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KR20160003547A - Method for analyzing prenatal genetic information from blood or plasma of pregnant woman using digital PCR - Google Patents

Method for analyzing prenatal genetic information from blood or plasma of pregnant woman using digital PCR Download PDF

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KR20160003547A
KR20160003547A KR1020150034810A KR20150034810A KR20160003547A KR 20160003547 A KR20160003547 A KR 20160003547A KR 1020150034810 A KR1020150034810 A KR 1020150034810A KR 20150034810 A KR20150034810 A KR 20150034810A KR 20160003547 A KR20160003547 A KR 20160003547A
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oligonucleotide
nucleotide sequence
target gene
gene
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황승용
김승준
연종필
김지훈
이승용
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바이오코아 주식회사
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Abstract

The present invention provides a technique which performs digital PCR on a maternal blood or plasma sample, which is detected that fetus-derived genes are present, amplifies a control gene, which is present in a maternal gene set and a fetal gene set in common, and has no relation to gene number abnormalities and a target gene having a relation to the gene number abnormalities by using the digital PCR, and then analyzes the gene number abnormalities by comparing results of digital PCR quantification of the target gene and the control gene when a result of digital PCR quantification of the control gene satisfies the Poissons distribution, in other words, when the control gene copy number per a well is statistically close to 1.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for analyzing fetal genetic information from blood or plasma of pregnant women using digital PCR,

The present invention relates to a method for analyzing genetic information of a fetus from blood or plasma of a pregnant woman using digital PCR. More particularly, the present invention relates to a method for analyzing genetic information of a fetus derived from a fetal gene And collecting and analyzing genetic information and applying it to diagnosis.

In addition, the present invention provides a basic result of fetal genetic information from a fetal-derived gene existing in a small amount in blood or plasma of a pregnant woman, and digital PCR is performed on a sample in which a fetal gene exists in blood or plasma of a pregnant woman To analyze the genetic information of the fetus so as to be able to diagnose early genetic diseases caused by abnormality in the number of genes in genetic diseases of the fetus.

In addition, the present invention provides a method for genetic analysis of abnormalities in the number of fetal genes from blood or plasma of a pregnant woman by performing digital PCR using a primer pair and a probe specific for the target gene with respect to the number of genes or more There is a problem that abnormalities in the number of genes related to a fetal-derived target gene existing in a small amount in the blood or plasma of a pregnant woman due to a background signal of a maternal-derived gene existing in large quantities are detected as undetected signals And a method for solving the problem.

Further, the present invention solves the problem of difficulty in combining the genotype analysis technique using the free glassy gene of the fetal cell and the digital PCR technique, and solves the problem of difficulty in separating only the signal derived from the fetal gene and the background signal of the maternal gene And to provide a technique that can satisfy the phasic distribution required in digital PCR technology and can reliably quantify results.

The probability of hereditary diseases in the fetus has increased rapidly in recent decades due to the aging of the marriage population and the aging of the childbearing age. Early detection of a pregnancy-related disorder, including genetic anomalies of the fetus, may provide early medical attention necessary for safety while reducing pain for pregnant women and the fetus, It is very important to diagnose abnormalities early. Genetic abnormalities that can occur in the fetus include abnormalities in the number of genes, for example, trisomy, translocation, deletion, redundancy, and insertion of chromosomes. If such chromosomal abnormalities are present, Structural abnormalities and functional abnormalities occur. These gene abnormalities are known to occur more frequently in older pregnant women.

The amniocentesis method is used to diagnose the genetic abnormality of the fetus. The amniocentesis method is used for examining the genetic abnormality of the fetus by collecting amniotic fluid containing the fetal tissue cells and examining the chromosomes or DNA in the fetal tissue cells to be. However, amniocentesis is an invasive test that involves risks as a prenatal diagnosis. There are risks such as bacterial infection of amniotic membrane caused by syringe, wound by syringe, leak of amniotic fluid, and in case of serious case, it may become miscarriage.

Therefore, there is a desperate need for a method which is not dangerous to both pregnant and fetus for genetic analysis of fetus and early diagnosis of disease. In this regard, it is known that 5 to 10% of genetic material in blood or plasma of pregnant women is a genetic material derived from fetus. In the fetal placental cells, apoptosis causes a short fragment of cellular DNA or RNA derived from fetus Into the blood of the pregnant woman. In the case of studying pediatric cancer or hereditary disease by analyzing various genotypes using the detected fetal-derived cell free genes, a non-invasive method is used as a method of detecting a fetus-derived cell free gene (cell free fetal DNA or RNA) It has the advantage of being safe. Therefore, a microarray method or a real time PCR method is used as a genotyping method using maternal plasma fetal DNA-derived free gene to replace or assist the conventional amniocentesis.

In order to non-invasively diagnose such fetal hereditary diseases, it is necessary to provide a technology capable of securing the genetic information of a fetus present in a small amount in the blood or plasma of a pregnant woman without missing and without error. However, since the research and diagnosis are proceeding without knowing the ratio of the fetal genetic information existing in the blood or plasma of the pregnant woman, there is a problem in the reliability of the analysis result of the analyzed fetal DNA-derived free gene. In other words, there is a difficulty in separating only the cell free gene derived from fetus from the blood of a pregnant woman, and there is a risk of loss of fetal genetic information which is very important in such a separation process. In addition, it is technically difficult and costly to isolate only the gene derived from fetus from the maternal gene, which is present in a large amount in the blood of a pregnant woman. In the process of amplifying a sample in blood or plasma of a pregnant woman, , The reliability of the results of analysis of the fetal genes is low and it is very likely to show false or false negative results.

In this connection, in Korean Patent Registration No. 10-1387582, in order to more effectively detect a cell glassy fetal nucleic acid present in an extremely small amount in a mother, a nucleic acid having a short continuous length starting from the 5 ' end of the fetal nucleic acid to be detected A primer capable of amplifying a portion of the DNA can be designed and a real-time PCR is performed using the primer as a target, thereby detecting a cell glassy fetal nucleic acid in the body.

However, the real-time PCR method adopted by the prior art is a non-invasive method. However, there are limitations in performing various types of tests at a time and it is necessary to use a standard curve to detect an abnormal number of genes of a fetus. Is required to a certain level or more. Real-time PCR also adopts the method of quantifying the number of gene copies by matching the Ct or Cp value of the standard curve with the Ct or Cp value of the test sample. Therefore, Secondary analysis is necessary.

Therefore, in the technical field of the present invention, considering the fact that the amount of the fetal-derived gene that can be obtained from blood or plasma samples of pregnant women is extremely smaller than that of the maternal gene, There is a need for a method that can easily carry out the test with a high degree of reliability even at a very low concentration while the genotype analysis process for the target gene is simple.

The digital PCR method, in which the resultant signal has a value of "0" or "1" and the analysis method is digital, analyzes the large-volume sample, tests various samples at once, There is an advantage that various inspection items can be performed at one time. Digital PCR (digital PCR) is a technique that allows absolute quantification of DNA samples using a single molecular counting method that does not require a standard curve. More accurate quantitation is performed by PCR reaction on one droplet per well (See Gudrun Pohl and le-Ming Shih, Principle and applications of digital PCR, Expert Rev. Mol. Diagn. 4 (1), 41-47 (2004)).

Digital PCR was performed by distributing each droplet containing a sample gene template, an amplification primer and a fluorescence probe prepared to be diluted to an average number of 0.5 to 1 number of copies into one well and performing emulsion PCR, 1 "because the sample of the number of gene copies is distributed and indicates the signal after amplification, and the wells without fluorescence signal are counted as" 0 "since zero samples are distributed and no amplification occurs. Absolute quantification can be performed by counting.

In digital PCR, data reliability is guaranteed only when the number of gene copies per well in which fluorescence signals appear is one. In other words, the quantification in digital PCR is based on the poisson distribution, and if there is or is not a signal per well, it is calculated as a digital value 1 or 0 and a ratio of positive (value of 1) and negative (value of 0) , And the ratio of positive to positive total and negative sum is far out of the number of gene copies of the Poisson distribution, it means that the number of copies of the gene per well exceeds one, and thus the reliability of the data can be secured. I will not. For this reason, in digital PCR, quantification after amplification does not satisfy the phasic distribution. If the number of copies per well exceeds one, it is important to dilute the gene sample to satisfy the phasic distribution, When the number of copies is close to 1, it is important to quantify the corrected value (for example, using the Poisson 9 program).

However, in spite of the ease of quantitative analysis, large-volume analysis, and sample processing ability, the digital PCR method is required to correct the quantitative analysis to secure the data reliability of digital PCR. There are technological difficulties in fitting the dog.

In particular, as described above, in carrying out a genetic analysis for abnormalities in the number of genes derived from fetuses from blood or plasma of pregnant women, digital PCR using a primer pair and a probe specific for the target gene concerning the number of genes or more There is a problem that abnormalities in the number of genes related to a fetal-derived target gene existing in a small amount in the blood or plasma of a pregnant woman due to a background signal of a large amount of maternal-derived gene are detected as undetected signals . This problem is becoming a barrier that makes it difficult to combine genetic analysis technology using fetal DNA-derived cell free genes and digital PCR technology.

Therefore, when analyzing the number of genes in the fetus using a DNA derived from a fetus in a trace amount in the blood or plasma of a pregnant woman based on the digital PCR technique, it is difficult to separate only the signals derived from the fetal gene, It can be said that it is required to provide a technology that can reliably solve quantization results by satisfying the phasic distribution required in the digital PCR technique while solving the problem caused by the background signal of genes.

Korean Registered Patent No. 10-1387582 (Apr. 24, 2014)

Gudrun Pohl and le-Ming Shih, Principle and applications of digital PCR, Expert Rev. Mol. Diagn. 4 (1), 41-47 (2004) (disclosed in 2004)

It is an object of the present invention to provide a method for diagnosing and collecting characteristic genetic information from a fetal-derived gene existing in a small amount in blood or plasma of a pregnant woman using digital PCR.

The object of the present invention is also to provide a method for obtaining genetic information of a fetus from a fetal-derived gene existing in a small amount in blood or plasma of a pregnant woman, To analyze the genetic information of the fetus so as to provide a method for early diagnosis of genetic diseases caused by abnormality in the number of genes in genetic diseases of the fetus.

It is a further object of the present invention to provide a method and apparatus for genetic analysis of abnormalities in the number of fetal genes from blood or plasma of a pregnant woman by using a primer pair and a probe specific for a target gene, When the PCR is performed, abnormalities in the number of genes related to the fetal-derived target gene existing in a small amount in blood or plasma of a pregnant woman due to a background signal of a maternal-derived gene existing in a large amount are not detected or detected as an unidentified signal And to provide a method for solving the problem.

Furthermore, it is an object of the present invention to solve the difficulties of the difficulty of grafting the genomic analysis technique using the free glassy gene of a fetal cell and the digital PCR technique, and to solve the difficult problem of separation of only the signal derived from the fetal gene, It is to solve the problem caused by the signal and to provide a technique which can satisfy the phasic distribution required in digital PCR technology and can reliably quantify the result.

In order to solve the above technical problem, the present invention provides a method for quantitatively determining the ratio of maternal gene amplification amount to fetal gene amplification amount by real-time PCR amplification of a specific target gene in blood or plasma samples of a pregnant woman, The present invention provides a technique for identifying the presence of genetic fragments derived from a fetus and collecting genetic information of the fetus stably on the basis thereof to enhance the possibility of early diagnosis of genetic diseases of the fetus.

The present invention also relates to a method for detecting a fetal chromosomal abnormality by performing digital PCR on a blood or plasma sample of a pregnant woman in which a fetal gene is detected to be present, When the comparison target gene (control gene) and the target gene related to the gene number abnormality are amplified by digital PCR, and the digital PCR quantification result of the gene to be compared (control gene) satisfies the Poisson distribution, that is, When the number of control gene copies per well is close to one, a technique for analyzing the number of genes can be provided by comparing the result of digital PCR measurement of the target gene with the result of digital PCR quantification of the control gene.

First, terms used in the specification of the present invention will be described as follows.

The term " real-time PCR "used in the specification of the present invention refers to the application of a fluorescent material to a PCR technique, in which amplification of a target gene existing in a specimen during the reaction, Of the target gene in real time and quantitatively analyze it to quickly and accurately analyze whether the target gene is amplified or not. This real-time PCR is divided into two methods, one using SYBR Green and the other using double-labeled probes.

The SYBR green technique combines the SYBR green dye into the DNA amplified during real-time PCR amplification and binds to the double-stranded DNA synthesized by the PCR reaction to generate a fluorescent signal. The fluorescent signal is detected, It is a method that can measure the presence and the amount of amplification product from it. The real-time PCR technique using a dual labeled probe is a method using a double-labeled probe in which a fluorescent substance is labeled at the 5 'end and a quencher is labeled at the 3' end, Through the fluorescence signal from the labeled probe, it is possible to confirm whether or not the double-labeled probe is annealed with the PCR amplification product of the target gene, and the presence or absence of the target gene and the amount of the amplification product generated therefrom can be measured.

The term " digital PCR "used in the specification of the present invention means that a single molecule counting method is applied to the result of PCR amplification of one gene copy in one droplet per well, It is a technology that can be done. Digital PCR can analyze up to 3,072 samples in one well plate and analyze up to four well plates at the same time. Therefore, it is possible to analyze a large sample and to inspect various samples. In addition, digital PCR is a technique that does not require analysis using a standard curve and can easily quantify the number of wells representing a fluorescence signal by digital value (Gudrun Pohl and Lee Ming Shih, Principle and Applications of Digital PCR, Expert Rev. Mol. Diagn. 4 (1), 41-47 (2004)).

The term "target gene" used in the specification of the present invention is a gene having a mutation useful for genotyping analysis among the genes in the sample to be analyzed, for example, a marker gene used for gene detection or individual identification, A gene having a chromosome number greater than or equal to the number of genes, a gene having a genetically significant mutation, a gene having STR (short tandem repeat), or a gene having a single nucleotide polymorphism have. On the other hand, a "control gene" refers to a gene that is known to be unrelated to the number of genes in both the maternal gene set in the mother's blood or plasma and the fetal derived gene set.

Meanwhile, the control gene, the target gene and the related chromosomes described in the embodiments of the present invention should be understood as examples, and the method of analyzing the genetic information of the fetus from the blood or plasma of the pregnant woman using the digital PCR of the present invention may be various objects Or samples and various target genes and chromosomes, or as an underlying technology applicable to abnormal gene number analysis.

The method of analyzing the genetic information of the fetus from the blood or plasma of a pregnant woman using the digital PCR of the present invention,

(a) analyzing whether a fetal-derived gene is present in a pregnant blood or plasma sample, and

(b) performing a digital PCR by distributing a sample detected as a result of the analysis of the step (a) to the wells of the well plate, wherein a set of maternal genes and a gene set common to fetal genes Performing a digital PCR on a control gene that is not related to a gene number abnormality and a target gene related to abnormal gene number,

(c) analyzing the digital PCR amplification product of the control gene obtained in the step (b), analyzing whether the number of control gene copies per well is statistically close to one,

(d) if it is determined that the number of control gene copies per well is statistically more than 1 as a result of the analysis in the step (c), the sample is further diluted and the steps (b) and (c) Repeating,

(e) If it is determined that the number of control gene copies per well is close to one statistically as a result of the analysis in the step (c), the digital PCR amplification product of the target gene is quantified, and the digital PCR Calculating a ratio of a digital PCR quantification value of the target gene to a quantification value;

(f) determining that the number of genes in the fetal-derived target gene is greater than or equal to the number of genes if the ratio calculated in step (e) is 1.5 or more.

In the method of analyzing fetal gene information from the blood or plasma of a pregnant woman in one embodiment of the present invention, the fact that the number of control gene copies per well is statistically close to one means that the result of digital PCR measurement on the control gene This means that the quantification results of digital PCR are statistically significant. For example, not only when the number of copies per well is one, but also when the number of copies per well falls within a range of approximately 1.01 to 1.59, more preferably within a range of approximately 1.01 to 1.39, It is considered to be close.

A method for analyzing fetal genetic information from blood or plasma of a pregnant woman in an embodiment of the present invention, wherein the digital PCR quantitative value of the control gene in step (e) is an average number of copies of the control gene per well, The digital PCR quantification value of the gene may be an average number of copies of the target gene per well.

In the method of analyzing genetic information of a fetus from blood or plasma of a pregnant woman according to an embodiment of the present invention, the dilution of the sample in step (d) may be performed stepwise from about 1000 times to 5000 times desirable.

According to the present invention, it is possible to solve the conventional problems that make it difficult to combine the genotype analysis technique using the fetal DNA-derived cell free gene and the digital PCR technique. According to the conventional method, in order to apply the digital PCR technique to the genotype analysis of a fetal-derived cell free gene, it is necessary to set the number of copies of the target gene related to the fetal genetic disease to one per well, but this is technically very difficult.

For example, in order to distribute one template of a target gene to each well, blood or plasma of a pregnant woman is diluted and then quantified by performing digital PCR, and statistical conditions such as a phasic distribution of one target gene copy number per well However, since the quantification result of digital PCR is the sum of the amplification result of the target gene template of the pregnant woman and the amplification result of the target gene template of the fetus, the fetal target gene template It is difficult to distinguish only the amplification result of In addition, when a sample is diluted in order to distribute one target gene template of the fetus to each well, there is a problem that since the amount of the gene of the fetus is very small, it is lost in the dilution process or the analytical signal is weak, . On the other hand, the method of the present invention has the above-described constitution, thereby solving the problem of separation of only the signal derived from the fetal gene and the problem caused by the background signal of the maternal gene, Can be satisfied and the quantitative results can be reliably provided.

As described above, various control genes and target genes and related chromosomes can be applied to the method of the present invention. Various control primers and various primers for amplifying the target gene and various probes for detecting the amplification products can also be used as the gene Can be designed using target sequences.

As an example without limiting the present invention, the control gene comprises a control gene having the nucleotide sequence of SEQ ID NO: 33, a gene on chromosome 2, and a control gene having the nucleotide sequence of SEQ ID NO: 37, May be used.

