Method and kit for simultaneously determining copy number of gene and gene variant

Abstract

The present invention relates to a real-time quantitative polymerase chain reaction (qPCR) method and kit for simultaneously determining the classification of specific gene variants and the copy number of a gene. More specifically, the present invention relates to an efficient qPCR method and kit for simultaneously determining the variant of a target gene and the copy number of a gene by performing real-time quantitative PCR on a reference gene having a known copy number and the target gene in one reactor in order to determine the copy number of the target gene.

Description

Method and kit for simultaneous determination of gene copy number and genetic variant type

[0001] The present invention relates to a real-time quantitative polymerase chain reaction (qPCR) method and kit for simultaneously determining the identification of specific genetic variants and the copy number of a gene.

[0002] More specifically, the present invention relates to an efficient qPCR method and kit for determining the copy number of a gene of interest by performing real-time quantitative PCR (qPCR) on a reference gene of known copy number and a target gene in one reaction vessel to determine the copy number of the gene of interest and simultaneously determine a variant of the target gene.

[0003] Extensive human genome research has revealed that genetic variations, including single nucleotide polymorphisms (SNPs), which vary across races and individuals, are closely linked to drug pharmacodynamics. Therefore, information on genetic variations associated with the pharmacodynamics of prescribed medications will be invaluable to physicians in enabling personalized treatment and precision medicine.

[0004] Since the launch of the Precision Medicine Initiative in the United States in 2015, extensive pharmacogenomics research has been underway, and overseas, the development of personalized diagnostic products utilizing these findings is actively underway. However, currently leading product development technologies, such as next-generation sequencing (NGS) and DNA chip-based technologies, have shown a high false positive rate and are equipment-dependent, making it difficult to produce meaningful clinical trial results and expand the market. Therefore, alternative technologies are needed to overcome these challenges.

[0005] Various studies on the human genome have revealed that copy number variations (CNVs) are widespread, accounting for approximately 12% of the genome. CNVs are structural mutations in the genome that occur through mechanisms such as insertions, deletions, or duplications of large genes. CNVs in various genes have been found to be associated with genetic diseases, cancer, metabolic disorders, and other disorders.

[0006] Genetic diseases associated with CNVs are known to be associated with neurodegenerative disorders such as Parkinson's disease (PD) and early-onset Alzheimer's disease (AD), autoimmune diseases such as systemic lupus erythromatosus (SLE), rheumatoid arthiritis (RA), Crohn's disease, type 2 diabetes, obesity, and susceptibility to HIV infection, as well as drug metabolism and toxicity.

[0007] In addition, recent research results have revealed that CNVs cause changes in gene expression levels in various cancer types and are involved in the development and progression of cancer, so accurate interpretation of CNVs is required for cancer mechanism and genetic research (Shao et al. BMC Medical Genetics 2019; 20: 175).

[0008] The most important genes associated with known CNVs are those involved in drug metabolism. CNVs in these genes are linked to drug ADME (absorption, distribution, metabolism, excretion), which is related to individual drug response. Drug ADME affects pharmacokinetics and pharmacodynamics, which in turn leads to differences in drug efficacy and safety. CNVs in genes of Phase I enzymes associated with drug metabolism, such as CYP2A6 (Cytochrome P450 Family 2 Subfamily A Member 6), CYP2D6 (Cytochrome P450 2D6), GSTT1 (glutathione S-transferase theta 1), GSTM1 (Glutathione S-Transferase Mu 1), SULT1A1 (Sulfotransferase Family 1A Member 1), and Phase II enzymes, such as UGT2B17 (UDP Glucuronosyltransferase Family 2 Member B17), have been reported to be associated with drug metabolism.

[0009] In particular, CYPs, such as CYP2D6 and CYP2A6, are distributed in the liver, blood, etc. and are responsible for about 90% of phase I drug metabolism, and are enzymes that are also involved in the synthesis or metabolism of hormones, cholesterol, vitamin D3, etc., and are a very important enzyme group in the human body. Many SNPs (more than 2,000 reported) have been discovered in human CYPs (57 genes), and many of these variants are associated with drug metabolism, and there are differences in variation patterns by race. Drug metabolism can occur not only due to variations in metabolism-related enzymes and the resulting differences in activity, but also depending on the number of genes encoding the enzymes. Recent research results have reported that variations in drug metabolism appear in a very complex pattern due to various copy number variations (CNVs) in addition to simple variations in drug-metabolizing genes such as CYP.

[0010] Depending on the differences in genetic variants (In / Del, SNP, etc.), the number of CNVs and mutation patterns, as well as the complete deletion of the gene, drug metabolism can occur in a very complex manner, such as UM (ultra-rapid metabolizer), RM (rapid metabolizer), PM (poor metabolizer), IM (intermediate metabolizer), and NM (normal metabolizer) (Wilkinson, GR N Engl J Med 2005; 352: 2211-2221).

