount is 0.5 ng, the deviation from 2 is larger than when the genomic DNA input amount is 2.0 ng, and the error bar becomes larger. Was. On the other hand, when six detection regions and six reference regions are used (Nt, Nr = 6, 6), the copy number of the target region is 2 even if the input amount of genomic DNA is 0.5 ng. Was close enough and the error bars were small. The copy number was closer to 2 and the error bar was smaller when the genomic DNA input amount was 2.0 ng than when the genomic DNA input amount was 0.5 ng. In FIG. 4, the same tendency as in FIG. 3 was observed.

  From these results, it was shown that the copy number of the target region can be measured more accurately than when only one detection region and one reference region were used by setting the detection region and the reference region to two or more, respectively. The effect of improving the accuracy of copy number detection of the target region by increasing the number of the detection region and the reference region was more remarkable as the input amount of the genomic DNA was smaller.

[Example 2]
The effect of the amount of genomic DNA input to the reaction system on the accuracy of CNV analysis was examined.

  Using the AKT1 gene region (human chromosome 14) as a target region, three detection regions (AKT1-R1 to AKT1-R3) were set in the AKT1 gene region. For detection of the AKT1-R1 region, a primer probe set “AKT1 CNV” including a forward primer and a reverse primer for PCR amplification of the region and a FAM-labeled probe for detecting an amplification product (product number: A104, RIKEN GENESIS (Manufactured by Sharp Corporation). For detection of the AKT1-R2 region, a primer probe set “AKT1-2 CNV” including a forward primer and a reverse primer for PCR-amplifying the region and a FAM-labeled probe for detecting an amplification product (product number: A226, RIKEN GENESIS) was used. For detection of the AKT1-R3 region, a primer probe set “AKT1-3 CNV” including a forward primer and a reverse primer for PCR amplification of the region and a FAM-labeled probe for detecting an amplification product (product number: A227; RIKEN GENESIS) was used.

  The reference regions include a region (Ref-R7) in the RPP30 gene region (human chromosome 10), a region (Ref-R8) in the AQP5 gene region (human chromosome 12), and a TOP3A gene region (human chromosome 17). )) (Ref-R9). For detection of the Ref-R7 region, a primer probe set “RPP30 CNV (Reference)” including a forward primer and a reverse primer for PCR amplification of the region and a HEX-labeled probe for detecting an amplification product (product number: A121) And RIKEN GENESIS). For detection of the Ref-R8 region, a primer probe set “AQP5 CNV (Reference)” including a forward primer and a reverse primer for PCR amplification of the region and a HEX-labeled probe for detecting an amplification product (product number: A124) And RIKEN GENESIS). For detection of the Ref-R9 region, a primer probe set “TOP3A CNV (Reference)” including a forward primer and a reverse primer for PCR amplification of the region and a HEX-labeled probe for detecting an amplification product (product number: A126) And RIKEN GENESIS).

  The genomic DNA used as a template was the human genomic DNA NA18943 used in Example 1 and the human genomic DNA NA19089 (Coriell Institute). In each of the genomic DNAs, the copy number (Copy Number: CN) of the AKT1 gene is 2, and the copy number of each reference region is 2. The final concentration of the genomic DNA in the PCR mixture was 0.5 ng, 2 ng, or 20 ng. Genomic DNA was previously prepared using Covaris M220 (manufactured by Covaris) at 20 ° C. under the conditions of a reaction solution volume of 130 μL, Peak Incident Power (W) of 50, Duty Factor of 2%, and Cycles per Burst of 200. After processing for 25 seconds, the fragment was mixed with the PCR mixture.

<One detection area and one reference area are used (Nt, Nr = 1, 1)>
The AKT1-R1 region was set as a detection region, and the Ref-R8 region was set as a reference region.
The final concentration of each primer for amplifying each detection region and the reference region in the PCR mixture was 900 nM, and the final concentration of each fluorescently labeled probe for detecting an amplification product in each region was 250 nM.

<Using three detection areas and three reference areas (Nt, Nr = 3, 3)>
The AKT1-R1 region to the AKT1-R3 region were defined as the detection region, and the Ref-R7 region to the Ref-R9 region were defined as the reference region.
The final concentration of each primer for amplifying each detection region and the reference region in the PCR mixture was 300 nM, and the final concentration of each fluorescent-labeled probe for detecting an amplification product in each region was 83 nM.