The primer pair of SEQ ID NOS: 34 and 35 may be used as the primer pair for amplifying the control gene having the nucleotide sequence of SEQ ID NO: 33, and the probe for detecting the control gene amplification product having the nucleotide sequence of SEQ ID NO: At least one probe selected from the group consisting of oligonucleotides of SEQ ID NO: 36 and oligonucleotides complementary to these oligonucleotides may be used. 38 and 39 may be used as a pair of primers for amplifying the control gene having the nucleotide sequence of SEQ ID NO: 37, and a probe for detecting the control gene amplification product having the nucleotide sequence of SEQ ID NO: 37 At least one probe selected from the group consisting of oligonucleotides of SEQ ID NO: 40 and oligonucleotides complementary to these oligonucleotides may be used.

In the method of one embodiment of the present invention, a control gene having the nucleotide sequence of SEQ ID NO: 33 and a nucleotide sequence of the nucleotide sequence of SEQ ID NO: 37 using the mixture of the primer pairs of SEQ ID NOs: 34 and 35 and the primer pairs of SEQ ID NOs: The control gene having the nucleotide sequence is amplified together in the same well, and at least one probe selected from the group consisting of the oligonucleotide of SEQ ID NO: 36 and an oligonucleotide complementary to the oligonucleotide, and the oligonucleotide of SEQ ID NO: A control gene amplification product having the nucleotide sequence of SEQ ID NO: 33 and a control gene amplification product having the nucleotide sequence of SEQ ID NO: 37 using a mixture of at least one probe selected from the group consisting of oligonucleotides complementary to the nucleotide Detected together in the same well Can. As described above, when a primer pair and a detection probe specific to a plurality of control genes are mixed and divided into the same well, the amount of the sample to be distributed to the well plate can be increased, and inconvenience of layout design, inconvenience of the primer and probe dispensing process Can be improved.

In addition, the target gene may be at least one selected from the group consisting of a target gene on chromosome 21, a target gene on chromosome 13, and a target gene on chromosome 18. In addition, according to the method of one embodiment of the present invention, digital PCR is performed on at least two target genes selected from the group consisting of a target gene on chromosome 21, a target gene on chromosome 13, and a target gene on chromosome 18 Can be performed.

As a non-limiting example of the present invention, the target gene on chromosome 21 includes a target gene having the nucleotide sequence of SEQ ID NO: 21, a target gene having the nucleotide sequence of SEQ ID NO: 25, and a target gene having the nucleotide sequence of SEQ ID NO: At least one selected from the group constituted can be used.

The pair of primers of SEQ ID NOs: 22 and 23 may be used as the primer pair for amplifying the target gene having the nucleotide sequence of SEQ ID NO: 21, and the probe for detecting the target gene amplification product having the nucleotide sequence of SEQ ID NO: At least one probe selected from the group consisting of oligonucleotides of number 24 and oligonucleotides complementary to these oligonucleotides may be used. As a pair of primers for amplifying a target gene having the nucleotide sequence of SEQ ID NO: 25, a pair of primers of SEQ ID NOs: 26 and 27 may be used, and a probe for detecting a target gene amplification product having the nucleotide sequence of SEQ ID NO: At least one probe selected from the group consisting of oligonucleotides of SEQ ID NO: 28 and oligonucleotides complementary to these oligonucleotides may be used. As the primer pair for amplifying the target gene having the nucleotide sequence of SEQ ID NO: 29, a pair of primers of SEQ ID NOS: 30 and 31 may be used, and a probe for detecting the target gene amplification product having the nucleotide sequence of SEQ ID NO: At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 32 and an oligonucleotide complementary to the oligonucleotide may be used.

In the method of one embodiment of the present invention, the primer pair of SEQ ID NOS: 22 and 23, the pair of primers of SEQ ID NOS: 26 and 27, and the primer pair of SEQ ID NOS: 30 and 31, The target gene having the nucleotide sequence of SEQ ID NO: 25 and the target gene having the nucleotide sequence of SEQ ID NO: 29 are amplified together in the same well, and the oligonucleotide of SEQ ID NO: 24 and the complementary nucleotide sequence complementary to the oligonucleotide And at least one probe selected from the group consisting of an oligonucleotide complementary to the oligonucleotide of SEQ ID NO: 28 and at least one probe selected from the group consisting of the oligonucleotide of SEQ ID NO: 32 and the oligonucleotide of SEQ ID NO: Lt; RTI ID = 0.0 > oligonucleotides & A target gene amplification product having the nucleotide sequence of SEQ ID NO: 21, a target gene amplification product having the nucleotide sequence of SEQ ID NO: 25, and a target gene amplification product having the nucleotide sequence of SEQ ID NO: 29 using the mixture of at least one probe selected from the group consisting of Target gene amplification products with base sequences can be detected together in the same well. As described above, when a primer pair and detection probes specific for a plurality of target genes on chromosome 21 are mixed and dispensed in the same well, the amount of the sample to be distributed to the well plate can be increased and the inconvenience of layout design, The inconvenience of the dispensing process can be improved.

As a non-limiting example of the present invention, the target gene on chromosome 18 includes a target gene having the nucleotide sequence of SEQ ID NO: 57, a target gene having the nucleotide sequence of SEQ ID NO: 61, a target gene having the nucleotide sequence of SEQ ID NO: And a target gene having the nucleotide sequence of SEQ ID NO: 69 can be used.

The pair of primers of SEQ ID NOs: 58 and 59 may be used as the pair of primers for amplifying the target gene having the nucleotide sequence of SEQ ID NO: 57, and the probe for detecting the target gene amplification product having the nucleotide sequence of SEQ ID NO: At least one probe selected from the group consisting of oligonucleotides of number 60 and oligonucleotides complementary to these oligonucleotides may be used. As a pair of primers for amplifying a target gene having the nucleotide sequence of SEQ ID NO: 61, a pair of primers of SEQ ID NOS: 62 and 63 may be used, and a probe for detecting a target gene amplification product having the nucleotide sequence of SEQ ID NO: At least one probe selected from the group consisting of oligonucleotides of SEQ ID NO: 64 and oligonucleotides complementary to these oligonucleotides may be used. Further, as the primer pair for amplifying the target gene having the nucleotide sequence of SEQ ID NO: 65, a pair of primers of SEQ ID NOs: 66 and 67 may be used, and a probe for detecting the target gene amplification product having the nucleotide sequence of SEQ ID NO: At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 68 and an oligonucleotide complementary to such oligonucleotide may be used. The primer pair for amplifying the target gene having the nucleotide sequence of SEQ ID NO: 69 may be a primer pair of SEQ ID NOs: 70 and 71 and a probe for detecting the target gene amplification product having the nucleotide sequence of SEQ ID NO: 69 At least one probe selected from the group consisting of oligonucleotides of SEQ ID NO: 72 and oligonucleotides complementary to these oligonucleotides may be used.

In the method of one embodiment of the present invention, a primer pair of SEQ ID NOs: 58 and 59, a primer pair of SEQ ID NOs: 62 and 63, a pair of primers of SEQ ID NOs: 66 and 67, and a pair of primer pairs of SEQ ID NO: 70 and 71 The target gene having the nucleotide sequence of SEQ ID NO: 57, the target gene having the nucleotide sequence of SEQ ID NO: 61, the target gene having the nucleotide sequence of SEQ ID NO: 65, and the target gene having the nucleotide sequence of SEQ ID NO: Are amplified together in the same well, and at least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 60 and an oligonucleotide complementary to the oligonucleotide, an oligonucleotide of SEQ ID NO: 64 and an oligonucleotide complementary to the oligonucleotide At least one probe selected from the group consisting of SEQ ID NO: 68 At least one probe selected from the group consisting of a ribonucleotide and an oligonucleotide complementary to the oligonucleotide, and a mixture of an oligonucleotide of SEQ ID NO: 72 and at least one probe selected from the group consisting of an oligonucleotide complementary to the oligonucleotide , A target gene amplification product having the nucleotide sequence of SEQ ID NO: 61, a target gene amplification product having the nucleotide sequence of SEQ ID NO: 65, and a target gene amplification product having the nucleotide sequence of SEQ ID NO: A target gene amplification product having a nucleotide sequence of 69 can be detected together in the same well. As described above, when a primer pair specific for a plurality of target genes on chromosome 18 and detection probes are mixed and dispensed in the same well, the amount of sample to be distributed to the well plate can be increased and the inconvenience of layout design, The inconvenience of the dispensing process can be improved.

As a non-limiting example of the present invention, the target gene on chromosome 13 includes a target gene having the nucleotide sequence of SEQ ID NO: 41, a target gene having the nucleotide sequence of SEQ ID NO: 45, a target gene having the nucleotide sequence of SEQ ID NO: And a target gene having the nucleotide sequence of SEQ ID NO: 53 can be used.

The pair of primers of SEQ ID NOS: 42 and 43 may be used as the primer pair for amplifying the target gene having the nucleotide sequence of SEQ ID NO: 41, and the probe for detecting the target gene amplification product having the nucleotide sequence of SEQ ID NO: At least one probe selected from the group consisting of oligonucleotides of SEQ ID NO: 44 and oligonucleotides complementary to these oligonucleotides may be used. As a pair of primers for amplifying a target gene having the nucleotide sequence of SEQ ID NO: 45, a pair of primers of SEQ ID NOS: 46 and 47 may be used, and a probe for detecting a target gene amplification product having the nucleotide sequence of SEQ ID NO: At least one probe selected from the group consisting of oligonucleotides of SEQ ID NO: 48 and oligonucleotides complementary to these oligonucleotides may be used. 50 and 51 may be used as the primer pair for amplifying the target gene having the nucleotide sequence of SEQ ID NO: 49, and a probe for detecting the target gene amplification product having the nucleotide sequence of SEQ ID NO: 49 At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 52 and an oligonucleotide complementary to the oligonucleotide may be used. As a pair of primers for amplifying a target gene having the nucleotide sequence of SEQ ID NO: 53, a pair of primers of SEQ ID NOS: 54 and 55 may be used and a probe for detecting a target gene amplification product having the nucleotide sequence of SEQ ID NO: 53 At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 56 and an oligonucleotide complementary to the oligonucleotide may be used.

In the method of one embodiment of the present invention, a primer pair of SEQ ID NOs: 42 and 43, a pair of primers of SEQ ID NOs: 46 and 47, a pair of primers of SEQ ID NOs: 50 and 51, and a pair of primers of SEQ ID NOs: 54 and 55 The target gene having the nucleotide sequence of SEQ ID NO: 41, the target gene having the nucleotide sequence of SEQ ID NO: 45, the target gene having the nucleotide sequence of SEQ ID NO: 49, and the target gene having the nucleotide sequence of SEQ ID NO: Are amplified together in the same well, and at least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 44 and an oligonucleotide complementary to the oligonucleotide, an oligonucleotide of SEQ ID NO: 48 and an oligonucleotide complementary to the oligonucleotide At least one probe selected from the group consisting of SEQ ID NO: 52 At least one probe selected from the group consisting of a ribonucleotide and an oligonucleotide complementary to the oligonucleotide, and a mixture of an oligonucleotide of SEQ ID NO: 56 and at least one probe selected from the group consisting of an oligonucleotide complementary to the oligonucleotide The target gene amplification product having the nucleotide sequence of SEQ ID NO: 41, the target gene amplification product having the nucleotide sequence of SEQ ID NO: 45, the target gene amplification product having the nucleotide sequence of SEQ ID NO: 49, The target gene amplification products having the nucleotide sequence of SEQ ID NO: 53 can be detected together in the same well. Thus, when a primer pair specific for a plurality of target genes on chromosome 13 and detection probes are mixed and dispensed in the same well, the amount of the sample to be distributed to the well plate can be increased and the inconvenience of layout design, The inconvenience of the dispensing process can be improved.

In addition, the present invention provides a control gene composition for digital PCR quantitation, which is commonly present in a set of maternal-derived genes and a fetal-derived gene in a gene sample and is not related to the number of genes. The control gene composition for digital PCR quantification according to an embodiment of the present invention comprises at least one gene selected from the group consisting of the gene of SEQ ID NO: 37 on the chromosome 1 and the gene of SEQ ID NO: 33 on the chromosome 2, Amplified and quantified to obtain a reference quantification value for calculating the normalization value from the quantification value obtained after amplification of the target gene related to the number of existing genes or more. At this time, the normalization value is calculated to determine whether the number of genes of the target gene is abnormal.

In this regard, the present invention provides a probe composition for a control gene used for detecting and quantifying an amplification product of a control gene present in a gene sample.

The probe composition for a control gene according to an embodiment of the present invention comprises at least one oligonucleotide selected from the group consisting of oligonucleotides of SEQ ID NO: 36 and oligonucleotides complementary to the control gene having the nucleotide sequence of SEQ ID NO: At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 40 specific to a control gene having the nucleotide sequence of SEQ ID NO: 37 and at least one probe selected from the group consisting of an oligonucleotide complementary to the oligonucleotide, .

Further, the present invention provides a well plate used for amplifying a gene sample extracted from blood or plasma of a pregnant woman, and detecting and quantifying an amplification product.

The well plate of one embodiment of the present invention comprises at least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 36 specific to a control gene having the nucleotide sequence of SEQ ID NO: 33 and an oligonucleotide complementary to the oligonucleotide, At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 40 specific to a control gene having the nucleotide sequence of SEQ ID NO: 37 and at least one probe selected from the group consisting of oligonucleotides complementary to the oligonucleotide is divided Lt; / RTI > At this time, two or more probes may be individually dispensed into each well, or may be mixed together and dispensed into one well.

Wherein the well plate comprises at least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 24 and an oligonucleotide complementary to the oligonucleotide, which is specific to the target gene having the nucleotide sequence of SEQ ID NO: 21 on the chromosome 21, And at least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 28 and an oligonucleotide complementary to the oligonucleotide, which is specific to the target gene having the nucleotide sequence of SEQ ID NO: 25 on the chromosome of SEQ ID NO: At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 32 specific to the target gene having the nucleotide sequence of SEQ ID NO: 32 and at least one probe selected from the group consisting of an oligonucleotide complementary to the oligonucleotide The it may further include a dispensing well. At this time, two or more probes may be individually dispensed into each well, or may be mixed together and dispensed into one well. The probe specifically binds to the amplification product of the target gene on the chromosome 21 and is used to detect whether the number of genes is abnormal. For example, the above number of genes may be tri-chromosomal.

The well plate may further comprise at least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 60 specific to the target gene having the nucleotide sequence of SEQ ID NO: 57 on the chromosome 18 and an oligonucleotide complementary to the oligonucleotide, , At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 64 specific to the target gene having the nucleotide sequence of SEQ ID NO: 61 on the chromosome 18 and an oligonucleotide complementary to the oligonucleotide, and a sequence on the chromosome 18 At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 68 specific to the target gene having the nucleotide sequence of SEQ ID NO: 65 and an oligonucleotide complementary to the oligonucleotide, and a nucleotide sequence of SEQ ID NO: 69 on the chromosome 18 And at least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 72 specific for the target gene having a nucleotide sequence complementary to the oligonucleotide and at least one probe selected from the group consisting of oligonucleotides complementary to the oligonucleotide is dispensed . At this time, two or more probes may be individually dispensed into each well, or may be mixed together and dispensed into one well. The probe specifically binds to the amplification product of the target gene on the chromosome 18 and is used to detect whether the number of genes is abnormal. For example, the above number of genes may be tri-chromosomal.

The well plate may further comprise at least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 44 specific to the target gene having the nucleotide sequence of SEQ ID NO: 41 on chromosome 13 and an oligonucleotide complementary to the oligonucleotide , At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 48 specific to the target gene having the nucleotide sequence of SEQ ID NO: 45 on the chromosome 13 and an oligonucleotide complementary to the oligonucleotide, and a sequence on the chromosome 13 At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 52 and an oligonucleotide complementary to the oligonucleotide, which is specific to the target gene having the nucleotide sequence of SEQ ID NO: 49, and a nucleotide sequence of SEQ ID NO: 53 And at least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 56 specific to the target gene having a nucleotide sequence complementary to the target gene and at least one probe selected from the group consisting of oligonucleotides complementary to the oligonucleotide . At this time, two or more probes may be individually dispensed into each well, or may be mixed together and dispensed into one well. The probe specifically binds to the amplification product of the target gene on the chromosome 13 and is used to detect abnormalities in the number of genes. For example, the above number of genes may be tri-chromosomal.

The present invention also provides a probe composition for a target gene, which is used for detecting and quantifying a target gene amplification product associated with an abnormality in the number of genes present in a gene sample.

The probe composition for a target gene of an embodiment of the present invention is characterized in that it is selected from the group consisting of an oligonucleotide of SEQ ID NO: 24 specific to a target gene having a nucleotide sequence of SEQ ID NO: 21 on chromosome 21 and an oligonucleotide complementary to such oligonucleotide At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 28 and an oligonucleotide complementary to the oligonucleotide, which is specific to the target gene having the nucleotide sequence of SEQ ID NO: 25 on the chromosome 21, , At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 32 specific to a target gene having the nucleotide sequence of SEQ ID NO: 29 on the chromosome 21 and an oligonucleotide complementary to the oligonucleotide From comprises at least one probe selected. The probe specifically binds to the amplification product of the target gene on the chromosome 21 and is used to detect whether the number of genes is abnormal. For example, the above number of genes may be tri-chromosomal.

In addition, the probe composition for a target gene of an embodiment of the present invention comprises an oligonucleotide of SEQ ID NO: 60 specific to a target gene having the nucleotide sequence of SEQ ID NO: 57 on chromosome 18 and an oligonucleotide complementary to the oligonucleotide At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 64 specific for a target gene having a nucleotide sequence of SEQ ID NO: 61 on the chromosome 18 and an oligonucleotide complementary to the oligonucleotide At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 68 and an oligonucleotide complementary to the oligonucleotide, which is specific to the target gene having the nucleotide sequence of SEQ ID NO: 65 on the chromosome 18, At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 72 specific to the target gene having the nucleotide sequence of SEQ ID NO: 69 on the chromosome and at least one probe selected from the group consisting of oligonucleotides complementary to the oligonucleotide . The probe specifically binds to the amplification product of the target gene on the chromosome 18 and is used to detect whether the number of genes is abnormal. For example, the above number of genes may be tri-chromosomal.