[0011] CYP2D6, one of the most studied CYPs, is known to be involved in the metabolism of 20 to 25% of commercially available drugs (Trends Pharmacol Sci. 2004 Apr; 25(4):193-200. [PubMed: 15063083]), and its activity is known to vary greatly between individuals due to environmental and genetic factors. Genetically, CYP2D6 is known to have polymorphisms due to various factors such as SNPs, insertions, deletions, and CNVs resulting from them, which can cause decreased or increased drug metabolism.

[0012] In addition, recent research results have shown that the transcript of CYP2A7, a pseudogene in the expression of CYP2A6, acts as a decoy for microRNA (miR-126) (Nakano et al. Drug Metab Dispos. 2015: 43: 703-712), and the need to accurately measure the copy number of the pseudogene is also emerging.

[0013] Therefore, in addition to determining genotypes such as SNPs of genes associated with drug metabolism, cancer, and hereditary diseases, diagnosis of CNV patterns is very important for more accurately predicting drug metabolism according to the genotype of the target enzyme.

[0014] Technologies that accurately measure CNV of genes include next-generation sequencing (NGS), DNA chip technology, multiplex ligation-dependent probe amplification (MLPA), and qPCR technology.

[0015] The most common method for identifying genetic mutations and CNVs is NGS technology. This technology uses the number of reads (i.e., quantitative information about the target gene sequence range) from sequence analysis of the entire genome or multiple target gene groups to determine the copy number.

[0016] This method is suitable for analyzing multiple genes simultaneously, but it is not suitable for general laboratories because it requires expensive equipment and is costly and time-consuming to analyze a single drug or a small number of target genes. In addition, it has the disadvantage of being difficult to determine the exact copy number when the gene is homo-type.

[0017] Another CNV analysis technology is the Multiplex Ligation-dependent Probe Amplification (MLPA) technology developed by MRC Holland (Schoutenet et al. Nucleic Acids Res. 2002; 30(12);e57) (US Patent No. US 6,955,901 B2).

[0018] This technology requires expensive equipment, including a capillary fragment analyzer, sophisticated sample preparation, and skilled researchers, preventing its commercialization. Furthermore, this technology is not particularly suitable for identifying mutations.

[0019] The most useful method for identifying known genetic mutations is qPCR. This method utilizes a fluorescent probe specific to the mutation sequence. It is highly economical and, developed in kit form, simplifies sample preparation and analysis. Furthermore, the widespread availability of qPCR systems makes this method suitable for use in most laboratories conducting clinical analyses.

[0020] Recently, in order to improve the specificity and sensitivity in the qPCR method for mutation confirmation, the present inventors have invented a qPCR method introducing a com-probe that competes with the detection probe (Kim, JJ et al. Sci Rep. 2023; 13: 1036) and a method using discrimination boosting (db) oligonucleotides that increase the specificity of specific primers for alleles (Korean Patent Registration; 10-2275038) (Kim, JJ, et al. Sci Rep. 2021; 11: 19098).

[0021] The most widely known CNV measurement technique using qPCR is the TaqMan probe (Mayo P. et al. Current Protocols in Human Genetics, 2010; 2.13.1-2.13.10). A representative commercially available product is the TaqMan Copy Number Assays Kit (Applied Biosystems, Forest City, CA, USA). This kit uses the RNaseP TaqMan Copy Number Reference Assay as an internal control and CopyCaller Software (Applied Biosystems, Forest City, CA, USA) to determine the copy number.

[0022] This method is based on the relative quantification of the target gene with a reference gene such as RNaseP whose exact copy number is known. qPCR of the target gene and the reference gene is performed separately, the comparative ΔCt of the target gene is obtained, and the ΔΔCt is calculated by normalizing using a calculator and calculating the relative quantification (RQ) (RQ = 2 △△Ct) to determine the CNV (=2 x RQ) of the target gene (Livak KJ and Schmittgen TD Methods 2001; 25: 402-408).

[0023] This method is a copy number determination technique that has the advantages of the above-mentioned qPCR method, but because it measures the copy number by statistically calculating multiple measurement results determined from multiple PCR reaction groups, multiple measurements are required, the accuracy is somewhat low, and in particular, there is the inconvenience of having to test the variant through a separate reaction, and especially, when the gene is heterotype, there is a disadvantage in that the copy number of each allele within the diplotype cannot be determined. For example, this method cannot distinguish between the Tx1 / Gx2 type and the Tx2 / Gx1 type in the case of a heterotype with a total copy number of 3.

[0024] The above various copy number determination methods require expensive equipment, skilled techniques, and a large number of reactions, so a simple, economical, and highly accurate method for determining CNV and simultaneously measuring variants is needed.

[0025] The present invention aims to provide a simple qPCR method for determining the copy number of a gene along with determining the genotype (variant) of the gene. More specifically, the present invention provides a qPCR method capable of identifying genetic mutations such as SNPs and In-Del in a single reaction tube, and provides a method for comparing and analyzing the copy number of a target gene by simultaneously performing qPCR with a reference gene of known copy number (e.g., CANX, TBP, YWHAZ, ATP5F18, UBC, etc., including RNaseP and having no other pseudogenes in the chromosome) (Fig. 2a).