  The results of the copy number of the target region (AKT1 gene region) calculated by performing ddPCR are shown in FIGS. FIG. 5 shows the results using NA18943 as the template genomic DNA, and FIG. 6 shows the results using NA19089 as the template genomic DNA. In the figure, “Target (1), Reference (1)” is the result when one detection area and one reference area are used (Nt, Nr = 1, 1), and “Target (3), Reference (1)” “(3)” is the result when three detection areas and three reference areas are used (Nt, Nr = 3, 3). Numerical values in each figure show the calculated copy number of the target region.

  As shown in FIGS. 5 and 6, as in Example 1, in this example using the AKT1 gene region as the target region, the number of detection regions was three compared with the case where one detection region and one reference region were used. When the three reference regions were used, the copy number of the target region was closer to the true value of 2, and the error bar was smaller. In addition, the deviation width and error bar of the copy number of the target region from 2 became larger as the input amount of genomic DNA to the PCR mixture (reaction system) was smaller. These results also show that setting the number of detection areas and reference areas to 2 or more can improve the detection accuracy of the copy number of the target area compared to the case where only one detection area and one reference area are used. Was done.

[Example 3]
The influence of the number of detection regions and reference regions on the accuracy of CNV analysis was examined.

The AKT1 gene region was set as the target region, and the AKT1-R1 region to AKT1-3 region were set as the detection regions, as in Example 2, and the primer and probe sets for amplifying and detecting these were used in Example 2. Was used.
As the reference region, the Ref-R7 region to the Ref-R9 region were used as in Example 2, and the primer and probe sets used in Example 2 for amplifying and detecting these regions were used.

  As the genomic DNA used as a template, the human genomic DNAs NA18943 and NA19089 used in Example 2 were used. The final concentration of genomic DNA in the PCR mixture was 0.5 ng. The genomic DNA was fragmented by previously treating with Covaris M220 (manufactured by Covaris) in the same manner as in Example 2.

<One detection area and one reference area are used (Nt, Nr = 1, 1)>
The AKT1-R1 region was set as a detection region, and the Ref-R8 region was set as a reference region.
The final concentration of each primer for amplifying each detection region and the reference region in the PCR mixture was 900 nM, and the final concentration of each fluorescently labeled probe for detecting an amplification product in each region was 250 nM.

<Two detection areas and two reference areas are used (Nt, Nr = 2, 2)>
The AKT1-R1 region and the AKT1-R3 region were set as detection regions, and the Ref-R8 region and Ref-R9 region were set as reference regions.
The final concentration of each primer for amplifying each detection region and the reference region in the PCR mixture was 300 nM, and the final concentration of each fluorescent-labeled probe for detecting an amplification product in each region was 83 nM.

<Using three detection areas and three reference areas (Nt, Nr = 3, 3)>
The AKT1-R1 region to the AKT1-R3 region were defined as the detection region, and the Ref-R7 region to the Ref-R9 region were defined as the reference region.
The final concentration of each primer for amplifying each detection region and the reference region in the PCR mixture was 300 nM, and the final concentration of each fluorescent-labeled probe for detecting an amplification product in each region was 83 nM.

  The results of the copy number of the target region (AKT1 gene region) calculated by ddPCR are shown in FIGS. 7 and 8. FIG. 7 shows the results obtained by using NA18943 as the template genomic DNA, and FIG. 8 shows the results obtained by using NA19089 as the template genomic DNA. In the figure, “Target (1), Reference (1)” is the result when one detection area and one reference area are used (Nt, Nr = 1, 1), and “Target (2), Reference (1)” “(2)” is a result when two detection areas and two reference areas are used (Nt, Nr = 2, 2), and “Target (3), Reference (3)” is three detection areas. , Three reference areas (Nt, Nr = 3, 3). Numerical values in each figure show the calculated copy number of the target region.

  As shown in FIGS. 7 and 8, it was confirmed that the error bar became smaller as the number of detection regions and reference regions increased, regardless of which genomic DNA was used as a template. These results indicate that the accuracy of copy number detection of the target region can be improved by increasing the number of detection regions and reference regions.