The probe composition for a target gene according to an embodiment of the present invention comprises an oligonucleotide of SEQ ID NO: 44 specific to a target gene having the nucleotide sequence of SEQ ID NO: 41 on chromosome 13 and an oligonucleotide complementary to the oligonucleotide At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 48 specific for a target gene having the nucleotide sequence of SEQ ID NO: 45 on chromosome 13 and an oligonucleotide complementary to such oligonucleotide And at least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 52 and an oligonucleotide complementary to the oligonucleotide, which is specific to the target gene having the nucleotide sequence of SEQ ID NO: 49 on the chromosome 13, At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 56 specific to the target gene having the nucleotide sequence of SEQ ID NO: 53 on the chromosome and at least one probe selected from the group consisting of oligonucleotides complementary to the oligonucleotide . The probe specifically binds to the amplification product of the target gene on the chromosome 13 and is used to detect abnormalities in the number of genes. For example, the above number of genes may be tri-chromosomal.

Meanwhile, in the present invention, one end of the above-mentioned probe, for example, the 5 'terminus is Cy5, Cy3, x-Rhodamine, Texas Red, SYBR green, FAM, VIC, JOE, NED, HEX Such as IABkFQ, DABCYL, TAMRA, ECLIPSE, BHQ (BHQ-1, BHQ-2, BHQ-3, etc.) and the like at the other end of the probe, for example, It can be labeled with minerals. However, it should be understood that the present invention is not limited thereto, and a variety of fluorescent materials and minerals known in the art can be used.

The present invention can be applied to diagnosis by collecting and analyzing characteristic genetic information from a fetal-derived gene existing in a small amount in blood or plasma of a pregnant woman using digital PCR.

In addition, the present invention provides a basic result of fetal genetic information from a fetal-derived gene existing in a small amount in blood or plasma of a pregnant woman, and digital PCR is performed on a sample in which a fetal gene exists in blood or plasma of a pregnant woman By analyzing the genetic information of the fetus, it can be used to diagnose early genetic diseases caused by abnormality in the number of genes in genetic diseases of the fetus.

Further, the present invention relates to a method for genetic analysis of an abnormality in the number of genes of a fetus from blood or plasma of a pregnant woman by performing a digital PCR on the blood of a pregnant woman by a background signal of a maternal- It is possible to solve the problem that an abnormality with respect to the number of genes related to the fetal-derived target gene existing in a small amount in the plasma is not detected or is detected as an unidentified signal. That is, the present invention is advantageous in that a genetic analysis process for a specific target gene of a fetus can be easily performed using a fetal-derived gene present in a trace amount in a sample obtained from the blood of a pregnant woman, but the test can be performed with high reliability even at a very low concentration .

Therefore, the present invention is capable of solving the barriers that make it difficult to combine the genotype analysis technique using the fetal DNA-derived cell free gene and the digital PCR technology, and it is also possible to solve the difficulty of separating only the signal derived from the fetal gene, It is possible to solve the problem caused by the signal and to satisfy the phasic distribution required in the digital PCR technique and to provide a reliable technique for quantitative results.

FIG. 1 is a graph showing the results of real-time PCR quantitation of the APP gene by each sample and dividing the result by the results for a short amplicon (length 67 bp) and a long amplicon (length 180 bp).
Figure 2 is a graph showing the results of analysis using the CopyCaller ® software, the software analyzes the results of the TaqMan ® assay copy number (Copy Number Assays).
FIG. 3 is a schematic view showing the positions of a target gene and a control gene on chromosome 21 selected to perform gene analysis on a fetal-derived gene.
FIG. 4 is a diagram schematically showing the position of a target gene on chromosome 13 and a target gene on chromosome 18, which are selected to perform gene analysis on a fetal gene.
FIG. 5 is a photograph showing the result of performing PCR amplification on a corresponding target gene and a control gene using each pair of primers designed in Example 3-1, followed by electrophoresis. FIG.
FIGS. 6 to 24 are graphs showing real-time PCR performed on the corresponding target genes and control genes using the respective primer pairs and detection probes designed in Example 3-1.
25 is a digital PCR layout performed in Example 4-1.
26 is a diagram showing a digital PCR heat map as a result of performing digital PCR in Example 4-1.
FIG. 27 is a graph quantitatively showing the number of copies of each target gene and the number of copies of each control gene for each sample by analyzing fluorescence values measured in each well of FIG. 26.
28 is a digital PCR layout performed in Example 4-2.
29 is a diagram showing a digital PCR heat map as a result of performing digital PCR in Example 4-2.
FIG. 30 is a graph quantitatively showing the copy number of each target gene and the copy number of each control gene for each sample by analyzing fluorescence values measured in each well of FIG. 29.
31 is a digital PCR layout performed in Example 4-3.
FIG. 32 is a diagram showing a digital PCR heat map as a result of performing the digital PCR in Example 4-3. FIG.
FIG. 33 is a graph quantitatively showing the copy number of each target gene and the copy number of each control gene for each sample by analyzing fluorescence values measured in each well of FIG. 32; FIG.
34 is a digital PCR layout performed in Example 4-4.
35 is a diagram showing a digital PCR heat map as a result of performing the digital PCR in Example 4-4.
36 is a graph quantitatively showing the copy number of each target gene and the copy number of each control gene for each sample by analyzing fluorescence values measured in each well of FIG.

Hereinafter, the present invention will be described in more detail based on the embodiments of the present invention. It should be understood that the following embodiments of the present invention are only for embodying the present invention and do not limit or limit the scope of the present invention. It will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. The references cited in the present invention are incorporated herein by reference.

Example

Example 1: Measurement of the ratio of maternal-derived gene amount to fetal-derived gene amount for a specific gene in a pregnant blood sample

Example 1-1: Design of primers and probes

It is known that the fetal DNA in blood or plasma of a pregnant woman is smaller in size than the maternal DNA. Thus, when real-time PCR amplifies small-sized fetal-derived DNA and larger-sized maternal-derived DNA with respect to a specific gene, fetal-derived DNA and maternal-derived DNA present in blood or plasma of a pregnant woman And the ratio of the amount of these genes can be obtained therefrom.

Therefore, in this embodiment, an amyloid precursor protein (APP) gene (GenBank accession No. NM_000484), a beta -actin gene and an SRY gene (GenBank accession No. NM_000484) are used as specific genes commonly present in fetal DNA and maternal- , A pair of primers capable of amplifying the fetal DNA and the maternal DNA associated with the specific gene, and a probe having a fluorescent label to confirm the amplification reaction product were designed (see Table 1 below). These primers and probes were synthesized by requesting Bioneer (Daejeon, Republic of Korea).

Primer pairs and probes for real-time PCR amplification for specific genes Amplicon Length Fluorescent probe Forward primer Reverse primer Target
gene (5'-FAM, 3'-TAMRA) (5 '- > 3') (5 '- > 3') 67 bp ACCCCAGAGGAGCGCCACCTG
(SEQ ID NO: 1) TCAGGTTGACGCCGCTGT
(SEQ ID NO: 2) TTCGTAGCCGTTCTGCTGC
(SEQ ID NO: 3) APP 180 bp TCTATAAATGGACACCGATGGGTAGT (SEQ ID NO: 4) 83 bp CCCACTTCTCTCTAAGGAGAATGGCCC (SEQ ID NO: 5) TACAGGAAGTCCCTTGCCAT
(SEQ ID NO: 6) CCTGTGTGGACTTGGGAGAG
(SEQ ID NO: 7) beta -actin 170 bp CACGAAGGCTCATCATTCAA
(SEQ ID NO: 8) 67 bp TTCCGCTGCCCTGAGGCACT
(SEQ ID NO: 9) AGAGCTACGAGCTGCCTGAC
(SEQ ID NO: 10) CCATCTCTTGCTCGAAGTCC
(SEQ ID NO: 11) 169 bp GGCAGGACTTAGCTTCCACA
(SEQ ID NO: 12) 79 bp CGCCTTCCGACGAGGTCGAT
(SEQ ID NO: 13) TACAGGCCATGCACAGAGAG
(SEQ ID NO: 14) CAATTCTTCGGCAGCATCTT
(SEQ ID NO: 15) SRY 185 bp GGTAAGTGGCCTAGCTGGTG
(SEQ ID NO: 16) 90 bp TCTTGCGCCTCTGATCGCGA
(SEQ ID NO: 17) AACGCATTCATCGTGTGGT
(SEQ ID NO: 18) CAGCTGCTTGCTGATCTCTG
(SEQ ID NO: 19) 189 bp TTCCGACGAGGTCGATACTT
(SEQ ID NO: 20)

For reference, in Table 1, in order to evaluate the ratio of the amount of these genes through quantitative comparison of fetal DNA and maternal DNA using real-time PCR, it is considered that the size of fetal DNA is smaller than the size of DNA of the mother Primer pairs were designed. In particular, as shown in Table 1, in order to accurately evaluate the amount of the maternal gene and the amount of the gene derived from the fetus and to unify the analysis conditions at the time of quantitative evaluation, a short amplicon from the fetal DNA and a maternal- The detection probes for the set of long amplicones and forward primers were designed to be common and reverse primer only. Each detection probe is double-labeled with a fluorescent substance and a small-molecule substance to quantitatively analyze the result of a real-time PCR amplification reaction. In the present embodiment, the fluorescent probe, FAM, was labeled at the 5 'end of each detection probe, and TAMRA, which is a small mineral, was labeled at the 3' end.

As shown in Table 1, in the case of the APP target gene, a short amplicon (length 67 bp) from the fetal DNA and the maternal DNA was obtained from the primer pairs of SEQ ID NOS: 2 and 3, From the primer pair, a long amplicon (length of 180 bp) from the maternal DNA is obtained.

In the case of the? -actin gene, a short amplicon (length 83 bp) from the fetal DNA and the maternal DNA was obtained from the primer pairs of SEQ ID NOS: 6 and 7, and from the primer pairs of SEQ ID NOS: 6 and 8, (Length 170 bp) is obtained. From the primer pairs of SEQ ID NOs: 10 and 11, a short amplicon (length 67 bp) from the fetal DNA and the maternal DNA was obtained. From the primer pairs of SEQ ID NOs: 10 and 12, the long amplicon (Length 169 bp) is obtained.

For the SRY gene, a short amplicon (length 79 bp) from the fetal DNA and the maternal DNA was obtained from the primer pairs of SEQ ID NOS: 14 and 15, and from the primer pairs of SEQ ID NOS: 14 and 16, A long amplicon (length 185 bp) is obtained. From the primer pairs of SEQ ID NOs: 18 and 19, a short amplicon (length 90 bp) from the fetal DNA and the maternal DNA was obtained. From the primer pairs of SEQ ID NOs: 18 and 20, the long amplicon (Length 189 bp) is obtained.

Example 1-2: Real-time PCR using pregnant blood sample

Genomic DNA (gDNA) extracted from the blood or plasma of pregnant women or the blood of pregnant women was used for blind test from the general hospital gynecology department. Each sample is given an arbitrary sample number as shown in Table 4 below, and then the same sample number is used for the extracted gDNA sample corresponding to each sample. Plasma was separated from blood samples of pregnant women. For plasma samples (usage: 1 ml), gDNA was extracted using QIAamp Circulating Nucleic Acid Kit (Qiagen Cat # 55114) from QIAGEN, Germany. The gDNA extraction procedure was performed according to the manufacturer's manual of QIAamp Circulating Nucleic Acid Kit. This will be described in detail as follows.

First, 100 μL of proteinase K was added to a 50 mL centrifugal butt to remove protein components present in the pregnant plasma sample and to increase the purity of the sample. Then, 1 ml of pregnant plasma was added to a 50 ml centrifuge tube to which Proteinase K was added, and an ACL buffer containing 1.0 μg of carrier RNA was added, followed by vortexing Plasma samples were mixed with the ACL buffer. After mixing the mixed plasma sample with the ACL buffer for 30 minutes at 60 ° C, 3.6 ml of an ACB buffer (lysate buffer) was added to the centrifuge tube and subjected to vortexing to obtain an ACB buffer mixture And then reacted on ice for 5 minutes.

A vacuum pump system was used to increase the yield of gDNA extracted by increasing the adsorption of membranes present on the columns of the QIAamp Circulating Nucleic Acid Kit. Using a vacuum pump system, a QIAamp Mini column and a 20 ml tube extender were installed to allow the ACB buffer mixture to flow through the QIAamp mini column. After operating the vacuum pump system and flowing the ACB buffer mixture, the vacuum pump system was adjusted to zero and the tube extender removed. This ensures that as much reaction material as possible is bound to the membrane of the QIAamp mini column.

Then, 600 μL of ACW1 buffer, which is the buffer to be firstly washed with the QIAamp mini column-bound reactant, was pumped into the QIAamp mini column by operating the vacuum pump system, and the vacuum pump system was adjusted to a value of 0, The extender has been removed. This process cleans the DNA bound to the QIAamp mini column membrane. In addition, a secondary washing operation is performed to obtain high-purity DNA. The second wash solution, 750 μL of ACW2 buffer, was run through a vacuum pump system into the QIAamp mini-column, adjusted to a vacuum pump system reading of 0 and the tube extender removed. Through this process, the DNA bound to the QIAamp mini column membrane is further washed, and high-purity DNA can be obtained.

750 μL of the last wash solution (96% to 100%) of ethanol was pumped into the QIAamp mini-column by operating the vacuum pump system, the vacuum pump system was adjusted to 0 and the tube extender was removed. Then, the extracted DNA was purified by removing the ethanol component to a maximum degree, and then the lid of the QIAamp mini column was closed and centrifuged at 20,000 g and 14,000 rpm for 3 minutes. The lid of the QIAamp mini column was opened and allowed to dry for 10 minutes at 56 ° C. Then, 20 μL of AVE buffer was added to the QIAamp mini-column and reacted at room temperature for 3 minutes, then the lid of the QIAamp mini column was closed and centrifuged at 20,000 g and 14,000 rpm for 1 minute. Thus, a gDNA sample corresponding to each of the blood or plasma samples of the pregnant woman provided for the blind test was finally obtained.

Real-time PCR was performed to quantify a gDNA sample provided for blind testing or a gDNA sample separated from a blood or plasma sample through the above procedure. Real-time PCR was performed in this example using the 7900HT Fast Real Time PCR System (Life Technologies, USA). Each primer pair and a detection probe for obtaining and detecting each ampiclone shown in Table 1, a DNA template prepared as described above, and a real-time PCR master mix such as a real-time PCR master mixture Real-time PCR was performed under the real-time PCR amplification conditions shown in Table 3 below after the composition was as shown in Table 2 below. On the other hand, the master mix for PCR amplification of the PCR amplification composition, for example, PCR buffer, dNTP, DNA polymerase been made by Ajay et al., In this embodiment, TaqMan ® Universal Master Mix Ⅱ by real-time PCR Master Mix, no UNG ( Life Technologies, Cat. 4440040) was used.

Real-time PCR amplification composition No. PCR amplification composition Volume ([mu] l) One A sample with a DNA template One 2 Primer (5 pM) Forward primer 0.5 Reverse primer 0.5 3 Detection probe One 4 Distilled water 7 5 TaqMan ® Universal Master Mix II, no UNG 10 Sum 20

Real-time PCR amplification conditions No. step Temperature time cycle One Pre-denaturation 95 ℃ 10 minutes 2 denaturalization 95 ℃ 15 seconds 50 cycles 3 Annealing 60 ° C 60 seconds 4 extension 72 ℃ 60 seconds

In this example, real-time PCR was repeated for the respective DNA templates prepared for the blind test under the real-time PCR amplification composition of Table 2 above under the real-time PCR condition of Table 3 above. Each time the real-time PCR amplification reaction was repeated, the fluorescence value was measured at the FAM wavelength, and the fluorescence intensity for the reaction cycle was analyzed using SDS software. The real-time PCR quantification results for the APP genes are summarized for each sample, and the results are shown in Fig. 1, which is divided into the results for the short amplicon (length 67 bp) and the results for the long amplicon (length 180 bp). If the R2 value is 0.99 or more, it means that the reliability value is high, so that the standard curve of FIG. 1 has high stability and the obtained quantitative result is highly reliable.

The result of quantitative real-time PCR for a short amplicon (for example, length 67 bp) obtained for each sample and the quantitative result of real-time PCR for a long amplicon (for example, length 180 bp) Substitute in Equation 1 to obtain the ratio.

Equation 1

Rate = Short Amplicon (length 67bp) Amplicon / length (length 180bp) Amount

Then, by substituting the ratio value obtained in the above equation (1) into the following equation (2), the content of the fetal-derived gene existing in pregnant blood or plasma can be obtained for each sample.

Equation 2

(%) = (Ratio value obtained from formula (1) -1.1188) /0.065

For example, the amount of the 67 bp amplicon for the APP gene, which is judged to be contained in the pregnant blood plasma to plasma intracellular embryo-derived gene set, and the amount of the 180 bp amplicon compared to the 67 bp amplicon The ratio of the gene derived from the cell free embryo can be determined through comparison, and the content of the gene derived from the fetal gene existing in the pregnant blood or plasma can be deduced by using Equation (2).

Table 4 below shows the quantitative results of real-time PCR on short amplicon (67 bp in length), quantitative results of real-time PCR on long amplicons (length 180 bp), their ratio, and the ratio of these to the fetal- The results are shown in the form of a table for each sample.