[0026] To this end, the present inventors have invented a method for determining the copy number by performing qPCR reflecting the copy number of the target gene and simultaneously performing qPCR amplification of a reference gene and comparing them by using a forward and reverse primer set capable of specifically amplifying only the target gene in a single reaction tube and also using a probe capable of detecting the target gene. At this time, the variant type can be determined simultaneously by using an allele-specific probe reflecting the sequence variation (allele type) of the target gene (Fig. 2a).

[0027] Mutant sequence typing can be determined by checking the presence or absence of allele-specific probe signals, and the total copy number of the target gene and / or the copy number of the allele can be calculated by comparing it to the Ct of a reference gene of known copy number (e.g., RNaseP, etc.) (Fig. 2a).

[0028] As described above, in order to increase the specificity of qPCR in the qPCR method of the present invention invented by the present inventors, PCR using an allele-specific primer or an ARMS primer can be performed, and in order to increase the discrimination in detecting minor alleles, an oligonucleotide composed of any sequence that partially or entirely forms a double strand as invented by the present inventors (Kim, JJ, et al. Sci Rep. 2021; 11: 19098) (hereinafter used interchangeably with "db-Oligo" in the description and drawings of the present invention) can be added, and in order to increase the specificity of the probe in the qPCR method of the present invention, a non-specific signal suppressing Com-probe (hereinafter used interchangeably with "Com-probe" in the description and drawings of the present invention) that is complementary to the non-mutated target base sequence, binds preferentially to the non-mutated target base sequence rather than the mutated target base sequence, and has a structure in which the 3' end is not extended by a nucleic acid polymerase (Kim, JJ et al. Sci Rep. 2023; 13: 1036) can be added (Fig. 1a).

[0029] The oligonucleotide composed of any sequence that forms a double strand partially or entirely to increase the discrimination in detecting minor alleles invented by the present inventors refers to an oligonucleotide that is not complementary to a forward primer, a reverse primer, and a specific target gene for a target gene and can reversibly bind to a DNA polymerase, and the sequence is not specified, and refers to an oligonucleotide composed of any sequence that forms a double strand partially or entirely within a single strand or with another strand. In the present specification and drawings, it is used interchangeably with "db-Oligo". When this oligonucleotide is used in qPCR, the specificity and sensitivity of qPCR in detecting minor alleles are significantly improved (Kim, JJ, et al. Sci Rep. 2021; 11: 19098).

[0030] In addition, the Com-probe for suppressing non-specific signals invented by the inventors of the present invention will be described in more detail. The fluorescent hydrolysis probe has a sequence identical to the mutated base sequence of the target gene, and differs from the non-mutated base sequence of the target gene by at least one, preferably one to three, and more preferably one or two bases. In general, when the probe has one or more mismatches with the target gene, the hybridization robustness is weak, and the probe is displaced before hydrolysis by the 3' nuclease of the enzyme occurs during the polymerization process of DNA polymerase. However, when an excess of non-mutated target gene is present, hydrolysis of a small amount of non-displaced fluorescent probe occurs, resulting in non-specific signals. In the present invention, in order to suppress the generation of non-specific signals generated from an excess of non-mutated target gene, a Com-probe having a sequence identical to the non-mutated target gene can be added. At this time, the Comprobbe does not contain a fluorescent substance and is not linked to a fluorescent substance. Since the Comprobbe does not displace after binding to the non-mutant target gene (wild type, WT) present in excess in the sample, non-specific signal generation by the fluorescent hydrolysis probe can be suppressed. In addition, since the added Comprobbe mismatches the sequence of the mutant target gene, it does not affect the hydrolysis of the fluorescent hydrolysis probe matching the mutant target gene sequence, thereby significantly facilitating the detection of mutants by qPCR (Kim, JJ et al. Sci Rep. 2023; 13: 1036).

[0031] The diplotype of the target gene can be clearly determined by determining the exact copy number of the target gene through qPCR implemented by the above method, measuring variants (allele types) such as SNPs, and determining the copy number of each gene.

[0032]

[0033] To explain the method for determining the copy number of the present invention in more detail, the CN Ct values ​​(CN Ct values) corresponding to the copy numbers 1, 3, 5, and 6 of the virtual target gene are calculated based on the Ct values ​​of copy numbers 2 (CN2) and 4 (CN4) obtained in each individual experiment, and by substituting these, a quantitative analysis curve is created to obtain the function y = A x ln(x) + B, and by substituting the Ct value (y) of the target gene, the copy number (x) of the target gene is calculated, and the copy number can be determined as an approximate value (Fig. 13b).

[0034] In order to obtain each CN Ct value in this way, the deviation of Ct values ​​from the same two gene copies in the qPCR reaction of the reference gene and the target gene as an internal control, dCt(TCN - IC1) and It is desirable to perform PCR so that dCt(AS - IC1) satisfies the condition of 0 ± 0.1 (b in Fig. 2). "dCt(TCN - IC1)" refers to the Ct value of TCN - the Ct value of IC1, and also dCt(AS - IC1) refers to the Ct value of AS - the Ct value of IC1, and “IC1” refers to Internal Control 1.