[Example 4]
When the copy number of the target genomic DNA was 2.0 to 3.0 (CN = 2.0 to 3.0), the influence of the number of detection regions and reference regions on the accuracy of CNV analysis was examined.

The AKT1 gene region was set as the target region, and the AKT1-R1 region to AKT1-3 region were set as the detection regions, as in Example 2, and the primer and probe sets for amplifying and detecting these were used in Example 2. Was used.
As the reference region, the Ref-R7 region to the Ref-R9 region were used as in Example 2, and the primer and probe sets used in Example 2 for amplifying and detecting these regions were used.

  The genomic DNA used as a template was the human genomic DNA NA18943 used in Example 1 and the human genomic DNA HCC1143 (obtained from ATCC). The final concentration of genomic DNA in the PCR mixture was 0.5 ng. The genomic DNA was fragmented by previously treating with Covaris M220 (manufactured by Covaris) in the same manner as in Example 2.

<Preparation of each genomic DNA with CN = 2.0 to 3.0>
Using the AKT1-R1 region to AKT1-3 region as the detection region and the Ref-R7 region to Ref-R9 region as the reference region, the copy number of the AKT1 gene of HCC1143 of the human genomic DNA is determined by ddPCR, and NA18943 and HCC1143 of the human genomic DNA are appropriately determined. Were mixed at various ratios to prepare human genomic DNA with CN = 2.0, 2.2, 2.4, 2.6, 2.8, or 3.0.

<One detection area and one reference area are used (Nt, Nr = 1, 1)>
The AKT1-R1 region was set as a detection region, and the Ref-R8 region was set as a reference region.
The final concentration of each primer for amplifying each detection region and the reference region in the PCR mixture was 900 nM, and the final concentration of each fluorescently labeled probe for detecting an amplification product in each region was 250 nM.

<Using three detection areas and three reference areas (Nt, Nr = 3, 3)>
The AKT1-R1 region to the AKT1-R3 region were defined as the detection region, and the Ref-R7 region to the Ref-R9 region were defined as the reference region.
The final concentration of each primer for amplifying each detection region and the reference region in the PCR mixture was 300 nM, and the final concentration of each fluorescent-labeled probe for detecting an amplification product in each region was 83 nM.

  The results of the copy number of the target region (AKT1 gene region) calculated by performing ddPCR are shown in FIG. In the figure, “Target (1), Reference (1)” is the result when one detection area and one reference area are used (Nt, Nr = 1, 1), and “Target (3), Reference (1)” “(3)” is the result when three detection areas and three reference areas are used (Nt, Nr = 3, 3). The numerical values in the figure indicate the calculated copy number of the target region.

  When the copy number of the AKT1 gene of the genomic DNA used was 2.0 to 3.0, the detection region was larger than the case where one detection region and one reference region were used (Nt, Nr = 1, 1). When three and three reference areas were used (Nt, Nr = 3, 3), the copy number was closer to the true value and the error bar was smaller. In particular, when one detection region and one reference region are used (Nt, Nr = 1, 1), error bars are greatly overlapped with each other, so that the copy number of the target region is 2.0, The case where the copy number of the target region was 2.2 to 3.0 could not be distinguished. On the other hand, when three detection regions and three reference regions are used (Nt, Nr = 3, 3), the copy number of the target region is 2.0 and the copy number of the target region is 2 0.8 to 3.0 means that the error bars do not overlap and the genomic DNA has a target region copy number of 2.0 and the genomic DNA has a target region copy number of 2.8 to 3.0. Were distinguishable. From these results, it was shown that by increasing the number of the detection region and the reference region, the resolution when detecting the copy number of the target region was increased, and the accuracy of the detection of the target region was improved. Similar results were observed when the amount of genomic DNA input to the PCR mixture (reaction system) was 2.0 ng (not shown).

[Example 5]
The influence of the number of detection regions and reference regions on the accuracy of CNV analysis was examined.

The AKT1 gene region was set as the target region, and the AKT1-R1 region to AKT1-3 region were set as the detection regions, as in Example 2, and the primer and probe sets for amplifying and detecting these were used in Example 2. Was used.
As the reference region, the Ref-R7 region to the Ref-R9 region were used as in Example 2, and the primer and probe sets used in Example 2 for amplifying and detecting these regions were used.