Sample
number 67 bp sheep 180 bp sheep ratio Fetal DNA% Sample
No. 67 bp sheep 180 bp sheep ratio Fetal DNA% 24 19.08056 9.790299 1.948925 12.77115 38 17.02248 16.8546 1.00996 0 36 26.99327 12.52466 2.15521 15.94477 39 14.4778 12.4566 1.16226 0.66861 3 11.12497 9.32045 1.193608 1.150892 40 12.23988 10.11207 1.210423 1.409581 4 8.520723 7.1852 1.185871 1.031862 41 9.523374 10.97833 0.86747 0 5 14.76195 11.57453 1.275382 2.408954 46 7.422427 7.21746 1.028399 0 6 12.48913 11.34698 1.100657 0 47 10.41614 8.4564 1.231746 1.737632 34 18.78969 16.4453 1.142557 0.365492 48 9.18421 11.6727 0.786811 0 12 16.21022 11.456 1.414998 4.556892 50 4.708003 3.641657 1.292819 2.677215 14 15.87964 8.4676 1.875341 11.63909 F1 5823.917 5057.577 1.151523 0.503433 19 15.88142 11.4556 1.386345 4.116077 F22 6962.107 4632.537 1.502871 5.90879 22 19.49134 16.80018 1.160186 0.636708 G1 45837.89 39213.56 1.16893 0.771224 33 46.00689 41.4642 1.109557 0

As shown in Table 4, the blind test samples (for example, F1 sample, F22 sample, and G1 sample), which are calculated to contain a predetermined amount of the fetal gene, It can be used for digital PCR analysis related to abnormal gene number.

Example 2: Performing Copy Number Assays and Copies Variation (CNV) Test

Copy count determination and copy number variation test can be performed to re-confirm whether the content of fetal DNA derived from the real-time PCR quantitation result in Example 1 is reliable. The TaqMan ® assay copy number (Copy Number Assays) and copy number variation tests known in the art is performed as follows.

(1) DNA extraction

Genomic DNA (gDNA) is used as a template for the TaqMan ® Copy Number Assays.

(2) Checking the amount of DNA

Standard curves of genomic DNA or plasmid DNA templates are made using quantitative PCR. The amount of DNA is measured using the UV absorbance (A260 / A280). For human gDNA, the absorbance ratio of A260 / A280 is 1.7 or higher.

(3) DNA dilution and plate preparation

Dilute the DNA so that the same amount of gDNA can be added to each well of the plate.

(4) Sample type and number

A gDNA sample in which the number of copies of the target template is unknown, a control sample (No Template Controls) (NTC) which shows a background fluorescence value as a control sample with no template and is used to check the contamination of the experiment, Use a calibrator sample to know the number of copies of the template.

(5) Reaction preparation for liquid gDNA

The amount of gDNA to be assayed is determined based on the amount of reaction and the number of experiments. The total amount is determined taking into account the amount consumed when transferring the reaction solution (see Table 5 below).


 Final reaction solution volume Amount of gDNA required per well 5 ng / [mu] L (5x) The volume of addition of the stock solution of gDNA per well 384-well plate 10 μL 10 ng 2 μL 96-well plates 20 μL 20 ng 4 μL

TaqMan ® assay copy number (Copy Number Assays) and mix the reagents and TaqMan ® reference copy number assays (Copy Number Reference Assays) reagent. Shake it gently and centrifuge to collect the reaction solutions. TaqMan ® genotype ping master mix to complete the reaction mixture (see Table 6, below).

The reaction solution mixture Volume per well (μL) 384-well plate 96-well plates 2 × TaqMan ® Geno Type Ping Master Mix 5.0 10.0 TaqMan ® copy number assay reagent (20 ×) 0.5 1.0 Refer to TaqMan ® Copy Number Assay reagent (20 ×) 0.5 1.0 Water without nuclease 2.0 4.0 Total volume 8.0 16.0

Place the reaction solution in a microcentrifuge tube so that the whole reaction solution mixture is well mixed, and dispense it into a prepared reaction well plate.

A diluted gDNA sample is prepared, and the diluted gDNA is added to the well-dispensed well plate. Alternatively, the gDNA may be firstly dispensed and the reaction mixture added. The gDNA and the reaction mixture are mixed well, and the reaction well plate is sealed and centrifuged.

Add 2 μL of gDNA (5 ng / μL) per well for 384-well plates as shown in Table 5 above and 4 μL of gDNA (5 ng / μL) per well for 96-well plate was added to the reaction is conducted according to the manual of the manufacturer TaqMan ® under the conditions shown in Table 7 and analyzing the results.

step Temperature time Hold 95 ℃ 10 minutes cycle
(40 cycles) 95 ℃ 15 seconds 60 ° C 60 seconds

(6) Copy Number Variation (CNV) test

The results of the TaqMan ® assay copy number (Copy Number Assays) performed in the order as described above for analysis software, for example, were analyzed by using the CopyCaller ® software (Fig. 2). As a result of the analysis, the copy number results for all the samples and the control with no template (NTC) were derived, and considering that female pregnant women can not have the Y chromosome gene, It can be determined that the fetal DNA is present in the sample in which the result for the Y chromosome gene is detected in the sample (the sample shown in Table 4).

Example 3 Preparation of Fetal Derived Gene Using Digital PCR

Example 3-1: Selection of target gene and design of primer pair and probe

First, in order to perform a gene analysis for a fetal gene, for example, a gene abnormality such as a trisomy, the following gene sequence on the chromosome 21 is used as a target gene sequence (see FIG. 3) , And primer pairs and probes were designed based on this.

(1) Target gene sequence (chromosome 21): NCBI's GenBank Accession No. NC_018932.2 (SEQ ID NO: 21)

A primer pair for amplifying the target gene of SEQ ID NO: 21 and a probe for detecting the signal of the amplification product are as follows. The 5 'end of the detection probe was labeled with FAM and the 3' end with TAMRA. However, the present invention is not limited thereto, and it is a matter of course that various fluorescent substances and minerals known in the art can be labeled at the 5 'end and the 3' end, respectively.

Forward primer 21-01_73 F: 5'-ATGCTGTTCATGGAGGATGCT-3 '(SEQ ID NO: 22)

Reverse primer 21-01_73 R: 5'-TCTATCTCCCACTCTCTCCAACTAATC-3 '(SEQ ID NO: 23)

Detection probe 21-01_73 P: 5'-AGGAAGATGAGAGAAAATTAGTA-3 '(SEQ ID NO: 24)

(2) Target gene sequence (chromosome 21): NCBI's GenBank Accession No. NC_000021.9 (SEQ ID NO: 25)

A primer pair for amplifying the target gene of SEQ ID NO: 25 and a probe for detecting a signal of the amplification product are as follows. The 5 'end of the detection probe was labeled with FAM and the 3' end with TAMRA. However, the present invention is not limited thereto, and it is a matter of course that various fluorescent substances and minerals known in the art can be labeled at the 5 'end and the 3' end, respectively.

Forward primer 21-03_73 F: 5'-GCACTGGGAATTTATTAGCACAAA-3 '(SEQ ID NO: 26)

Reverse primer 21-03_73 R: 5'-GCTAAGATATGGTAGAGTCCCTTCTGA-3 '(SEQ ID NO: 27)

Detection probe 21-03_73 P: 5'-TCATTCTTTGCAACTCAAA-3 '(SEQ ID NO: 28)

(3) Target gene sequence (chromosome 21): NCBI's GenBank Accession No. NC_018932.2 (SEQ ID NO: 29)

A primer pair for amplifying the target gene of SEQ ID NO: 29 and a probe for detecting the signal of the amplification product are as follows. The 5 'end of the detection probe was labeled with FAM and the 3' end with TAMRA. However, the present invention is not limited thereto, and it is a matter of course that various fluorescent substances and minerals known in the art can be labeled at the 5 'end and the 3' end, respectively.

Forward primer 21-05_58 F: 5'-TGATGGGCTTGCGCTTATC-3 '(SEQ ID NO: 30)

Reverse primer 21-05_58 R: 5'-TGGGCTCCACAAGAAATCAGA-3 '(SEQ ID NO: 31)

Detection probe 21-05_58 P: 5'-TTGTCCCTTCTCCC-3 '(SEQ ID NO: 32)

Next, in order to carry out a gene analysis on the fetal gene, the sequence of the control gene serving as a standard for calculating the normalization value for the amount of amplification of the target gene as described above is referred to as the first gene on chromosome 1 and the second gene on chromosome 2 (See Fig. 3).

(4) Control gene sequence (chromosome 2): NCBI's GenBank Accession No. NC_018913.2 (SEQ ID NO: 33)

The primer pair for amplifying the control gene of SEQ ID NO: 33 and the probe for detecting the signal of the amplification product are as follows. The 5 'end of the detection probe was labeled with VIC and the 3' end with TAMRA. However, the present invention is not limited thereto, and it is a matter of course that various fluorescent substances and minerals known in the art can be labeled at the 5 'end and the 3' end, respectively.

Forward primer 02-03_68 F: 5'-GAGGGACTCTGCCATTACTTAGCT-3 '(SEQ ID NO: 34)

Reverse primer 02-03_68 R: 5'-CAAGCCCATTCACGTTTGG-3 '(SEQ ID NO: 35)

Detection probe 02-03_68 P: 5'-AGCACAGAGGAGTGCC-3 '(SEQ ID NO: 36)

(5) Control gene sequence (chromosome 1): NCBI's GenBank Accession No. NC_018912.2 (SEQ ID NO: 37)

A pair of primers for amplifying the control gene of SEQ ID NO: 37 and a probe for detecting the signal of the amplification product are as follows. The 5 'end of the detection probe was labeled with VIC and the 3' end with TAMRA. However, the present invention is not limited thereto, and it is a matter of course that various fluorescent substances and minerals known in the art can be labeled at the 5 'end and the 3' end, respectively.

Forward primer 01-05_56 F: 5'-CATGGGCCCCACATGGT-3 '(SEQ ID NO: 38)

Reverse primer 01-05_56 R: 5'-GCCAGTGTGCCTGAAGCATA-3 '(SEQ ID NO: 39)

Detection probe 01-05_56 P: 5'-CTAGTTTCCCCGGCTCA-3 '(SEQ ID NO: 40)

Meanwhile, the control genes, target genes and chromosomes exemplified in this embodiment should be understood as an example, and the method of the present invention for analyzing genetic information of a fetus from blood or plasma of a pregnant woman using digital PCR may be applied to various individuals or samples , As a base technology applicable to analysis of genotypes or abnormality in gene numbers for various target genes and chromosomes.

A primer pair for amplifying the target gene and the control gene as described above and a probe for detecting the amplified product from the primer pair were synthesized by Biona (Daejeon, Republic of Korea).

Example 3-2: Evaluation of primer and probe performance for fetal-derived gene analysis

First, in order to confirm whether the corresponding target gene and the control gene were amplified normally using each primer pair designed in Example 3-1, the PCR composition was set as shown in Table 8 below, and PCR amplification PCR amplification was performed. On the other hand, PCR amplification master mix for PCR amplification of the composition, for example, PCR buffer, dNTP, DNA polymerase been made by Ajay et al., In this embodiment, TaqMan ® Universal Master Mix Ⅱ, no UNG (Life Technologies to the PCR master mix , Cat. 4440040) was used.

PCR amplification composition No. PCR amplification composition Volume ([mu] l) One Sample with DNA template or control without mold (NTC) One 2 Primer (5 pM) Forward primer 0.5 Reverse primer 0.5 3 Distilled water 8 4 TaqMan ® Universal Master Mix II, no UNG 10 Sum 20

PCR amplification conditions No. step Temperature time cycle One Pre-denaturation 95 ℃ 10 minutes 2 denaturalization 95 ℃ 30 seconds 40 cycles 3 Annealing 60 ° C 60 seconds

The PCR amplified product under the above composition and conditions was subjected to electrophoresis to confirm bands. As a result, it was confirmed that the target gene and the control gene corresponding to each primer pair were amplified normally (see FIG. 5).

Next, real-time PCR was performed on the target gene and the control gene using the respective primer pairs and probes designed in Example 3-1. In this example, real-time PCR was performed using a 7900HT Fast Real Time PCR System (Life Technologies, USA). The samples prepared in Example 1-2 (F1 sample confirmed to contain a fetal gene, F22 sample , G1 sample) and a real-time PCR master mixture were prepared as shown in Table 10 below, and real-time PCR amplification was carried out under real-time PCR amplification conditions shown in Table 11 below. PCR was performed.

Real-time PCR amplification composition No. PCR amplification composition Volume ([mu] l) One Sample with DNA template or control without mold (NTC) One 2 Primer (5 pM) Forward primer 0.5 Reverse primer 0.5 3 Detection probe One 4 TaqMan ® Universal Master Mix II, no UNG 5 5 Distilled water 2 Sum 10

Real-time PCR amplification conditions No. step Temperature time cycle One Activation 50 ℃ 2 minutes 2 Hold 95 ℃ 10 minutes 3 denaturalization 95 ℃ 15 seconds 40 cycles 4 Annealing 60 ° C 60 seconds

The results of real-time PCR under real-time PCR composition and conditions as described above are shown in Figs. 6 to 24. In the case of the F1 sample, the F22 sample and the G1 sample, the target gene and the control gene are normally expressed in real time Amplification curves indicating that PCR amplification was performed were confirmed (Figures 6 to 19) and no significant amplification curves were observed for template-free control (NTC) (Figures 20-24). Therefore, it was confirmed that the primer pair and the probe designed in Example 3-1 were suitable for the specific amplification and detection of the corresponding target gene and the control gene contained in the sample.

Example 4 Analysis of Fetal Derived Gene Using Digital PCR

Example 4-1: Digital PCR performed on a sample confirmed to contain a fetal gene

The F1 sample, the F22 sample, the G1 sample, the G2 sample (the sample in which the DNA extraction was once added to the G1 sample) and the control without the template, which were confirmed to contain a predetermined amount of the fetal gene from the gDNA samples prepared in Example 1-2, (NTC) were distributed to each well on an assigned array of OpenArray ( R) well plates (Life Technologies, USA) according to the digital PCR layout of Figure 25, and each well on each sample- Each primer pair and a corresponding probe (total of 5 sets) were dispensed (see Fig. 25). At this time, the digital PCR composition per well was as shown in Table 12 below, and 20-fold dilutions of each sample of 20 ng / ul were used because they did not know the sweet spot of the sample for digital PCR.

Digital PCR amplification was performed using an AccuFill device and a QuantStudio 12K Flex device (Life Technologies, USA). Digital PCR amplification was performed according to the manufacturer's manual under the digital PCR amplification conditions shown in Table 13 below. On the other hand, digital PCR amplified digital PCR amplification master mix for one composition are, for example, PCR buffer, dNTP, as a DNA polymerase consisting of a kinase, such as, in the present embodiment, a digital PCR master mix TaqMan ® Universal Master Mix Ⅱ, no UNG (Life Technologies, Cat. 4440040) was used.

Digital PCR amplification composition No. PCR amplification composition Volume ([mu] l) One Samples with DNA templates or NTC One 2 Primer (5 pM) Forward primer 0.5 Reverse primer 0.5 3 Detection probe (5 pM) One 4 TaqMan ® Universal Master Mix II, no UNG 3

Digital PCR amplification conditions No. step Temperature time cycle One Hold 95 ℃ 20 seconds 2 denaturalization 95 ℃ 1 second 40 cycles
3 Annealing 60 ° C 20 seconds

In FIG. 26, the result of performing digital PCR in this embodiment is shown as a digital PCR heat map. As shown in Fig. 26, the target gene (the gene of SEQ ID NO: 21, SEQ ID NO: 25 or SEQ ID NO: 29) or the control gene (the gene of SEQ ID NO: 33 or SEQ ID NO: 37) The fluorescence signal was generated in the well where the amplification occurred and the fluorescence signal was not detected in the well where amplification did not occur.

27, fluorescence values measured in each well of the digital PCR layout were analyzed by execution of QuantStudio 12K Flex software and DigitalSuite software provided in the QuantStudio 12K Flex device, and the fluorescence values of each target gene of Example 3-1 (SEQ ID NO: 21, SEQ ID NO: 25 or SEQ ID NO: 29) and the number of copies of each control gene (SEQ ID NO: 33 or SEQ ID NO: 37). As a result of this copy number analysis, it was confirmed that the number of copies of the control gene per well or the number of copies of the target gene was excessively large and did not satisfy the phasic distribution. Therefore, it was analyzed that additional sample dilution and determination of the proper concentration were necessary.

Example 4-2: Digital PCR performed on diluted F1 and F22 samples

Each of the F1 sample and the F22 sample, which were confirmed to contain a predetermined amount of the fetal gene from the gDNA samples prepared in Example 1-2, were placed into an allocated array of OpenArray 占 well plate (Life Technologies, USA) according to the digital PCR layout of Fig. And each primer pair designed in Example 3-1 and corresponding probes (total of 5 sets) were dispensed into each well on the array for each sample (see Fig. 28).

The digital PCR composition per well was as shown in Table 14 below, and each sample of 20 ng / ul was diluted 100-fold to select the sweet spot of the sample for digital PCR. On the other hand, the digital PCR device and PCR conditions were the same as in Example 4-1.

Digital PCR amplification composition No. PCR amplification composition Volume ([mu] l) One A sample with a DNA template One 2 Primer (20 pM) Forward primer 0.25 Reverse primer 0.25 3 Detection probe (5 pM) One 4 TaqMan ® Universal Master Mix II, no UNG 2.5

In FIG. 29, the result of performing digital PCR in the present embodiment is shown as a digital PCR heat map. (The gene of SEQ ID NO: 21, SEQ ID NO: 25 or SEQ ID NO: 29) or the control gene (the gene of SEQ ID NO: 33 or SEQ ID NO: 37) for each sample on the digital PCR layout of FIG. The fluorescence signal was generated in the well where the amplification occurred and the fluorescence signal was not detected in the well where amplification did not occur.

30, the fluorescence values measured in each well of the digital PCR layout were analyzed by execution of QuantStudio 12K Flex software and DigitalSuite software provided in the QuantStudio 12K Flex device, and the fluorescence values of each target gene (SEQ ID NO: 21, SEQ ID NO: 25 or SEQ ID NO: 29) and the number of copies of each control gene (SEQ ID NO: 33 or SEQ ID NO: 37). As a result of the analysis of the number of copies, a stable result value was obtained rather than the case of Example 4-1. However, in order to confirm the number of genes of the fetal-derived target gene, the number of copies of the control gene per well still exceeded the Poisson distribution Respectively. Therefore, it was analyzed that additional sample dilution and determination of the proper concentration were necessary.