[0035] Also, in performing PCR, it is more preferable to perform PCR so that the Ct value of another internal standard (IC) probe (CN4; IC2) of the reference gene reflects the copy number of the target gene and twice the copy number, and the deviation from the Ct value of CN2, i.e., dCt(CN2;IC1- CN4;IC2) satisfies the condition of 1.0 ± 0.1 (Fig. 2b). dCt(CN2;IC1- CN4;IC2) refers to the difference between the Ct value of the internal control 1 with a copy number of 2 and the Ct value of the internal control 2 with a copy number of 4. At this time, CN4; IC2 is preferably composed of two probes that emit the same fluorescence and bind to two different regions so that dCt(CN2;IC1- CN4;IC2) becomes 1.0 ± 0.1, and such two probes are indicated as IC2.1 and IC2.2 in the present specification and drawings, etc.

[0036] In this way, the Ct values ​​obtained from the experimental results of samples artificially prepared using standard plasmids for each copy number performed in one reaction group are converted to dCt (Ct of the target gene - Ct of the reference gene IC1) to synchronize the target gene and the reference gene, and then a quantitative curve for each copy number is obtained (b and c in Fig. 3), and the CN Ct coefficients are determined to obtain artificial Ct values ​​for copy numbers 1, 3, 5, and 6 (Fig. 5, Table 5).

[0037] Theoretically, if we explain the CN Ct factor assuming 100% PCR efficiency, the regression analysis curve equation for the change in Ct value according to the change in copy number, y = -1.443 x ln(x) + 1 (b in Fig. 8), is used to obtain the CN1 Ct factor [dCt(CN1 - CN2) / dCt(CN2 - CN4)x 100 = 100%], CN3 Ct factor [dCt(CN2 - CN3) / dCt(CN2 - CN4)x 100 = 58.5%]; CN5 Ct coefficient [dCt(CN4 - CN5) / dCt(CN2 - CN4)x 100 = 32.2%], CN6 Ct coefficient [dCt(CN4 - CN6) / dCt(CN2 - CN4)x 100 = 58.6%] are calculated (a in Table 7 or Table 10), and by applying these, CN1 Ct value = Ct CN2 + 100% of dCt, CN3 Ct value = Ct CN2 - 58.5% of dCt, CN5 Ct value = Ct CN4 - 32.2% of dCt, CN6 Ct value = Ct CN4 - 58.6% of dCt can be obtained (Fig. 9). At this time, dCt is the difference between the Ct values ​​of CN2 and CN4 of the reference gene (dCt (CN2-CN4) )am.

[0038] The CN Ct coefficient and CN Ct values ​​for experimental values ​​can be obtained in the same manner as the theoretical calculation (Fig. 5, Table 5).

[0039] Using the CN Ct coefficients calculated using the method described above, various information such as the Ct values ​​of CN 1, 3, 5, and 6 and the CN2, CN4, allele, and total copy number obtained from the experiment can be expressed in a single table (Table 6), and a Ct judgment table (plot) that is schematically expressed for easy visual reading can be obtained (Fig. 6b).

[0040] By applying the CN Ct value obtained by the theoretical or experimental method described above, the regression analysis curve (standard quantitative curve) equation, y = A x ln(target gene copy number x) + B, can be derived, and the Ct value y of the analyzed target gene can be substituted to obtain the copy number x of the target gene (Table 10, Table 11).

[0041] The number of replicates can be determined by calculating them from individual tests (Table 13), or by calculating them from the average test value of 3 to 10 replicate tests (Table 14).

[0042] The judgment table of the present invention can simultaneously display the Ct value of the target gene derived from qPCR performance, the Ct value of the signal corresponding to the allele, the Ct value corresponding to CN2 of the reference gene, and the Ct value corresponding to CN4 of the reference gene (a in FIG. 6), and can display the CN1 Ct value, CN3 Ct value, CN5 Ct value, and CN6 Ct value calculated here (b in FIG. 6).

[0043] The CN Ct coefficient presented in the present invention can be used as a constant value without having to be derived for each individual test, and this enables the generation of quantitative curves for CN1, CN2, CN3, CN4, CN5, and CN6 for each individual reaction, thereby enabling the accurate determination of the copy number for each allele of the target gene diplotype.

[0044] The Ct determination table (Ct plot) proposed in the present invention displays each analyzed sample on a single axis, thereby enabling the results of multiple samples to be more clearly distinguished by sample copy number and diploid type. In addition, through the calculation of test results and CN Ct values, the genotype and copy number of the sample can be clearly identified, and if necessary, even the results of pseudogene analysis can be visualized to provide simple information, facilitating the determination of multiple samples (Figs. 11 and 12).

[0045] The present invention provides a simple qPCR method and kit capable of simultaneously providing copy number and genetic variant information of a target gene by performing qPCR of a reference gene and a target gene in one reaction tube and comparing and calculating the Ct value of the reference gene.