  As a genomic DNA used as a template, the human genomic DNA NA18943 used in Example 1 was used. The final concentration of genomic DNA in the PCR mixture was 0.5 ng. The genomic DNA was fragmented by previously treating with Covaris M220 (manufactured by Covaris) in the same manner as in Example 2.

<One detection area and three reference areas are used (Nt, Nr = 1, 3)>
The AKT1-R1 region was set as a detection region, and the Ref-R7 to Ref-R9 regions were set as reference regions.
The final concentration of each primer for amplifying each detection region and the reference region in the PCR mixture was 300 nM, and the final concentration of each fluorescent-labeled probe for detecting an amplification product in each region was 83 nM.

<Using three detection areas and three reference areas (Nt, Nr = 3, 3)>
The AKT1-R1 region to the AKT1-R3 region were defined as the detection region, and the Ref-R7 region to the Ref-R9 region were defined as the reference region.
The final concentration of each primer for amplifying each detection region and the reference region in the PCR mixture was 300 nM, and the final concentration of each fluorescent-labeled probe for detecting an amplification product in each region was 83 nM.

  By performing ddPCR, the calculated average value (CNV) of copy number of the target region (AKT1 gene region), [average value of copy number + standard error] (CNV max), [average value of copy number−standard error] ( Table 1 shows CNV min) and the range of variation (2 × standard error: [CNV max] − [CNV min]). In the table, “Target (1), Reference (3)” is the result when one detection area and three reference areas are used (Nt, Nr = 1, 3), and “Target (3), Reference (3)” “(3)” is the result when three detection areas and three reference areas are used (Nt, Nr = 3, 3).

  In the case where one detection region and three reference regions are used (Nt, Nr = 1, 3) and in the case where three detection regions and three reference regions are used (Nt, Nr = 3, 3), the target region The number of copies (CNV) was the same value, but the error bar uses three detection areas and three reference areas rather than one detection area and three reference areas (Nt, Nr = 1, 3) (Nt, Nr = 3, 3) was smaller by 24%. These results indicate that the detection accuracy of the copy number of the target region can be improved by detecting (multiplex) not only the reference region but also the target region in a plurality of regions.

[Example 6]
When nucleic acid amplification was performed by real-time PCR, the influence of the number of detection regions and reference regions on the accuracy of CNV analysis was examined.

The AKT1 gene region was set as the target region, and the AKT1-R1 region to AKT1-3 region were set as the detection regions, as in Example 2, and the primer and probe sets for amplifying and detecting these were used in Example 2. Was used.
As the reference region, the Ref-R7 region to the Ref-R9 region were used as in Example 2, and the primer and probe sets used in Example 2 for amplifying and detecting these regions were used.

  As a genomic DNA used as a template, the human genomic DNA NA18943 used in Example 1 was used. The final concentration of genomic DNA in the PCR mixture was 0.5 ng. The genomic DNA was fragmented by previously treating with Covaris M220 (manufactured by Covaris) in the same manner as in Example 2.

<One detection area and three reference areas are used (Nt, Nr = 1, 3)>
The AKT1-R1 region was set as a detection region, and the Ref-R7 to Ref-R9 regions were set as reference regions.
The final concentrations of the primers for amplifying the detection region and each reference region in the PCR mixture were each 300 nM, and the final concentrations of the fluorescently labeled probes for detecting the amplification products in each region were each 83 nM.

<Using three detection areas and three reference areas (Nt, Nr = 3, 3)>
The AKT1-R1 region to the AKT1-R3 region were defined as the detection region, and the Ref-R7 region to the Ref-R9 region were defined as the reference region.
The final concentration of each primer for amplifying each detection region and the reference region in the PCR mixture was 300 nM, and the final concentration of each fluorescent-labeled probe for detecting an amplification product in each region was 83 nM.

  Real-time PCR was performed, and the calculated average copy number (CNV) of the target region (AKT1 gene region), [average copy number + 2 SD] (CNV max), [average copy number−2 SD] (CNV min) ) And the range of variation ([CNV max]-[CNV min]) are shown in Table 2. In the table, “Target (1), Reference (3)” is the result when one detection area and three reference areas are used (Nt, Nr = 1, 3), and “Target (3), Reference (3)” “(3)” is the result when three detection areas and three reference areas are used (Nt, Nr = 3, 3).