Example 4-3: Digital PCR performed on diluted G1 samples and male control samples

A G1 sample, which was confirmed to contain a predetermined amount of a fetal gene from the gDNA samples prepared in Example 1-2, and a genomic DNA sample (male control sample) of a normal male genetic disorder 31 into a respective well on an assigned array of OpenArray ( R) well plates (Life Technologies, USA) according to the digital PCR layout of FIG. 31, and each primer pair designed in Example 3-1 and And the corresponding probes (total of 5 sets) were dispensed (see Fig. 31).

The digital PCR composition per well was as shown in Table 15 below, and each sample of 20 ng / ul was diluted 100-fold to select the sweet spot of the sample for digital PCR. On the other hand, the digital PCR device and PCR conditions were the same as in Example 4-1.

Digital PCR amplification composition No. PCR amplification composition Volume ([mu] l) One A sample with a DNA template One 2 Primer (20 pM) Forward primer 0.25 Reverse primer 0.25 3 Detection probe (5 pM) One 4 TaqMan ® Universal Master Mix II, no UNG 2.5

In FIG. 32, the result of digital PCR performed in this embodiment is shown as a digital PCR heat map. As shown in Fig. 32, the target gene (the gene of SEQ ID NO: 21, SEQ ID NO: 25 or SEQ ID NO: 29) or the control gene (the gene of SEQ ID NO: 33 or SEQ ID NO: 37) The fluorescence signal was generated in the well where the amplification occurred and the fluorescence signal was not detected in the well where amplification did not occur.

33 shows fluorescence values measured in each well of the digital PCR layout by the execution of QuantStudio 12K Flex software and DigitalSuite software provided in the QuantStudio 12K Flex device, and the fluorescence values measured in each well of each of the samples were measured for each target gene (SEQ ID NO: 21, SEQ ID NO: 25 or SEQ ID NO: 29) and the number of copies of each control gene (SEQ ID NO: 33 or SEQ ID NO: 37). As a result of the analysis of the number of copies, a stable result was obtained rather than the case of Example 4-1. However, in order to confirm the number of genes of the fetal-derived target gene for a normal male gene, the number of copies of the control gene per well is still excessively large, Distribution was not satisfied. Therefore, it was analyzed that additional sample dilution and determination of the proper concentration were necessary.

Example 4-4: Performing a digital PCR on an additional diluted G1 sample and a male control sample

A G1 sample, which was confirmed to contain a predetermined amount of a fetal gene from the gDNA samples prepared in Example 1-2, and a genomic DNA sample (male control sample) of a normal male genetic disorder 34 in accordance with the digital PCR layout of OpenArray ® well plate (Life Technologies, USA), and each well on an array of each sample was loaded with the respective primer pairs designed in Example 3-1 and And a corresponding probe (total of 5 sets) was dispensed (see Fig. 34).

The digital PCR composition per well was as shown in Table 16 below, and each sample of 20 ng / ul was diluted 1000-fold to select the sweet spot of the sample for digital PCR. On the other hand, the digital PCR device and PCR conditions were the same as in Example 4-1.

Digital PCR amplification composition No. PCR amplification composition Volume ([mu] l) One A sample with a DNA template One 2 Primer (20 pM) Forward primer 0.25 Reverse primer 0.25 3 Detection probe (5 pM) One 4 TaqMan ® Universal Master Mix II, no UNG 2.5

In FIG. 35, the result of digital PCR performed in this embodiment is shown as a digital PCR heat map. As shown in Fig. 35, the target gene (the gene of SEQ ID NO: 21, SEQ ID NO: 25 or SEQ ID NO: 29) or the control gene (the gene of SEQ ID NO: 33 or SEQ ID NO: 37) The fluorescence signal was generated in the well where the amplification occurred and the fluorescence signal was not detected in the well where amplification did not occur.

36, the fluorescence values measured in each well of the digital PCR layout were analyzed by execution of QuantStudio 12K Flex software and DigitalSuite software provided in the QuantStudio 12K Flex device, and the fluorescence values of each target gene (SEQ ID NO: 21, SEQ ID NO: 25 or SEQ ID NO: 29) and the number of copies of each control gene (SEQ ID NO: 33 or SEQ ID NO: 37).

As a result of the analysis of the number of copies, a stable overall result was obtained, and it was confirmed that the copy number of the control gene of SEQ ID NO: 33 and the copy number of the control gene of SEQ ID NO: 37 were close to one statistically. That is, in this embodiment, the control gene (SEQ ID NO: 33 or SEQ ID NO: 37) of each sample may be regarded as being statistically distributed about 1 per well, thus satisfying the Poisson distribution. Therefore, a digital PCR amplification product of each of the target genes (SEQ ID NO: 21, SEQ ID NO: 25 or SEQ ID NO: 29) was quantitated, and each quantified value was compared with the respective control genes (SEQ ID NO: 33 or SEQ ID NO: 37) The number of genes of the fetal-derived target gene relative to the normal male gene can be confirmed. The results are shown in Table 17 below.

The control gene of SEQ ID NO: 37
(Chromosome 1) Sample
number The control gene of SEQ ID NO: 33
(Chromosome 2) 1.65264 2.516783987 G1 2.112308677 1.387042 1.117781206 0.938141276 1.323355 1.110677064 1.03092 0.901134 MC 1.15633 1.181313 0.844723 1.21053 1.346902 1.17708

As shown in Table 17, in the case of the normal male sample (MC), the normalization value for the control gene (the gene on chromosome 1) of SEQ ID NO: 37 and the control gene (the gene on chromosome 2) 1, it can be confirmed that there is no variation in the number of genes of the target gene (gene on chromosome 21).

However, in the case of the G1 sample, the normalization values for the control gene of SEQ ID NO: 37 (gene on chromosome 1) and the control gene of SEQ ID NO: 33 (gene on chromosome 2) were calculated to be 1.65264 and 1.387042, respectively, Gene on the chromosome of the mouse). This means that the number of chromosome 21 genes is about 1.5 times larger than the number of chromosome 1 genes and the number of chromosome 2 genes known to be abnormal in the fetus. Thus, it can be assumed that the fetus has a trisomy, in which the chromosome number 21 of the chromosome 21, ie, the triplet of chromosome 21 (normal diploid), is derived from the embryo fetus, The risk of developing Down syndrome is high.

As described above, the method of the present invention can solve the problem caused by the difficulty of separation of only the signals derived from the fetal gene and the problem caused by the background signal of the pregnant woman, while satisfying the phasic distribution required in the digital PCR technique, And it is possible to solve the conventional problem that makes it difficult to apply the digital PCR technique to the genotyping technology using the fetal DNA-derived cell free gene.

Example 5: Preparation of trisomy of chromosome 21, chromosome 18 and chromosome 13 using digital PCR

Example 5-1: Preparation of sample to be tested from pregnant plasma sample and real-time PCR

The pregnant women in the general hospitals in Korea were given blood plasma for blind test. An arbitrary sample number such as MO, M6, M7, M9, S8, S8_1, S2, S4, S5, and S6 is assigned to each sample, and then the same sample number is used for the extracted gDNA sample corresponding to each sample. Using a QIAamp Circulating Nucleic Acid Kit (Qiagen Cat # 55114) from QIAGEN, Germany, a gDNA sample was extracted from a plasma sample (use amount: 1 ml) to be subjected to a trisomy of the fetal gene Respectively. The gDNA extraction process was performed according to the method described in Example 1-2.

The gDNA extracted from each plasma sample was analyzed for the content of the gene derived from fetus in the same manner as in Example 1-2. That is, the real-time PCR for the APP (Amyloid Precursor protein) gene (GenBank accession No. NM_000484), the β-actin gene, and the SRY gene was carried out using the primer pairs and the fluorescence- , And the content of the gene derived from the fetus was calculated from the real - time PCR quantification result. The fetal-derived gene content was calculated in the same manner as in Example 1-2.

Table 18 below shows the quantitative results of real-time PCR for short amplicons (67 bp in length), quantitative results of real-time PCR for long amplicons (length 180 bp), their ratio, and the ratio of their The results of the calculation of the content are shown for each sample.

Sample number 67 bp sheep 180 bp sheep ratio Fetal DNA% M0 33.58 21.43 1.57 6.90 M6 31.20 20.89 1.49 5.77 M7 32.70 26.48 1.23 1.78 M9 32.63 21.86 1.49 5.77 S8 12.35 8.26 1.50 5.79 S8_1 1.45 0.97 1.50 5.79 S2 6.94 4.59 1.51 6.03 S4 4.87 2.85 1.71 9.06 S5 28.66 17.92 1.60 7.30 S6 12.57 7.08 1.78 10.11

As shown in Table 18, the blind test sample (for example, MO sample, M6 sample, M9 sample, S8 sample, S8_1 sample, S2 sample, S4 Samples, S5 samples, and S6 samples) were used for digital PCR analysis related to the number of later fetal genes, ie, trisomy, since the fetal gene was sufficiently present.

Example 5-2: Selection of target gene for analysis of the number of genes of chromosome 13 and design of primer pair and probe

In addition to the chromosome 21 described in Example 3-1, the gene sequence on the chromosome 13 was further amplified in order to carry out an analysis of the gene abnormality such as a trisomy, (See FIG. 4), and a primer pair and a probe were designed based on the target gene sequence. The target gene sequence on chromosome 13, the primer pair to amplify it, and the detection probe are as follows.

(1) Target gene sequence (chromosome 13): NCBI's GenBank Accession No. NC_018924.2 (SEQ ID NO: 41)

The primer pair for amplifying the target gene of SEQ ID NO: 41 and the probe for detecting the signal of the amplification product are as follows. The 5 'end of the detection probe was labeled with FAM and the 3' end with TAMRA. However, the present invention is not limited thereto, and it is a matter of course that various fluorescent substances and minerals known in the art can be labeled at the 5 'end and the 3' end, respectively.

Forward primer 13_1_2_62F: 5'-CAAAGGCAACGCGTCTTTC-3 '(SEQ ID NO: 42)

Reverse primer 13_1_2_62R: 5'-GGCAGAGTGGATGTTTTTGCA-3 '(SEQ ID NO: 43)

Detection probe 13_1_2_62P: 5'-AGCCAGGCAGTCTGTA-3 '(SEQ ID NO: 44)

(2) Target gene sequence (chromosome 13): NCBI's GenBank Accession No. NC_018924.2 (SEQ ID NO: 45)

The primer pair for amplifying the target gene of SEQ ID NO: 45 and the probe for detecting the signal of the amplification product are as follows. The 5 'end of the detection probe was labeled with FAM and the 3' end with TAMRA. However, the present invention is not limited thereto, and it is a matter of course that various fluorescent substances and minerals known in the art can be labeled at the 5 'end and the 3' end, respectively.

Forward primer 13_2_1_69F: 5'-TTCTACCAGAAAGGAATGAAGAACAG-3 '(SEQ ID NO: 46)

Reverse primer 13_2_1_69R: 5'-CCATTTGATATTTGTCTGCATTGTG-3 '(SEQ ID NO: 47)

Detection probe 13_2_1_69P: 5'-ACCTTCAGGAATTGAG-3 '(SEQ ID NO: 48)

(3) Target gene sequence (chromosome 13): NCBI's GenBank Accession No. NC_018924.2 (SEQ ID NO: 49)

The primer pair for amplifying the target gene of SEQ ID NO: 49 and the probe for detecting the signal of the amplification product are as follows. The 5 'end of the detection probe was labeled with FAM and the 3' end with TAMRA. However, the present invention is not limited thereto, and it is a matter of course that various fluorescent substances and minerals known in the art can be labeled at the 5 'end and the 3' end, respectively.

Forward primer 13_3_1_62F: 5'-TGTCCTGCTCGCGAGTGA-3 '(SEQ ID NO: 50)

Reverse primer 13_3_1_62R: 5'-TCGTCTAGCTCCTTCAGGATCTCT-3 '(SEQ ID NO: 51)

Detection probe 13_3_1_62P: 5'-CTGTCCTTCTTGCCCC-3 '(SEQ ID NO: 52)

(4) Target gene sequence (chromosome 13): NCBI's GenBank Accession No. NC_018924.2 (SEQ ID NO: 53)

The primer pair for amplifying the target gene of SEQ ID NO: 53 and the probe for detecting the signal of the amplification product are as follows. The 5 'end of the detection probe was labeled with FAM and the 3' end with TAMRA. However, the present invention is not limited thereto, and it is a matter of course that various fluorescent substances and minerals known in the art can be labeled at the 5 'end and the 3' end, respectively.

Forward primer 13_4_2_69F: 5'-GCAGAAAATGAATGATAGCATGGA-3 '(SEQ ID NO: 54)

Reverse primer 13_4_2_69R: 5'-CCACCAAGGTCCTGAGATCCT-3 '(SEQ ID NO: 55)

Detection probe 13_4_2_69P: 5'-ACCTCAAACAAGGAAGAG-3 '(SEQ ID NO: 56)

On the other hand, the chromosome 13 and related target genes exemplified in the present embodiment should be understood as an example of a chromosome and related target genes to be subjected to a gene number abnormality such as a trichrome (trisomy) The method of the present invention for analyzing genetic information of a fetus from blood or plasma of a pregnant woman should be understood as a base technique applicable to various individuals or samples and genotyping or abnormal analysis of gene numbers for various target genes and chromosomes.

A primer pair for amplifying each of the target genes as described above and a probe for detecting the amplification product amplified from the primer pair were synthesized by requesting Bioneer (Daejeon, Republic of Korea).

Example 5-3: Selection of Target Gene for Analysis of Abnormal Number of Chromosome 18 and Designing of Primer Pair and Probe

In addition to the chromosome 21 described in Example 3-1, the gene sequence on the chromosome 18 was further amplified to perform gene analysis for the fetal gene, for example, a trisomy, (See FIG. 4), and a primer pair and a probe were designed based on the target gene sequence. The target gene sequence on chromosome 18, the primer pair to amplify it, and the detection probe are as follows.

(1) Target gene sequence (chromosome 18): NCBI's GenBank Accession No. NC_018929.2 (SEQ ID NO: 57)

A primer pair for amplifying the target gene of SEQ ID NO: 57 and a probe for detecting a signal of the amplification product are as follows. The 5 'end of the detection probe was labeled with FAM and the 3' end with TAMRA. However, the present invention is not limited thereto, and it is a matter of course that various fluorescent substances and minerals known in the art can be labeled at the 5 'end and the 3' end, respectively.

Forward primer 18_1_2_57F: 5'-CTGCTGGGCCCCCTTT-3 '(SEQ ID NO: 58)

Reverse primer 18_1_2_57R: 5'-GGGTTACTTGGGCAGAATGTCA-3 '(SEQ ID NO: 59)

Detection probe 18_1_2_57P: 5'-TGCTTCATGTCCTCTTG-3 '(SEQ ID NO: 60)

(2) Target gene sequence (chromosome 18): NCBI GenBank Accession No. NC_018929.2 (SEQ ID NO: 61)

A primer pair for amplifying the target gene of SEQ ID NO: 61 and a probe for detecting a signal of the amplification product are as follows. The 5 'end of the detection probe was labeled with FAM and the 3' end with TAMRA. However, the present invention is not limited thereto, and it is a matter of course that various fluorescent substances and minerals known in the art can be labeled at the 5 'end and the 3' end, respectively.

Forward primer 18_2_2_58F: 5'-GCCCCGTTCACGTCAGTTT-3 '(SEQ ID NO: 62)

Reverse primer 18_2_2_58R: 5'-TGGTACTGGCTTCGCTTTCTC-3 '(SEQ ID NO: 63)

Detection probe 18_2_2_58P: 5'-ACGTCTGCAACGGG-3 '(SEQ ID NO: 64)

(3) Target gene sequence (chromosome 18): NCBI GenBank Accession No. NC_018929.2 (SEQ ID NO: 65)

The primer pair for amplifying the target gene of SEQ ID NO: 65 and the probe for detecting the signal of the amplification product are as follows. The 5 'end of the detection probe was labeled with FAM and the 3' end with TAMRA. However, the present invention is not limited thereto, and it is a matter of course that various fluorescent substances and minerals known in the art can be labeled at the 5 'end and the 3' end, respectively.

Forward primer 18_3_2_62F: 5'-AGCTGGAAGTCGAGTGTGCTACT-3 '(SEQ ID NO: 66)

Reverse primer 18_3_2_62R: 5'-CTGCCGGAAGTTCAGTTTGTC-3 '(SEQ ID NO: 67)

Detection probe 18_3_2_62P: 5'-AACTCAGGAGATTTGG-3 '(SEQ ID NO: 68)

(4) Target gene sequence (chromosome 18): NCBI's GenBank Accession No. NC_018929.2 (SEQ ID NO: 69)

A primer pair for amplifying the target gene of SEQ ID NO: 69 and a probe for detecting a signal of the amplification product are as follows. The 5 'end of the detection probe was labeled with FAM and the 3' end with TAMRA. However, the present invention is not limited thereto, and it is a matter of course that various fluorescent substances and minerals known in the art can be labeled at the 5 'end and the 3' end, respectively.

Forward primer 18_5_1_67F: 5'-CAGGTCGTACAATTATGGCAAAAT-3 '(SEQ ID NO: 70)

Reverse primer 18_5_1_67R: 5'-AGGTCTGGGTTCTCGCTGAA-3 '(SEQ ID NO: 71)

Detection probe 18_5_1_67P: 5'-TGGCCTTATTTTGTTTTTAG-3 '(SEQ ID NO: 72)

On the other hand, the chromosome 18 and the related target genes exemplified in this embodiment should be understood as examples of chromosomes and related target genes to be analyzed for the number of genes, such as trisomy, The method of the present invention for analyzing genetic information of a fetus from blood or plasma of a pregnant woman should be understood as a base technique applicable to various individuals or samples and genotyping or abnormal analysis of gene numbers for various target genes and chromosomes.