[0046] Figure 1 is a diagram showing the establishment of qPCR formats and conditions for measuring mutations and CNVs in CYP2A6. Figure 1a shows the composition and arrangement of primers and probes for three qPCR formats (①, ②, ③) for detection tests of CYP2A6 mutations and pseudogene CYP2A7, and Figure 1b shows the test results (Ct values) for each qPCR format test and the qPCR amplification plots for each qPCR format test.

[0047] Figure 2 is a diagram showing the format of qPCR for mutation and CNV determination and the prerequisites for multiplex qPCR performance. a shows the positions and configurations of primers and probes for qPCR synchronization with the target gene (CYP2A6), the reference gene (RNaseP), and the prerequisites (prerequistes) of qPCR for CNV and mutation measurement, the test results (qPCR), each signal value (Ct & dCt values), and the judgment using them.

[0048] Figure 3 is a diagram showing the change in dCt for the reference gene (RNaseP) according to the change in the copy number of CYP2A6 (-48T) (homo).

[0049] a. qPCR amplification curve according to the change in copy number of CYP2A6 (-48T),

[0050] b. Regression analysis curve of dCt(FAM - Cy5) according to change in CYP2A6 (-48T) copy number,

[0051] c. Regression analysis curve of dCt(JOE - CY5) according to changes in CYP2A6 (-48T) copy number.

[0052] Figure 4 is a qPCR amplification curve according to the change in copy number of CYP2A6(-48T / G)(heterozygote).

[0053] Figure 5 is a diagram explaining the calibration curve of the number of copies and dCt values ​​and the CN factors.

[0054] Figure 6 shows a Ct curve according to the change in copy number and a Ct judgment table (Ct plot) applying the calculated Ct value for each copy number. (a) is a vertical Ct graph of qPCR signal according to copy number, and (b) is a Ct graph applying the calculated CN Ct value for use as a copy number counter.

[0055] Figure 7 shows the change in dCt for the reference gene (RNaseP) according to the change in CYP2A7 copy number. a is a qPCR amplification curve according to the change in CYP2A7 copy number, and b is a graph of dCt values ​​(FAM - Cy5) according to the change in CYP2A6 (-48T) copy number.

[0056] Figure 8 is a comparison of the CN coefficient calculated from experimental values ​​and the CN coefficient calculated from theoretical values. a is the dCt according to the number of replicates obtained from the experiment. (FAM-Cy5) a shows the graph and CN coefficient of dCt(FAM - Cy5) according to the change in theoretical copy number, and b shows the graph and CN coefficient of dCt(FAM - Cy5).

[0057] Figure 9 is a diagram showing the results of SNP and CNV tests using a standard plasmid, as well as the Ct graph and the determination of diplotypes. The copy number was measured using the values ​​calculated from Table 9, and the diplotypes were determined from this. Test numbers 1 to 5; the tests and results of Example 5 (Table 3); Test numbers 6 to 14; the tests and results of Example 6 (Table 4); and Test numbers 15 to 20; the tests and results of Example 10 (Table 9) were used.

[0058] Figure 10 shows qPCR amplification curves for measuring mutations and copy number of CYP2A6 and pseudogene CYP2A7 in human genomic DNA. Genomic DNA was used at 2 ng, and other qPCR conditions were the same as in Example 13. Only one representative example of each of the three replicate tests is presented.

[0059] Figure 11 is a diagram showing the results of SNP and CNV determination using human genomic DNA, along with a Ct graph and diplotype determination using the results. (a) is a table of copy number and diplotype determination, (b) shows the results of three replicate qPCR tests for each sample, and (c) shows the average value of the tests.

[0060] Figure 12 is a diagram showing the results of SNP and CNV determination using human genomic DNA, along with a Ct graph and diplotype determination. The results of three replicate qPCR tests of the sample are shown, and c represents the average value of the tests.

[0061] Figure 13 shows a formula for calculating the number of copies and an example of the calculation (human genome DNA sample 1). a shows a graph formula derived using the Ct value of sample 1 in Figure 8, and b shows a calculation method using the derived graph formula.

[0062] One aspect of the present invention is

[0063] a) A step of simultaneously adding a) a target gene-specific primer, a target gene-specific probe, and a probe capable of distinguishing alleles of the target gene, and b) a reference gene-specific primer and probe capable of amplifying and identifying a reference gene, to one reaction zone, and synchronizing the amplification of the gene to obtain an amplification experimental value (Ct value) using qPCR;

[0064] b) A step of determining the copy number Ct factor (used interchangeably with “CN Ct factor”) from the amplification experimental value and / or theoretical amplification value;

[0065] d) A step of determining the Ct values ​​of target gene copy numbers 1, 3, 5 and / or 6 from the amplification experimental values ​​of reference gene copy numbers 2 and 4 using the determined copy number Ct coefficients; and

[0066] a) A step of comparing the amplification experimental value of the target gene with the test copy number of the reference gene and the Ct values ​​of the copy numbers 1, 3, 5 and / or 6 of the target gene determined in step c) above to determine the total copy number of the target gene and the genotype of the allele; relates to a method for simultaneously determining the copy number and genotype of a gene, including:

[0067] In addition, one aspect of the present invention relates to a method for simultaneously determining the copy number and genotype of a gene, wherein a primer and probe set capable of specifically amplifying the pseudogene of the target gene is added to a) in step a).