  Regarding the copy number of the target region, the case where one detection region and three reference regions are used (Nt, Nr = 1, 3) and the case where three detection regions and three reference regions are used (Nt, Nr = 3, 3) In both cases, the deviation widths from the true value (2) were almost the same, but the error bar was smaller than the detection area than one detection area and three reference areas (Nt, Nr = 1, 3). In the case where three reference regions and three reference regions were used (Nt, Nr = 3, 3), it was 30% smaller. From these results, even when the nucleic acid amplification reaction is performed by real-time PCR instead of digital PCR, detection of not only the reference region but also the target region in a plurality of regions (multiplex) enables detection of the copy number of the target region. It was shown that the accuracy could be improved.

50: reagent kit, 51: reagent container containing a primer set for amplifying the first detection region, 52 ... reagent container containing a primer set for amplifying the second detection region, 53: amplifying the reference region Reagent container containing primer set, 54: package insert, 55: packing box.

Claims (17)

A method for detecting the copy number of a target region in genomic DNA,
An amplification step of amplifying each of a plurality of detection regions per target region and a reference region in a region other than the target region in the genomic DNA,
A detection step of detecting the copy number of the target region in the genomic DNA based on the amplification result obtained in the amplification step,
A method for detecting the copy number of a target region in genomic DNA, comprising:   The method for detecting the copy number of a target region in genomic DNA according to claim 1, wherein a plurality of reference regions are amplified in the amplification step.   The method for detecting a copy number of a target region in genomic DNA according to claim 2, wherein the plurality of reference regions are present on two or more chromosomes.   The method of detecting a copy number of a target region in genomic DNA according to claim 3, wherein the plurality of reference regions are present on different chromosomes.   The method for detecting the copy number of a target region in genomic DNA according to any one of claims 1 to 4, wherein the detection region and the reference region are present on different chromosomes. A method for detecting the copy number of a target region in genomic DNA,
An amplification step of amplifying the first detection region and the second detection region in the target region, and a reference region in a region other than the target region in the genomic DNA,
A detection step of detecting a copy number of the target region in the genomic DNA based on the amplification result;
Including
A method for detecting a copy number of a target region in genomic DNA, wherein the first detection region and the second detection region are present on the same chromosome.   The method for detecting the copy number of a target region in genomic DNA according to claim 6, wherein the reference region includes a first reference region and a second reference region.   The method of detecting a copy number of a target region in genomic DNA according to claim 7, wherein the first reference region and the second reference region are present on different chromosomes. Before the amplification step, the method includes a fragmentation step of fragmenting the genomic DNA,
The method for detecting the copy number of a target region in genomic DNA according to any one of claims 1 to 8, wherein in the amplification step, an amplification reaction is performed using the fragment obtained in the fragmentation step as a template.   In the fragmentation step, the genomic DNA is formed into a nucleic acid fragment containing two or more detection regions, a nucleic acid fragment containing two or more reference regions, and a nucleic acid fragment containing both the detection region and the reference region. The method for detecting the copy number of a target region in genomic DNA according to claim 9, wherein a treatment with a restriction enzyme is carried out so as not to cause a copy.   The method for detecting a copy number of a target region in genomic DNA according to claim 9, wherein the detection region is cleaved by a restriction enzyme in the fragmentation step.   The method for detecting the copy number of a target region in genomic DNA according to any one of claims 1 to 11, wherein the detection region is 3 or more.   The method for detecting the copy number of a target region in genomic DNA according to any one of claims 1 to 12, wherein the amplification is performed by digital PCR or real-time PCR.   The method for detecting the copy number of a target region in genomic DNA according to any one of claims 1 to 13, wherein the target region is in a HER2 gene region or an AKT1 gene region. Amplify a plurality of detection regions in a target region in genomic DNA using a plurality of detection primer sets,
Amplify a reference region in a region other than the target region in the genomic DNA using a reference primer set,
A copy number detection method for a target region in genomic DNA, wherein the copy number of the target region in the genomic DNA is detected based on the amplification result of the detection region and the amplification result of the reference region.   A reagent kit comprising a plurality of primer sets for amplifying the plurality of detection regions, respectively, and being used in the method for detecting a copy number of a target region in genomic DNA according to any one of claims 1 to 15.   17. The reagent kit according to claim 16, further comprising a primer set for amplifying the reference region.

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