A primer pair for amplifying each of the target genes as described above and a probe for detecting the amplification product amplified from the primer pair were synthesized by requesting Bioneer (Daejeon, Republic of Korea).

Example 6: Digital PCR for analysis of trisomy of chromosome 21, chromosome 18 and chromosome 13

Example 6-1: Digital PCR for analysis of trisomy of chromosome 21

M0 sample, M6 sample, and M9 sample, which were confirmed to contain a predetermined amount of the fetal gene from the gDNA samples prepared in Example 5-1, and genomic DNA samples of male genetic abnormality control sample was subjected to digital PCR in the same manner as described in Example 4. [ A digital PCR amplification apparatus, and a pair of primers to be dispensed per well and a corresponding detection probe (three target genes on chromosome 21, one chromosome on the chromosome 2) A pair of primers for amplification and a pair of detection probes corresponding to the control gene and one control gene on chromosome 1, totaling 5 sets) were the same as those in Example 4, and the digital PCR amplification composition was as shown in Table 19 below .

Digital PCR amplification composition PCR amplification composition Volume ([mu] l / well) (384 wells) A sample with a DNA template One Primer (20 pM) Forward primer 0.25 Reverse primer 0.25 Detection probe (2.5 pM) One TaqMan ® Digital PCR Master Mix 2.5 Sum 5

The software as described in Example 4 was performed, and fluorescence values measured in each well of the digital PCR layout were analyzed in the same manner as in Example 4, and the respective target genes of chromosome 21 (SEQ ID NO: 21, SEQ ID NO: No. 25 or SEQ ID NO: 29) and the number of copies of each control gene (the gene of SEQ ID NO: 33 or SEQ ID NO: 37). That is, a digital PCR amplification product of each target gene (SEQ ID NO: 21, SEQ ID NO: 25 or SEQ ID NO: 29) was quantified for each sample to be examined and each quantified value was compared with each control gene 33 or SEQ ID NO: 37) was calculated. From this, it was confirmed that the number of genes of the fetal-derived target gene for the normal male gene, that is, the chromosome 3 of the chromosome 21 (see Table 20).

The control gene of SEQ ID NO: 37
(Chromosome 1) Sample
number The control gene of SEQ ID NO: 33
(Chromosome 2) 1.360 1.117677348 M0 0.736625849 1.206 1.086434891 1.516032336 1.874456107 1.364799482 2.996 2.922318152 M6 2.003473315 2.111 3.323963935 2.278825941 2.742352911 2.050155311 1.632 1.440920821 M9 0.896090445 1.015 1.85241346 1.151989294 1.603982108 0.997501391

Analysis of the above results shows that the normalization value for the control gene (the gene on chromosome 1, the gene on chromosome 2) in the case of a normal male sample (Human Male Control) is close to 1 and the target gene (the gene on chromosome 21) It is confirmed that there is no variation in the number of genes. In the case of the M6 sample, normalization values for the control gene of SEQ ID NO: 37 (gene on chromosome 1) and the control gene of SEQ ID NO: 33 (gene on chromosome 2) were calculated to be 2.996 and 2.111, respectively, Gene) mutation in the gene. This means that the number of chromosome 21 in the fetus is about 1.5 times greater than the number of chromosome 1 in the chromosome 1 and the number of chromosome 2 in the fetus. . Thus, it can be assumed that the fetus has a trisomy, in which the chromosome 21 is triploid (normal diploid), and that the risk of developing fetal Down syndrome is high, .

In the case of the M0 sample and the M9 sample, the number of the chromosome 21 gene is found to be less than 1.5 times the number of the chromosome 1 gene and the number of the chromosome 2 gene. Analysis of the M0 sample and the M9 sample on the basis of chromosome 21 can be deduced that the chromosome 21 of the fetus is a normal diploid. Thus, the risk of developing Down syndrome in the fetus of the mother who provided the M0 sample and the M9 sample can be predicted to be low.

On the other hand, the results of digital PCR quantification and interpretation of the samples were in agreement with the clinical results relating to the chromosome 21 abnormality confirmed from the provider side of each sample.

Example 6-2: Digital PCR for analysis of trisomy of chromosome 13

M6 samples and M9 samples confirmed to contain a predetermined amount of the fetal gene from the gDNA samples prepared in Example 5-1 and genomic DNA sample (male control sample) Were subjected to digital PCR in the same manner as described in Example 6-1. The sample distribution system, digital PCR amplification composition, digital PCR amplification conditions, and digital PCR amplification equipment for each array of the well plate were the same as in Example 6-1. The pair of primers to be dispensed per well and the corresponding detection probes include a primer pair for amplification for each of four target genes on chromosome 13, one control gene on chromosome 2 and one control gene on chromosome 1, Corresponding detection probes (total 6 sets) were used.

In addition, a digital PCR amplification product of each target gene (SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 49 or SEQ ID NO: 53) was determined for each sample to be examined in the same manner as in Example 6-1 , And quantified values were normalized to digital PCR quantitative values of respective control genes (SEQ ID NO: 33 or SEQ ID NO: 37). From this, it was confirmed that the number of genes of the fetal-derived target gene for the normal male gene, ie, the chromosome 13 of the chromosome 13 (see Table 21).

The control gene of SEQ ID NO: 37
(Chromosome 1) Sample
number The control gene of SEQ ID NO: 33
(Chromosome 2) 1.3894 1.8988 M6 1.9130 1.4016 1.0001 1.0095 1.2542 1.2660 1.4046 1.4178 0.9782 3.6907 M9 3.7847 1.0033 0.0604 0.0621 0.0891 0.0916 0.0728 0.0748

Analysis of the above results suggests that the number of chromosome 13 gene is less than 1.5 times the number of chromosome 1 gene and the number of chromosome 2 gene, which are known to have no more than the number of genes, in the case of M6 sample and M9 sample. Analysis of chromosome 13 on M6 and M9 samples suggests that chromosome 13 in the fetus is a normal diploid. Thus, the risk of developing thrombotic thyroid 13 in a fetus that provided M6 and M9 samples could be predicted to be low.

On the other hand, the results of digital PCR quantification and interpretation of the samples were in agreement with the clinical results relating to the chromosome 13 abnormality confirmed from the provider side of each sample.

Example 6-3: Digital PCR for analysis of trisomy of chromosome 18

M0 and M6 samples and M9 samples, which were confirmed to contain a predetermined amount of the fetal gene from the gDNA samples prepared in Example 5-1, and genomic DNA samples (male control sample) was subjected to digital PCR in the same manner as described in Example 6-1. The sample distribution system, digital PCR amplification composition, digital PCR amplification conditions and digital PCR amplification equipment for each array of the well plate were the same as in Example 6-1. The pair of primers to be dispensed per well and the corresponding detection probes include pairs of amplification primers for each of four target genes on chromosome 18, one control gene on chromosome 2 and one control gene on chromosome 1, Corresponding detection probes (total 6 sets) were used.

In addition, a digital PCR amplification product of each target gene (SEQ ID NO: 57, SEQ ID NO: 61, SEQ ID NO: 65 or SEQ ID NO: 69) was determined for each sample to be examined in the same manner as in Example 6-1 , And quantified values were normalized to digital PCR quantitative values of respective control genes (SEQ ID NO: 33 or SEQ ID NO: 37). From this, it was confirmed that the number of genes of the fetal-derived target gene for the normal male gene, that is, the chromosomal nature of the chromosome 18 (see Table 22).

The control gene of SEQ ID NO: 37
(Chromosome 1) Sample
number The control gene of SEQ ID NO: 33
(Chromosome 2) 1.2205 1.5103 M0 1.4506 1.1380 1.2971 1.2459 1.0208 0.9805 1.0539 0.8752 1.1626 1.2608 M6 0.8312 0.7285 0.9659 0.6367 0.7232 0.4768 1.7006 0.9693 0.5499 0.5114 M9 1.0204 1.0563 0.6370 1.2710 0.4447 0.8874 0.6066 1.0465

In the case of M0 sample, M6 sample, and M9 sample, the number of chromosome 13 gene is known to be less than 1.5 times the number of chromosome 1 gene and the number of chromosome 2 gene . Analysis of the M0 sample, the M6 sample, and the M9 sample based on the chromosome 13 can be deduced that the chromosome 13 of the fetus is a normal diploid. Thus, the risk of developing trisomy 18 in the fetus of the mother who provided the M0 sample, the M6 sample, and the M9 sample may be predicted to be low.

On the other hand, the results of digital PCR quantification and interpretation of the samples were in agreement with the clinical results relating to the chromosome 18 abnormality confirmed from the provider side of each sample.

Example 7: Confirmation of cross-reactivity of target gene detection probes

In this embodiment, it was confirmed whether the probes for detecting any one trisomy of any sample to be inspected represent false-positive signals for the other trisomy of the other samples.

For example, it is possible to specifically bind to an amplification product of a target gene (SEQ ID NO: 21, SEQ ID NO: 25, or SEQ ID NO: 29) of a chromosome 21 in a sample having a trisomy 21, (A probe of SEQ ID NO: 24, SEQ ID NO: 28 or SEQ ID NO: 32) used in the present invention is a target gene of the chromosome 18 in another sample having the trisomy 18 (SEQ ID NO: 57, SEQ ID NO: 61, SEQ ID NO: Or the gene of SEQ ID NO: 69) is not present in the amplification product. On the contrary, it specifically binds to the amplification product of the target gene (SEQ ID NO: 57, SEQ ID NO: 61, SEQ ID NO: 65 or SEQ ID NO: 69) of the chromosome 18 in any one sample having trisomy 18, (SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68 or SEQ ID NO: 72) used in the detection of the target gene of the chromosome 21 in the other sample having the trisomy 21 No 25 or SEQ ID NO: 29) in the absence of a response to the amplification product.

In order to confirm whether these detection probes were cross-reacted, a sample confirmed to contain a predetermined amount of the fetal-derived gene among the gDNA samples prepared in Example 5-1 was an S8 sample having 21 trisomy and 18 Samples D3_1 and D8-3 were obtained from a male general control sample and a genomic DNA sample (male control sample) of a genetically normal male from a general hospital in the same manner as described in Example 6-1. Respectively. However, in the present embodiment, experiments were performed using two well plates in a digital PCR instrument at the same time. That is, for the first well plate in which the samples are distributed for each array, a pair of primers for amplification for each of three target genes on chromosome 21, one control gene on chromosome 2, and one control gene on chromosome 1 (Corresponding to a total of 5 sets) were reacted in a divided manner. For the second well plate, four target genes on the chromosome 18, one control gene on the chromosome 2 and one control gene on the chromosome 1 The amplification primer pair and the corresponding detection probes (total of 6 sets) were reacted by dispensing. Then, in the same manner as in Example 6-1, the digital PCR amplification products of the three target genes on the chromosome 21 on each of the samples dispensed to the first well plate were quantified, and 18 DNA PCR products of four target genes on chromosome 1 were quantified. And the value of each target gene quantification value was normalized to the digital PCR quantification value of each control gene (SEQ ID NO: 33 or SEQ ID NO: 37) (see Table 23).

Sample
number Detection probe The control gene of SEQ ID NO: 33
(Chromosome 2) The control gene of SEQ ID NO: 37
(Chromosome 1) S8
(Trisomy 21) 21-01_73 P
(SEQ ID NO: 24) 1.9705 2.1385 3.1077 3.3728 21-03_73 P
(SEQ ID NO: 28) 2.2469 3.5437 21-05_58 P
(SEQ ID NO: 32) 2.1982 3.4669 18_1_2_57P
(SEQ ID NO: 60) 1.0238 0.9401 1.6147 1.4827 18_2_2_58P
(SEQ ID NO: 64) 1.0148 1.6005 18_3_2_62P
(SEQ ID NO: 68) 1.0423 1.6439 18_5_1_67P
(SEQ ID NO: 72) 0.6795 1.0716 S8_1
(Trisomy 18) 21-01_73 P
(SEQ ID NO: 24) 1.2800 1.2902 1.1388 1.1479 21-03_73 P
(SEQ ID NO: 28) 1.3055 1.1616 21-05_58 P
(SEQ ID NO: 32) 1.2850 1.1434 18_1_2_57P
(SEQ ID NO: 60) 4.0088 3.0080 3.5668 2.6764 18_2_2_58P
(SEQ ID NO: 64) 3.7724 3.3565 18_3_2_62P
(SEQ ID NO: 68) 2.1104 1.8777 18_5_1_67P
(SEQ ID NO: 72) 2.1406 1.9046

In the case of the S8 sample, probes (SEQ ID NO: 24, SEQ ID NO: 28 and SEQ ID NO: 32) that detect the amplification products of the target genes of the chromosome 21 (SEQ ID NO: 21, SEQ ID NO: 25 and SEQ ID NO: 29) , The values obtained by normalizing the quantitative values of the digital PCR performed on the control gene of SEQ ID NO: 33 (the gene on chromosome 2) and the control gene of SEQ ID NO: 37 (the gene on chromosome 1) were 2.1385 and 3.3728, respectively And it was confirmed that the number of genes of the target gene (gene on chromosome 21) was variable. On the other hand, probes (SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68 or SEQ ID NO: 64) which detect the amplification products of the target genes of the chromosome 18 (SEQ ID NO: 57, SEQ ID NO: 61, SEQ ID NO: (The gene on chromosome 2) of SEQ ID NO: 33 and the control gene of SEQ ID NO: 37 (the gene on chromosome 1), the value obtained by normalizing the quantitative value of the digital PCR performed using the probe of SEQ ID NO: Were calculated as 0.9401 and 1.4827, respectively, and it was confirmed that there was no variation in the number of genes of the target gene (gene on chromosome 18) . Therefore, in the case of the S8 sample, the chromosome 21 can be predicted to have a high risk of developing a three-chromosomal Down syndrome, and the chromosome 18 can be determined to be normal. Thus, chromosome 21 and 18 This was consistent with the clinical results of chromosomal anomalies.

In the case of the S8_1 sample, probes (SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 64, SEQ ID NO: 60, SEQ ID NO: (The gene on chromosome 2) and the control gene (the gene on chromosome 1) of SEQ ID NO: 33 and the quantitative value of the digital PCR performed using the probe of SEQ ID NO: 68 or SEQ ID NO: The normalized values were calculated as 3.0080 and 2.6764, respectively, and it was confirmed that the number of genes of the target gene (gene on chromosome 18) varied. On the other hand, probes (SEQ ID NO: 24, SEQ ID NO: 28 and SEQ ID NO: 32) that detect the amplification products of the target genes of the chromosome 21 (SEQ ID NO: 21, SEQ ID NO: 25 and SEQ ID NO: 29) The normalized value of the digital PCR performed on the control gene of SEQ ID NO: 33 (gene on chromosome 2) and the control gene of SEQ ID NO: 37 (gene on chromosome 1) was calculated as 1.2902 and 1.1479, (Gene on chromosome 21) was found to have no mutation in the number of genes . Therefore, in the case of the S8_1 sample, it is possible to preliminarily judge that the risk of occurrence of trisomy is high for the chromosome 18, and the chromosome 21 can be judged to be normal. Therefore, the chromosome 21 and 18 This was consistent with the clinical results of chromosomal anomalies.

That is, as a result of the above-mentioned cross-reaction, the detection probe (the probe of SEQ ID NO: 24, SEQ ID NO: 28 or SEQ ID NO: 32) specifically binding to the amplification product of the target gene for the tri- It was confirmed that there was no reactivity to the amplification product of the target gene of the chromosome 18 having the chromosome (SEQ ID NO: 57, SEQ ID NO: 61, SEQ ID NO: 65 or SEQ ID NO: 69) and did not show a false positive signal. In addition, a detection probe (a probe of SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68 or SEQ ID NO: 72) that specifically binds to the amplification product of a target gene for three chromosomal discrimination of chromosome 18 has three chromosomal It was confirmed that there was no reactivity to the amplification product of the target gene of the chromosome 21 (SEQ ID NO: 21, SEQ ID NO: 25 or SEQ ID NO: 29) and thus did not show a false positive signal.

Example 8: Digital PCR quantification using a mixture of detection probes specific for a plurality of target genes

Example 8-1: Each detection probe was divided into respective wells and subjected to digital PCR quantification

First, among the gDNA samples prepared in Example 5-1, S2 sample, S4 sample, S5 sample and S6 sample, which were confirmed to contain a predetermined amount of the fetal gene, and a normal male Digital PCR was performed in the same manner as described in Example 6-1 for a genomic DNA sample (male control sample). The sample distribution system, digital PCR amplification composition, digital PCR amplification conditions, and digital PCR amplification equipment for each array of the well plate were the same as in Example 6-1. The pair of primers to be dispensed per well and the corresponding detection probes include a primer pair for amplification for each of three target genes on chromosome 21, one control gene on chromosome 2 and one control gene on chromosome 1, Corresponding detection probes (total of 5 sets) were used.

Also, in the same manner as in Example 6-1, the digital PCR amplification products of the respective target genes (SEQ ID NO: 21, SEQ ID NO: 25 or SEQ ID NO: 29) were quantitated for each sample to be examined and quantified values And each value was normalized to a digital PCR quantitative value of each control gene (SEQ ID NO: 33 or SEQ ID NO: 37 gene). From this, it was confirmed that the number of genes of the fetal-derived target gene for the normal male gene, that is, the chromosome 3 of the chromosome 21 (see Table 24).

The control gene of SEQ ID NO: 33
(Chromosome 2) Sample
number The control gene of SEQ ID NO: 37
(Chromosome 1) 1.0041 1.2681 S2 0.2643 0.2093 0.8585 0.1790 0.8858 0.1846 1.9532 1.9106 S4 1.5300 1.5499 1.9760 1.5878 1.0138 1.5319 1.9989 2.1530 S5 3.0000 1.8551 1.9806 2.7599 1.8631 2.5961 1.2236 1.5102 S6 0.6461 0.5235 1.1608 0.4966 1.0000 0.4278

In the case of the S4 sample, the normalization values for the control gene of SEQ ID NO: 33 (chromosome 2) and the control gene of SEQ ID NO: 37 (chromosome 1) were calculated to be 1.9532 and 1.5499, respectively and for the S5 sample, 1.9989 and 1.8551 And these samples were confirmed to have mutation in the number of genes . This means that the number of chromosome 21 in the fetus is about 1.5 times greater than the number of chromosome 1 in the chromosome 1 and the number of chromosome 2 in the fetus. . Thus, it can be assumed that the fetus has a trisomy, a chromosome 21 chromosomal triploid (normal diploid), and that the risk of developing fetal Down syndrome in the pregnant women who provided S4 and S5 samples is high can do.