[0068] In addition, one aspect of the present invention relates to a method for simultaneously determining the copy number and genotype of a gene, wherein at least one of the target gene-specific primer and the pseudogene-specific primer is an AS primer (Allele specific primer) or an ARMS primer (Amplification Refractory Mutation System primer).

[0069] In addition, one aspect of the present invention relates to a method for simultaneously determining the copy number and genotype of a gene, further introducing a discrimination enhancing oligonucleotide that is not complementary to the primer and template in order to increase the discrimination of specific primer amplification in the qPCR step of the above a), is reversibly bindable to a qPCR DNA polymerase, and forms a double strand partially or completely within a single strand or with another strand; and a non-specific signal suppression probe that is complementary to the target base sequence of a non-mutated target gene in order to increase the discrimination of the specific probe, binds preferentially to the target base sequence of the non-mutated gene over the target base sequence of the mutated target gene, and has a structure in which the 3' end is not extended by a nucleic acid polymerase.

[0070] In addition, one aspect of the present invention relates to a method for simultaneously determining the copy number and genotype of a gene, in which qPCR is performed using fluorescent probes of different wavelengths as the target gene-specific probe and the allele-specific probe to confirm the copy number and allele copy number of the target gene in the qPCR step of the above a).

[0071] In addition, one aspect of the present invention relates to a method for simultaneously determining the copy number and genotype of a gene, which performs qPCR using fluorescent probes of different wavelengths to obtain amplification experimental values ​​of copy number 2 and copy number 4 of the reference gene in the qPCR step of the above a).

[0072] In addition, one aspect of the present invention relates to a method for simultaneously determining the copy number and genotype of a gene, wherein the method for determining the copy number Ct coefficient from the amplification experimental value of step b) is to derive a calibration curve using the experimental Ct value of a standard gene to determine the copy number Ct coefficient of copy numbers 1, 3, 5 and / or 6 of the target gene.

[0073] In addition, one aspect of the present invention relates to a method for simultaneously determining the copy number and genotype of a gene, wherein the method for determining the copy number Ct coefficient from the theoretical amplification value of step b) is performed using the mathematical formula y = -1.443 x ln(x) + 1 (wherein y is the Ct value of the target gene and x is the copy number of the target gene).

[0074] In addition, one aspect of the present invention relates to a method for simultaneously determining the copy number and genotype of a gene, wherein the method for determining the Ct value of the copy number of the target gene using the copy number Ct factor of the step d) is to obtain the difference (dCt(CN2-CN4)) between the Ct of the copy number 2 of the reference gene and the Ct of the copy number 4 obtained from the amplification experiment values ​​of the target gene and the reference gene of which the copy number is known, and to determine the Ct of the copy number of 1, 3, 5 and / or 6 of the target gene by applying the copy number Ct factor obtained above.

[0075] In addition, one aspect of the present invention relates to a method for simultaneously determining the copy number and genotype of a gene, wherein a method of comparing an amplification experimental value of a target gene to a Ct value of a copy number of a target gene is performed by applying the Ct values ​​of copy numbers 1, 3, 5 and / or 6 of the target gene, the CN2 Ct value of the target gene and the CN4 Ct value of the target gene to obtain a standard quantitative curve equation, y = A x ln(x) + B (wherein y is the Ct value of the target gene and x is the copy number of the target gene), and using this equation to obtain the copy numbers of the target gene and alleles.

[0076] In addition, one aspect of the present invention relates to a method for simultaneously determining the copy number and genotype of a gene, in which the amplification synchronization of the target gene and the reference gene is performed by positioning the target gene and the reference gene on one plasmid to perform qPCR.

[0077] In addition, one aspect of the present invention relates to a method for simultaneously determining the copy number and genotype of a gene, which performs qPCR by synchronizing the amplification of a target gene and a reference gene, and which performs the qPCR by adjusting qPCR conditions so that the Ct value deviations dCt(TCN - IC1) and dCt(AS - IC1) in the same two gene copy numbers in the qPCR reaction of the reference gene and the target gene satisfy 0 ± 0.1, respectively.

[0078] In addition, one aspect of the present invention relates to a method for simultaneously determining the copy number and genotype of a gene, in which qPCR is performed so that the copy number of the reference gene is twice the copy number of the target gene.

[0079] In addition, one aspect of the present invention relates to a method for simultaneously determining the copy number and genotype of a gene, which is performed by adjusting qPCR conditions so that the copy number of the reference gene is twice the copy number of the target gene and so that dCt(CN2;IC1-CN4;IC2) satisfies 1.0 ± 0.1.

[0080] In addition, one aspect of the present invention relates to a method for simultaneously determining the copy number and genotype of a gene, which uses two probes having different sequences of the same fluorescence simultaneously to obtain an amplification experimental value of copy number 4 in the qPCR step.

[0081] In addition, one aspect of the present invention relates to a method for simultaneously determining the copy number and genotype of a gene, in which a Ct value that is 1.0 less than the Ct value of the copy number 2 of the target gene is arbitrarily substituted to obtain an amplification experimental value of the copy number 4 of the target gene, and the Ct value of the copy number 4 is calculated. The general copy number is 2, and the Ct value of the copy number 4, which is twice that, can be arbitrarily substituted to obtain the Ct value of the copy number 2, and the Ct value of the copy number 4 can be calculated.

[0082] In addition, one aspect of the present invention relates to a method for preparing a simultaneous determination table of gene copy number and genotype, which displays qPCR Ct results and copy number Ct values ​​on one axis and displays target gene samples to be analyzed on another axis so that the genotype and copy number of one or more analysis samples can be visually confirmed.

[0083] In addition, one aspect of the present invention relates to a method for preparing a simultaneous determination table of gene copy number and genotype, which includes a Ct value, a Ct value by copy number, a copy number of a diploid genotype, and an allele type in the determination table.

[0084] In addition, one aspect of the present invention relates to a method for creating a simultaneous determination table of gene copy number and genotype, which includes a Ct value, a Ct value by copy number, a diplotype, an allele type, and a copy number of a pseudogene in the determination table.

[0085] In addition, one aspect of the present invention relates to a method for creating a simultaneous determination table of gene copy number and genotype, wherein the determination table reflects the result values ​​and / or calculation values ​​obtained from the simultaneous determination method of gene copy number and genotype.

[0086] In addition, one aspect of the present invention relates to a simultaneous determination table of gene copy number and genotype, which can visually confirm the genotype and copy number of one or more analysis samples by displaying the qPCR Ct result value and copy number Ct value on one axis and the target gene sample to be analyzed on the other axis.

[0087] In addition, one aspect of the present invention relates to a simultaneous determination table for gene copy number and genotype, which includes a Ct value, a Ct value by copy number, a copy number of a diploid genotype, and an allele type in the determination table.

[0088] In addition, one aspect of the present invention relates to a simultaneous determination table for gene copy number and genotype, which includes a Ct value, a Ct value by copy number, a diplotype, an allele type, and a copy number of a pseudogene in the determination table.

[0089] In addition, one aspect of the present invention

[0090] a) Target gene-specific primers and target gene-specific probes;

[0091] b) A specific probe capable of detecting and distinguishing the alleles of the target gene;

[0092] c) Reference gene-specific primers and reference gene-specific probes capable of amplifying and identifying the reference gene; and

[0093] a) A qPCR kit for simultaneous determination of the copy number and genotype of a target gene, including DNA polymerase.

[0094] In addition, one aspect of the present invention

[0095] a) Target gene-specific primers and target gene-specific probes;

[0096] b) A specific probe capable of detecting and distinguishing the alleles of the target gene;

[0097] c) Reference gene-specific primers and reference gene-specific probes capable of amplifying and confirming the reference gene;

[0098] a) Primers and probes capable of specifically amplifying the pseudogene of the target gene; and

[0099] b) A qPCR kit for simultaneous determination of the copy number and genotype of a target gene, including a DNA polymerase.

[0100] In addition, one aspect of the present invention is the kit

[0101] b) To enhance the discrimination of specific primer amplification, an oligonucleotide for enhancing discrimination that is not complementary to the primer and the target gene template, can reversibly bind to a DNA polymerase for qPCR, and forms a double strand partially or completely within a single strand or with another strand; and

[0102] The present invention relates to a qPCR kit for simultaneous determination of the copy number and genotype of a target gene, which further comprises a non-specific signal suppression probe having a structure that is complementary to the target base sequence of a non-mutant target gene, binds preferentially to the target base sequence of a non-mutant target gene over the target base sequence of a mutated target gene, and does not have a 3' end extended by a nucleic acid polymerase, in order to increase the distinguishability of a target gene-specific probe.

[0103] Below, the composition of the present invention is described in more detail, using specific embodiments and test examples. However, it will be apparent to those skilled in the art that the scope of the present invention is not limited to the description of the embodiments and test examples. Unless otherwise specified, the terminology used herein is used in the sense commonly used by those skilled in the art.

[0104] In addition, in the embodiments of the present invention, FAM, JOE, CFR610, and Cy5 were used as fluorescent substances for identification, but the fluorescent substances are not limited to the substances exemplified above, and it is obvious to a person having ordinary skill in the art to which the present invention pertains that the fluorescent substances labeled for each sequence may be replaced with different fluorescent substances.