On the other hand, the S2 sample and the S6 sample were confirmed to have a normalization value of less than 1.5, suggesting that the number of chromosome 21 was less than 1.5 times the number of chromosome 1 gene and the number of chromosome 2 gene . The S2 and S6 samples can be analyzed on the basis of chromosome 21 to estimate that the chromosome 21 of the fetus is a normal diploid. Thus, the risk of developing Down syndrome in the fetus of the pregnant woman who provided the S2 sample and the S6 sample can be predicted to be low.

Example 8-2: Quantification of a digital PCR using a probe mixture

On the other hand, when each detection probe is divided into each well, the number of wells used by the detection probe in the well plate increases as the number of detection probes increases. Therefore, the number of samples usable in one well plate is limited. Further, since many detection probes must be considered in designing a well plate layout, layout design is inconvenient, and there is a problem that the process of dividing the detection probe is complicated. Therefore, if the primer pair and the detection probes specific to the target genes for discrimination of three chromosomes of a specific chromosome are mixed and divided into one well, the above problems and inconveniences can be solved. In addition, when the primer pair and the detection probes specific to the control genes are mixed and divided into one well, the amount of the sample to be distributed to the well plate can be further increased, and the inconvenience of the layout design and the probe dispensing process can be further improved have.

This example was carried out to confirm whether the result of digital PCR quantification when using a mixture of such detection probes is consistent with the result of digital PCR quantification of Example 8-1. For this, digital PCR was carried out in the same manner as described in Example 8-1 except that the composition of the primer pair and the detection probe specific to the target gene of chromosome 21, the primer pair specific to the control gene and the detection probe Digital PCR was performed after changing the composition as follows (1) to (4) and dividing each mixture into each well:

(1) Detection probe mixture (5 pM) specific to the target gene of chromosome 21: 0.2 μL of detection probe 21-01_73 P (SEQ ID NO: 24), 0.4 μL of detection probe 21-03_73 P (SEQ ID NO: 28) Using 1.0 μL of 0.4 μL of probe 21-05_58 P (SEQ ID NO: 32) mixed;

(2) Amplification primer pair mixture (20 pM) specific to the target gene of chromosome 21: 0.1 μL of primer pair 21-01_73 (SEQ ID NO: 22 and SEQ ID NO: 23), primer pair 21-03_73 (SEQ ID NO: 26 and SEQ ID NO: 27) and 0.2 μL of primer pair 21-05_58 (SEQ ID NO: 30 and SEQ ID NO: 31);

(3) Detection probe mixture (5 pM) specific to the control genes of chromosome 2 and chromosome 1: 0.5 μL of detection probe 02-03_68 P (SEQ ID NO: 36), and detection probe 01-05_56 P ) With 1.0 μL mixed with 0.5 μL;

(4) Amplification primer pair mixture (20 pM) specific for the control genes of chromosome 2 and chromosome 1: 0.25 μL of primer pair 02-03_68 (SEQ ID NO: 34 and SEQ ID NO: 35), and primer pair 01-05_56 SEQ ID NO: 38 and SEQ ID NO: 39).

Also, according to the method described in Example 6-1, a digital PCR amplification product of all target genes on the chromosome 21 was quantitatively determined for each sample to be tested, and the quantified value was compared with the digital PCR quantification value of all the control genes The normalized value was calculated. From this, it was confirmed that the number of genes of the fetal-derived target gene for normal male genes, ie, the chromosomality of chromosome 21 (see Table 25).

Sample number Normalized values for all control genes S2 1.4740 S4 1.9219 S5 2.0183 S6 1.3859 MC 1.0468

Analysis of the results showed that the normalization value for the entire control gene was close to 1 in the case of a normal male sample (Human Male Control), indicating that there was no variation in the number of genes of the target gene (gene on chromosome 21). In the case of the S4 sample and the S5 sample, normalization values for the entire control gene were calculated as 1.9219 and 2.0183, respectively, and these samples were confirmed to have mutation in the number of genes . On the other hand, it was confirmed that the S2 sample and the S6 sample had a normalization value of less than 1.5. Therefore, it was confirmed that the digital PCR quantitative results of this example corresponded to the digital PCR quantitative results of Example 8-1. From this point of view, it is understood that the digital PCR quantitative method using the primer pair mixture and the corresponding detection probe mixture shows significant results and can be used for diagnosis of diseases related to chromosome number abnormality of the fetal gene.

Although the present invention has been described with reference to the above embodiments, the present invention is not limited thereto. It will be understood by those skilled in the art that modifications and variations may be made without departing from the spirit and scope of the invention, and that such modifications and variations are also contemplated by the present invention.

<110> BIOCORE CO., LTD. <120> Method for analyzing prenatal genetic information from blood or          plasma of pregnant woman using digital PCR <130> P14-0024KR <150> KR 10-2014-0081763 <151> 2014-07-01 <160> 72 <170> Kopatentin 1.71 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> APP Probe <400> 1 accccagagg agcgccacct g 21 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> APP forward primer <400> 2 tcaggttgac gccgctgt 18 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> APP reverse primer (67 bp) <400> 3 ttcgtagccg ttctgctgc 19 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > APP reverse primer (180 bp) <400> 4 tctataaatg gacaccgatg ggtagt 26 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Beta-actin probe 1 <400> 5 cccacttctc tctaaggaga atggccc 27 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta-actin forward primer 1 <400> 6 tacaggaagt cccttgccat 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> Beta-actin reverse primer 1 (83 bp) <400> 7 cctgtgtgga cttgggagag 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> Beta-actin reverse primer 1 (170 bp) <400> 8 cacgaaggct catcattcaa 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta-actin probe 2 <400> 9 ttccgctgcc ctgaggcact 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta-actin forward primer 2 <400> 10 agagctacga gctgcctgac 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> Beta-actin reverse primer 2 (67 bp) <400> 11 ccatctcttg ctcgaagtcc 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> Beta-actin reverse primer 2 (169 bp) <400> 12 ggcaggactt agcttccaca 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SRY probe 1 <400> 13 cgccttccga cgaggtcgat 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SRY forward primer 1 <400> 14 tacaggccat gcacagagag 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SRY reverse primer 1 (79 bp) <400> 15 caattcttcg gcagcatctt 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SRY reverse primer 1 (185 bp) <400> 16 ggtaagtggc ctagctggtg 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SRY probe 2 <400> 17 tcttgcgcct ctgatcgcga 20 <210> 18 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> SRY forward primer 2 <400> 18 aacgcattca tcgtgtggt 19 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SRY reverse primer 2 (90 bp) <400> 19 cagctgcttg ctgatctctg 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SRY reverse primer 2 (189 bp) <400> 20 ttccgacgag gtcgatactt 20 <210> 21 <211> 225 <212> DNA <213> Homo sapiens <400> 21 aaaactttct ttcttttttc ttttctattt ttaaaaccaa cttgcaatgc tgttcatgga 60 ggatgctcag gaagatgaga gaaaattagt aggattagtt ggagagagtg ggagatagac 120 gagacccccg ctagtaagat gttactttca tttacaaatc ctacagagag gcagaatagg 180 tggggtatag aaaaatgtca ggctgtcagt taccctttta aattg 225 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer 21-01_73 F <400> 22 atgctgttca tggaggatgc t 21 <210> 23 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer 21-01_73 R <400> 23 tctatctccc actctctcca actaatc 27 <210> 24 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Probe 21-01_73 P <400> 24 aggaagatga gagaaaatta gta 23 <210> 25 <211> 220 <212> DNA <213> Homo sapiens <400> 25 tgcaggtgag gtctttaaag tttcaatgaa agtttcttct ggatctacag aaaaaatttt 60 tttttttcaa tctaaaaact ggaaattcta gggtttttgt acattttgga tgcactggga 120 atttattagc acaaaatcat tctttgcaac tcaaaattca gaagggactc taccatatct 180 tagctcagag cacagaggag tgccttatcc ccacacttga 220 <210> 26 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer 21-03_73 F <400> 26 gcactgggaa tttattagca caaa 24 <210> 27 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer 21-03_73 R <400> 27 gctaagatat ggtagagtcc cttctga 27 <210> 28 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Probe 21-03_73 P <400> 28 tcattctttg caactcaaa 19 <210> 29 <211> 221 <212> DNA <213> Homo sapiens <400> 29 gaatcagaca gatggagatc aaaacgtcag ctttgctgct gatcagaagg aggtggctgg 60 atgtggttta tgccttcagg ttgatgggct tgcgcttatc tgcttgtccc ttctcccatc 120 tgatttcttg tggagcccat ggaggtaaag ttggtatttc caaatcagac attttgccca 180 aaattgctgc ccctctctgg ttctgagtgg aaagggaaga a 221 <210> 30 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward primer 21-05_58 F <400> 30 tgatgggctt gcgcttatc 19 <210> 31 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer 21-05_58 R <400> 31 tgggctccac aagaaatcag a 21 <210> 32 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Probe 21-05_58 P <400> 32 ttgtcccttc tccc 14 <210> 33 <211> 220 <212> DNA <213> Homo sapiens <400> 33 aggagttttt catttccaat ctaaaaacta gaagctctag cattttgtac attttttgtt 60 gttgcactgg aagtttaact attggcacaa aatcattctt caaaactcac agagggactc 120 tgccattact tagctcagag cacagaggag tgccttgtcc ccaaacgtga atgggcttgt 180 ggaggtaggc atgtggggtc ccctggcccc aggctggaga 220 <210> 34 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer 02-03_68 F <400> 34 gagggactct gccattactt agct 24 <210> 35 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer 02-03_68 R <400> 35 caagcccatt cacgtttgg 19 <210> 36 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Probe 02-03_68 P <400> 36 agcacagagg agtgcc 16 <210> 37 <211> 221 <212> DNA <213> Homo sapiens <400> 37 taggcgtgag cctgattctt aagagaaatg aaagaggggc accagctggg tggtctggga 60 actcggtttc actgctgtcc tcaacatggg ccccacatgg tcctagtttc cccggctcat 120 tatgcttcag gcacactggc tttcttgcct tcctcaaaca gcattggtct gttctgccct 180 cagatcttgg tatggctagc tcctcctcag tagtcagctc a 221 <210> 38 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Forward primer 01-05_56 F <400> 38 catgggcccc acatggt 17 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer 01-05_56 R <400> 39 gccagtgtgc ctgaagcata 20 <210> 40 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Probe 01-05_56 P <400> 40 ctagtttccc cggctca 17 <210> 41 <211> 242 <212> DNA <213> Homo sapiens <400> 41 agtatttatt ctttgataga tttaattaca agtcttcaga atgccagaga tatacaggat 60 atgcgaatta agaagaaaca aaggcaacgc gtctttccac agccaggcag tctgtatctt 120 gcaaaaacat ccactctgcc tcgaatctct ctgaaagcag cagtaggagg ccaagttccc 180 tctgcgtgtt ctcataaaca ggtatgtgtt tgtctacaat actgatggct tttatgacag 240 ag 242 <210> 42 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward primer 13_1_2_62F <400> 42 caaaggcaac gcgtctttc 19 <210> 43 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer 13_1_2_62R <400> 43 ggcagagtgg atgtttttgc a 21 <210> 44 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Probe 13_1_2_62P <400> 44 agccaggcag tctgta 16 <210> 45 <211> 234 <212> DNA <213> Homo sapiens <400> 45 tgctgggcga ataagaaaaa cctgattgac tagaaagcaa atgtaaatgt aatctccttt 60 cttttcagtg attctaccag aaaggaatga agaacagaac cttcaggaat tgagtcacaa 120 tgcagacaaa tatcaaatgg gagattgttg caaggaagag attgatgata gtattttcta 180 ctagccattg ggaagataaa aggagacaga agattgaagc ctttgccagc catt 234 <210> 46 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Forward primer 13_2_1_69F <400> 46 ttctaccaga aaggaatgaa gaacag 26 <210> 47 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer 13_2_1_69R <400> 47 ccatttgata tttgtctgca ttgtg 25 <210> 48 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Probe 13_2_1_69P <400> 48 accttcagga attgag 16 <210> 49 <211> 238 <212> DNA <213> Homo sapiens <400> 49 ccggggccgt gtgggttgtc ccggtgtcct gctcgcgagt gacgcctgtc cttcttgccc 60 ccagagatcc tgaaggagct agacgagtgc tacgagcgct tcagtcgcga gacagacggg 120 gcgcagaagc ggcggatgct gcactgtgtg cagcgcgcgc tgatccgcag ccaggagctg 180 ggcgacgaga agatccagat cgtgagccag atggtggagc tggtggagaa ccgcacgc 238 <210> 50 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward primer 13_3_1_62F <400> 50 tgtcctgctc gcgagtga 18 <210> 51 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer 13_3_1_62R <400> 51 tcgtctagct ccttcaggat ctct 24 <210> 52 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Probe 13_3_1_62P <400> 52 ctgtccttct tgcccc 16 <210> 53 <211> 231 <212> DNA <213> Homo sapiens <400> 53 tctactcgaa cacgaatgca aaagcagaaa atgaatgata gcatggatac ctcaaacaag 60 gaagagaaat gaggatctca ggaccttggt ggacactgtg tacacctctg gattcattgt 120 ctctcacaga tgtgactgta taactttccc aggttctgtt tatggccaca tttaatatct 180 tcagctcttt ttgtggatat aaaatgtgca gatgcaattg tttgggtgat t 231 <210> 54 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer 13_4_2_69F <400> 54 gcagaaaatg aatgatagca tgga 24 <210> 55 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer 13_4_2_69R <400> 55 ccaccaaggt cctgagatcc t 21 <210> 56 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Probe 13_4_2_69P <400> 56 acctcaaaca aggaagag 18 <210> 57 <211> 240 <212> DNA <213> Homo sapiens <400> 57 ggccatcaca ctgaccatcc tgctcggggt cttcatcttc tgctgggccc cctttgtgct 60 tcatgtcctc ttgatgacat tctgcccaag taacccctac tgcgcctgct acatgtctct 120 cttccaggtg aacggcatgt tgatcatgtg caatgccgtc attgacccct tcatatatgc 180 cttccggagc ccagagctca gggacgcatt caaaaagatg atcttctgca gcaggtactg 240                                                                          240 <210> 58 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Forward primer 18_1_2_57F <400> 58 ctgctgggcc cccttt 16 <210> 59 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer 18_1_2_57R <400> 59 gggttacttg ggcagaatgt ca 22 <210> 60 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Probe 18_1_2_57P <400> 60 tgcttcatgt cctcttg 17 <210> 61 <211> 222 <212> DNA <213> Homo sapiens <400> 61 ccgtggcgcg gcagccatcg cctgcccggt gctgattgtg cctcctgtgt gcccttctcc 60 tgtagaattc tctggtggtt gagatcccgc catttcggaa tcagaggata accagccccg 120 ttcacgtcag tttctacgtc tgcaacggga agagaaagcg aagccagtac cagcgtttca 180 cctaccttcc cgccaacggt aacgccatct ttctaaccgt aa 222 <210> 62 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward primer 18_2_2_58F <400> 62 gccccgttca cgtcagttt 19 <210> 63 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer 18_2_2_58R <400> 63 tggtactggc ttcgctttct c 21 <210> 64 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Probe 18_2_2_58P <400> 64 acgtctgcaa cggg 14 <210> 65 <211> 228 <212> DNA <213> Homo sapiens <400> 65 atttgtcact agtgtgggcg tattaggttt tgctggttac agtctgtaat atcatcaatg 60 ttcatgtcca tgttttgctt tccttctcag agctggaagt cgagtgtgct actcaactca 120 ggagatttgg agacaaactg aacttccggc agaaacttct gaatctgata tccaaactct 180 tctgctcagg aacctgactg catcaaaaac ttgcatgagg ggactcct 228 <210> 66 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward primer 18_3_2_62F <400> 66 agctggaagt cgagtgtgct act 23 <210> 67 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer 18_3_2_62R <400> 67 ctgccggaag ttcagtttgt c 21 <210> 68 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Probe 18_3_2_62P <400> 68 aactcaggag atttgg 16 <210> 69 <211> 238 <212> DNA <213> Homo sapiens <400> 69 caacaggtcg tacaattatg gcaaaataat ggccttattt tgtttttagc ttcagcgaga 60 acccagacct ttcccaaagc tcaggattct tcgaaaagtt gagaaaattg atgacttcaa 120 agctgaagac tttcagattg aagggtacaa tccgcatcca actattaaaa tggaaatggc 180 tgtttagggt gctttcaaag gagctcgaag gatattgtca gtctttaggg gttgggct 238 <210> 70 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer 18_5_1_67F <400> 70 caggtcgtac aattatggca aaat 24 <210> 71 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer 18_5_1_67R <400> 71 aggtctgggt tctcgctgaa 20 <210> 72 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe 18_5_1_67P <400> 72 tggccttatt ttgtttttag 20

Claims (40)

(a) analyzing whether a fetal-derived gene is present in a pregnant blood or plasma sample, and
(b) performing a digital PCR by distributing a sample detected as a result of the analysis of the step (a) to the wells of the well plate, wherein a set of maternal genes and a gene set common to fetal genes Performing a digital PCR on a control gene that is not related to a gene number abnormality and a target gene related to abnormal gene number,
(c) analyzing the digital PCR amplification product of the control gene obtained in the step (b), analyzing whether the number of control gene copies per well is statistically close to one,
(d) if it is determined that the number of control gene copies per well is statistically more than 1 as a result of the analysis in the step (c), the sample is further diluted and the steps (b) and (c) Repeating,
(e) If it is determined that the number of control gene copies per well is close to one statistically as a result of the analysis in the step (c), the digital PCR amplification product of the target gene is quantified, and the digital PCR Calculating a ratio of a digital PCR quantification value of the target gene to a quantification value;
(f) determining that the number of genes in the fetal-derived target gene is greater than or equal to 1.5 if the ratio calculated in the step (e) is equal to or more than 1.5, the genetic information of the fetus from the blood or plasma of the pregnant woman using digital PCR How to analyze. The method according to claim 1,
In the step (e), when the number of copies per well is within the range of 1.01 to 1.59 as well as the number of copies per well, it is determined that the number of control gene copies per well is close to one statistically How to. 3. The method according to claim 1 or 2,
Wherein the digital PCR quantification value of the control gene in step (e) is an average copy number of the control gene per well, and the digital PCR quantification value of the target gene is an average copy number of the target gene per well. 3. The method according to claim 1 or 2,
Wherein the dilution of the sample is performed stepwise from 1000 times to 5000 times in the step (d). 3. The method according to claim 1 or 2,
Wherein the control gene is at least one selected from the group consisting of a control gene having a nucleotide sequence of SEQ ID NO: 33, which is a gene on chromosome 2, and a control gene having a nucleotide sequence of SEQ ID NO: 37, which is a gene on chromosome 1 Way. 6. The method of claim 5,
34 and 35 as the primer pair for amplifying the control gene having the nucleotide sequence of SEQ ID NO: 33 and a probe for detecting the control gene amplification product having the nucleotide sequence of SEQ ID NO: 33, 36 and at least one probe selected from the group consisting of oligonucleotides complementary to these oligonucleotides is used. 6. The method of claim 5,
38 and 39 as the primer pair for amplifying the control gene having the nucleotide sequence of SEQ ID NO: 37 and the probe for detecting the control gene amplification product having the nucleotide sequence of SEQ ID NO: 37, Wherein at least one probe selected from the group consisting of oligonucleotides complementary to these oligonucleotides and oligonucleotides complementary to these oligonucleotides is used. 6. The method of claim 5,
The control gene having the nucleotide sequence of SEQ ID NO: 33 and the control gene having the nucleotide sequence of SEQ ID NO: 37 were mixed in the same well using the mixture of the primer pairs of SEQ ID NOS: 34 and 35 and the primer pairs of SEQ ID NOS: 38 and 39, And at least one probe selected from the group consisting of an oligonucleotide complementary to the oligonucleotide of SEQ ID NO: 36 and at least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 40 and an oligonucleotide complementary to the oligonucleotide The control gene amplification product having the nucleotide sequence of SEQ ID NO: 33 and the control gene amplification product having the nucleotide sequence of SEQ ID NO: 37 are detected together in the same well using a mixture of at least one selected probe. Way. 3. The method according to claim 1 or 2,
Wherein the target gene is at least one selected from the group consisting of a target gene on chromosome 21, a target gene on chromosome 13, and a target gene on chromosome 18. 10. The method of claim 9,
Wherein the digital PCR is simultaneously performed on at least two target genes selected from the group consisting of the target gene on chromosome 21, the target gene on chromosome 13, and the target gene on chromosome 18. 10. The method of claim 9,
The target gene on chromosome 21 is at least one selected from the group consisting of a target gene having the nucleotide sequence of SEQ ID NO: 21, a target gene having the nucleotide sequence of SEQ ID NO: 25, and a target gene having the nucleotide sequence of SEQ ID NO: 29 Lt; / RTI &gt; 12. The method of claim 11,
The primer pair of SEQ ID NOS: 22 and 23 is used as the primer pair for amplifying the target gene having the nucleotide sequence of SEQ ID NO: 21, and the probe for detecting the target gene amplification product having the nucleotide sequence of SEQ ID NO: Characterized in that at least one probe selected from the group consisting of oligonucleotides complementary to such oligonucleotides and oligonucleotides complementary to these oligonucleotides is used. 12. The method of claim 11,
26 and 27 as the primer pair for amplifying the target gene having the nucleotide sequence of SEQ ID NO: 25, and the probe for detecting the target gene amplification product having the nucleotide sequence of SEQ ID NO: 28 and at least one probe selected from the group consisting of oligonucleotides complementary to these oligonucleotides is used. 12. The method of claim 11,
The primer pair of SEQ ID NOS: 30 and 31 is used as the primer pair for amplifying the target gene having the nucleotide sequence of SEQ ID NO: 29, and the probe for detecting the target gene amplification product having the nucleotide sequence of SEQ ID NO: 32 and at least one probe selected from the group consisting of oligonucleotides complementary to such oligonucleotides is used. 12. The method of claim 11,
The target gene having the nucleotide sequence of SEQ ID NO: 21, the nucleotide sequence of SEQ ID NO: 25, the nucleotide sequence of SEQ ID NO: 25, and the primer pair of SEQ ID NO: A target gene having a base sequence and a target gene having the nucleotide sequence of SEQ ID NO: 29 are amplified together in the same well, and at least one selected from the group consisting of an oligonucleotide of SEQ ID NO: 24 and an oligonucleotide complementary to such oligonucleotide At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 28 and an oligonucleotide complementary to the oligonucleotide, and an oligonucleotide of SEQ ID NO: 32 and an oligonucleotide complementary to the oligonucleotide A note Using a mixture of one probe, the target gene amplification product having the nucleotide sequence of SEQ ID NO: 21, the target gene amplification product having the nucleotide sequence of SEQ ID NO: 25, and the target gene amplification product having the nucleotide sequence of SEQ ID NO: And the products are detected together in the same well. 10. The method of claim 9,
The target gene on the chromosome 18 comprises a target gene having the nucleotide sequence of SEQ ID NO: 57, a target gene having the nucleotide sequence of SEQ ID NO: 61, a target gene having the nucleotide sequence of SEQ ID NO: 65 and a target gene having the nucleotide sequence of SEQ ID NO: Gene. &Lt; / RTI &gt; 17. The method of claim 16,
58 and 59 as the primer pair for amplifying the target gene having the nucleotide sequence of SEQ ID NO: 57 and a probe for detecting the target gene amplification product having the nucleotide sequence of SEQ ID NO: 57, Characterized in that at least one probe selected from the group consisting of oligonucleotides complementary to these oligonucleotides and oligonucleotides complementary to these oligonucleotides is used. 17. The method of claim 16,
The primer pair of SEQ ID NOS: 62 and 63 is used as the primer pair for amplifying the target gene having the nucleotide sequence of SEQ ID NO: 61, and the probe for detecting the target gene amplification product having the nucleotide sequence of SEQ ID NO: 64 and at least one probe selected from the group consisting of oligonucleotides complementary to these oligonucleotides is used. 17. The method of claim 16,
66 and 67 as the primer pair for amplifying the target gene having the nucleotide sequence of SEQ ID NO: 65 and a probe for detecting the target gene amplification product having the nucleotide sequence of SEQ ID NO: 68 and at least one probe selected from the group consisting of oligonucleotides complementary to these oligonucleotides is used. 17. The method of claim 16,
As primer pairs for amplifying the target gene having the nucleotide sequence of SEQ ID NO: 69, primer pairs of SEQ ID NOs: 70 and 71 are used, and probes for detecting the target gene amplification product having the nucleotide sequence of SEQ ID NO: 72 and at least one probe selected from the group consisting of oligonucleotides complementary to these oligonucleotides is used. 17. The method of claim 16,
The nucleotide sequence of SEQ ID NO: 57 was amplified using the primer pair of SEQ ID NOs: 58 and 59, the primer pair of SEQ ID NOs: 62 and 63, the primer pair of SEQ ID NOs: 66 and 67, and the primer pairs of SEQ ID NOs: 65, the target gene having the nucleotide sequence of SEQ ID NO: 61, the target gene having the nucleotide sequence of SEQ ID NO: 65, and the target gene having the nucleotide sequence of SEQ ID NO: 69 are amplified together in the same well, At least one probe selected from the group consisting of oligonucleotides complementary to these oligonucleotides, at least one probe selected from the group consisting of oligonucleotides of SEQ ID NO: 64 and oligonucleotides complementary to these oligonucleotides, The oligonucleotide of SEQ ID NO: 68 and the oligonucleotide of SEQ ID NO: At least one probe selected from the group consisting of oligonucleotides complementary to the nucleotide and a mixture of at least one probe selected from the group consisting of an oligonucleotide complementary to the oligonucleotide and the oligonucleotide of SEQ ID NO: The target gene amplification product having the nucleotide sequence of SEQ ID NO: 57, the target gene amplification product having the nucleotide sequence of SEQ ID NO: 61, the target gene amplification product having the nucleotide sequence of SEQ ID NO: 65, Is detected together in the same well. &Lt; RTI ID = 0.0 &gt; 8. &lt; / RTI &gt; 10. The method of claim 9,
The target gene on chromosome 13 includes a target gene having the nucleotide sequence of SEQ ID NO: 41, a target gene having the nucleotide sequence of SEQ ID NO: 45, a target gene having the nucleotide sequence of SEQ ID NO: 49 and a target gene having the nucleotide sequence of SEQ ID NO: Gene. &Lt; / RTI &gt; 23. The method of claim 22,
The primer pairs of SEQ ID NOs: 42 and 43 are used as the primer pairs for amplifying the target gene having the nucleotide sequence of SEQ ID NO: 41, and the probes for detecting the target gene amplification product having the nucleotide sequence of SEQ ID NO: 44 and at least one probe selected from the group consisting of oligonucleotides complementary to these oligonucleotides is used. 23. The method of claim 22,
As primer pairs for amplifying the target gene having the nucleotide sequence of SEQ ID NO: 45, primer pairs of SEQ ID NOS: 46 and 47 are used, and probes for detecting the target gene amplification product having the nucleotide sequence of SEQ ID NO: Wherein at least one probe selected from the group consisting of oligonucleotides complementary to these oligonucleotides and oligonucleotides complementary to these oligonucleotides is used. 23. The method of claim 22,
50 and 51 as the primer pair for amplifying the target gene having the nucleotide sequence of SEQ ID NO: 49, and the probe for detecting the target gene amplification product having the nucleotide sequence of SEQ ID NO: 49, And at least one probe selected from the group consisting of oligonucleotides complementary to these oligonucleotides and at least one probe selected from the group consisting of oligonucleotides complementary to these oligonucleotides is used. 23. The method of claim 22,
54 and 55 as the primer pair for amplifying the target gene having the nucleotide sequence of SEQ ID NO: 53 and a probe for detecting the target gene amplification product having the nucleotide sequence of SEQ ID NO: 53, Wherein at least one probe selected from the group consisting of oligonucleotides complementary to the oligonucleotide of SEQ ID NO: 56 and oligonucleotides complementary to such oligonucleotides is used. 23. The method of claim 22,
The nucleotide sequence of SEQ ID NO: 41 was amplified using the primer pair of SEQ ID NOs: 42 and 43, the primer pair of SEQ ID NOs: 46 and 47, the primer pair of SEQ ID NOs: 50 and 51, and the primer pairs of SEQ ID NOs: The target gene having the nucleotide sequence of SEQ ID NO: 45, the target gene having the nucleotide sequence of SEQ ID NO: 49, and the target gene having the nucleotide sequence of SEQ ID NO: 53 were amplified together in the same well, At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 48 and an oligonucleotide complementary to such oligonucleotide, at least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 48 and an oligonucleotide complementary to the oligonucleotide, The oligonucleotide of SEQ ID NO: 52 and the oligonucleotide At least one probe selected from the group consisting of oligonucleotides complementary to the nucleotide and a mixture of at least one probe selected from the group consisting of an oligonucleotide complementary to the oligonucleotide and the oligonucleotide of SEQ ID NO: The target gene amplification product having the nucleotide sequence of SEQ ID NO: 41, the target gene amplification product having the nucleotide sequence of SEQ ID NO: 45, the target gene amplification product having the nucleotide sequence of SEQ ID NO: 49, Is detected together in the same well. &Lt; RTI ID = 0.0 &gt; 8. &lt; / RTI &gt; A control gene composition for use in obtaining a reference quantification value for calculating a normalization value from a quantification value obtained after amplification of a target gene related to an abnormal number of genes present in a gene sample,
A gene of SEQ ID NO: 37 on the chromosome 1, and a gene of SEQ ID NO: 33 on the chromosome 2. 29. A probe composition for a control gene, which is used for detecting and quantifying an amplification product of the control gene according to claim 28,
At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 36 and an oligonucleotide complementary to the oligonucleotide, which is specific to the control gene having the nucleotide sequence of SEQ ID NO: 33, and a control gene having the nucleotide sequence of SEQ ID NO: 37 And at least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 40 specific to the oligonucleotide and an oligonucleotide complementary to the oligonucleotide. As a well plate used to amplify a gene sample and to detect and quantify an amplification product,
At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 36 and an oligonucleotide complementary to the oligonucleotide, which is specific to the control gene having the nucleotide sequence of SEQ ID NO: 33, and a control gene having the nucleotide sequence of SEQ ID NO: 37 Wherein at least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 40 specific for said oligonucleotide and an oligonucleotide complementary to said oligonucleotide is dispensed. 31. The method of claim 30,
Wherein the two or more probes are individually dispensed into each well or mixed together and dispensed into one well. 31. The method of claim 30,
At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 24 specific to the target gene having the nucleotide sequence of SEQ ID NO: 21 on the chromosome 21 and an oligonucleotide complementary to the oligonucleotide and the nucleotide sequence of SEQ ID NO: At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 28 and an oligonucleotide complementary to the oligonucleotide specific to the target gene having the nucleotide sequence of SEQ ID NO: 25 and a nucleotide sequence of SEQ ID NO: 29 on the chromosome 21 At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 32 specific to the target gene and at least one probe selected from the group consisting of oligonucleotides complementary to the oligonucleotide Well plates that also. 33. The method of claim 32,
Wherein the two or more probes are individually dispensed into each well or mixed together and dispensed into one well. 31. The method of claim 30,
At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 60 specific to the target gene having the nucleotide sequence of SEQ ID NO: 57 on the chromosome 18 and an oligonucleotide complementary to the oligonucleotide and the nucleotide sequence of SEQ ID NO: At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 64 and an oligonucleotide complementary to the oligonucleotide, the nucleotide sequence of SEQ ID NO: 65 on the chromosome 18, At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 68 specific to the target gene and an oligonucleotide complementary to the oligonucleotide, and a probe specific for the target gene having the nucleotide sequence of SEQ ID NO: 69 on the chromosome 18 Wherein at least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 72 and at least one probe selected from the group consisting of oligonucleotides complementary to such an oligonucleotide is dispensed. 35. The method of claim 34,
Wherein the two or more probes are individually dispensed into each well or mixed together and dispensed into one well. 31. The method of claim 30,
An oligonucleotide of SEQ ID NO: 44 specific to the target gene having the nucleotide sequence of SEQ ID NO: 41 on the chromosome 13, at least one probe selected from the group consisting of oligonucleotides complementary to the oligonucleotide and the nucleotide sequence of SEQ ID NO: At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 48 and an oligonucleotide complementary to the oligonucleotide, the nucleotide sequence of SEQ ID NO: 49 on the chromosome 13 At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 52 specific for the target gene and an oligonucleotide complementary to the oligonucleotide, and at least one probe selected from the group consisting of the nucleotide sequence of SEQ ID NO: 53 on the chromosome 13 Wherein at least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 56 and at least one probe selected from the group consisting of oligonucleotides complementary to such an oligonucleotide is dispensed. 37. The method of claim 36,
Wherein the two or more probes are individually dispensed into each well or mixed together and dispensed into one well. A probe composition for a target gene, which is used for detecting and quantifying a target gene amplification product associated with an abnormality in the number of genes present in a gene sample,
At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 24 specific to the target gene having the nucleotide sequence of SEQ ID NO: 21 on the chromosome 21 and an oligonucleotide complementary to the oligonucleotide and the nucleotide sequence of SEQ ID NO: At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 28 and an oligonucleotide complementary to the oligonucleotide specific to the target gene having the nucleotide sequence of SEQ ID NO: 25 and a nucleotide sequence of SEQ ID NO: 29 on the chromosome 21 A target gene comprising at least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 32 specific to the target gene and at least one probe selected from the group consisting of an oligonucleotide complementary to the oligonucleotide A probe composition for electronic use. A probe composition for a target gene, which is used for detecting and quantifying a target gene amplification product associated with an abnormality in the number of genes present in a gene sample,
At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 60 specific to the target gene having the nucleotide sequence of SEQ ID NO: 57 on the chromosome 18 and an oligonucleotide complementary to the oligonucleotide and the nucleotide sequence of SEQ ID NO: At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 64 and an oligonucleotide complementary to the oligonucleotide, the nucleotide sequence of SEQ ID NO: 65 on the chromosome 18, At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 68 specific to the target gene and an oligonucleotide complementary to the oligonucleotide, and a probe specific for the target gene having the nucleotide sequence of SEQ ID NO: 69 on the chromosome 18 At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 72 and at least one probe selected from the group consisting of oligonucleotides complementary to such oligonucleotides. A probe composition for a target gene, which is used for detecting and quantifying a target gene amplification product associated with an abnormality in the number of genes present in a gene sample,
An oligonucleotide of SEQ ID NO: 44 specific to the target gene having the nucleotide sequence of SEQ ID NO: 41 on the chromosome 13, at least one probe selected from the group consisting of oligonucleotides complementary to the oligonucleotide and the nucleotide sequence of SEQ ID NO: At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 48 and an oligonucleotide complementary to the oligonucleotide, the nucleotide sequence of SEQ ID NO: 49 on the chromosome 13 At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 52 specific for the target gene and an oligonucleotide complementary to the oligonucleotide, and at least one probe selected from the group consisting of the nucleotide sequence of SEQ ID NO: 53 on the chromosome 13 And at least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 56 and at least one probe selected from the group consisting of oligonucleotides complementary to such oligonucleotides.
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