[0105]

[0106] Example 1: Construction of a standard plasmid for testing

[0107] RNaseP, a housekeeping gene, was secured as a reference gene for CNV measurement and constructed by introducing it into a plasmid vector as follows. The RNaseP gene was obtained by PCR amplification using human genomic DNA (Promega) as a template, Pfu DNA polymerase (Enzynomics, Korea), and the forward primer RNaseP-F (5'- GGTACCGCTGGCACTTTCTGTTATG-3', SEQ ID NO: 1) and the reverse primer RNaseP-R (5'- ACTAGTGGGAGAAGAAGGAAGGGA-3', SEQ ID NO: 2). This was inserted into the pTOP Blunt V2 vector and transformed into E. coli DH5α to obtain a plasmid with the RNaseP gene inserted. The RNaseP sequence (SEQ ID NO: 26) of the obtained plasmid was confirmed by base sequence analysis, and the confirmed pRNaseP plasmid was constructed. The constructed pRNaseP plasmid was digested with the restriction enzyme NotI and used in the experiment. The vector was constructed so that the CYP-related genes for CNV measurement were positioned downstream of the pTOP-RNaseP vector. The vector was obtained using Pfu DNA polymerase (Enzynomics, Korea) using human genomic DNA of CYP2A6, CYP2A7, and CYP2D6 (Promega) as a template.For CYP2A6, the primers CYP2A6-F (5'-CCACAGCCCTGCGGCACC-3', SEQ ID NO: 3) and CYP2A6-R (5'-TGGTCAACCCCCTGCCAC-3', SEQ ID NO: 4) were used, for CYP2A7, CYP2A7-F (5'-TCACCCTAATAAAACCCAGACCT-3', SEQ ID NO: 5) and CYP2A7-R (5'-GGACCCGGATGCTGAAACT-3', SEQ ID NO: 6) were used, and for CYP2D6, the primers CYP2D6-F (5'-ACAGTCAACACAGCAGGTTCA-3', SEQ ID NO: 7) and CYP2D6-R (5'-CTGTTTCATGTCCACGACCC-3', SEQ ID NO: 8) were used. The amplified products of each gene were inserted into the pTOP Blunt V2 vector to produce them. The sequences of the produced CYP2A6 (SEQ ID NO: 27 or SEQ ID NO: 28) and CYP2A7 (SEQ ID NO: 29) were confirmed, and the mutant (allele) pCYP2A6(-48T) was produced. The final plasmids obtained are pCYP2A6(-48T), pCYP2A6(-48G), and pCYP2A7. In addition, in order to match the copy numbers of the reference gene and the target gene to be analyzed in these plasmids, the reference gene, the RNaseP gene, was produced as one by linking them as follows. The pRNaseP vector was digested with KpnI / SpeI to obtain a fragment (456 bp), which was then cloned into each gene plasmid digested with KpnI / SpeI to obtain pRNaseP / CYP2A6(-48T), pRNaseP / CYP2A6(-48G), and pRNaseP / CYP2A7. These were digested with restriction enzymes to make a single strand, purified, and used as standard plasmids for CNV and mutation measurement.

[0108]

[0109] Example 2: Performing qPCR

[0110] qPCR (real-time quantitative PCR) was performed using a CFX Opus 96 Real-Time PCR System with 45 cycles of 95°C for 10 minutes, 95°C for 20 seconds, and 60°C for 40 seconds. The primers and probes used for qPCR for each target were of the type and amount indicated in each example. The reaction was performed with 3U of Taq polymerase (STexS-TaqII, GenoTech Korea) and buffer (5x STexS-DB1.2 Taq buffer or 5x GenoTaq buffer, GenoTech, Korea) in a total volume of 20 μl. The Ct value of the performed qPCR was determined at a threshold of 400. At this time, the plasmid was 10 4 copies / ul, and human genomic DNA was used at a concentration quantified as 1 ng / ul. Human genomic DNA was donated from the Daejeon Biomedical Regulation-Free Special Zone Human Material Bank (IRB file No. CNUH 2022-08-047).

[0111]

[0112] Example 3: Determining the qPCR format

[0113] As an example of the test of the present invention, we attempted to measure the copy number and determine alleles for CYP2A6*9 (-48T>G). In the human genome, there is a pseudogene CYP2A7 (SEQ ID NO: 29) that is very similar to CYP2A6 (SEQ ID NO: 27). Therefore, an appropriate qPCR format for clear distinction between the two genes is required. To this end, as shown in Table 1, a general qPCR using primers, probes, and buffers (format ①), a method applying specific primers to suppress the amplification of the pseudogene CYP2A7 (format ②), and further, the STexS PCR method (see Kim, JJ et al. Sci Rep. 2023; 13: 1036) (using STexS-DB1.2 Taq buffer (GenoTech, Korea)) (format ③) were used and compared (Fig. 1a). The results of qPCR performed using each of the pCYP2A6(-48T), pCYP2A6(-48G), and pCYP2A7 plasmids as templates in each qPCR format are as shown in Fig. 1b. The qPCR results of format ① showed almost similar qPCR Ct values ​​for all genes, indicating no differentiation. However, when a specific primer (CYP2A6-SF, SEQ ID NO: 12) whose 3'-terminal sequence can provide differentiation between CYP2A6 and CYP2A7 was used and a -48T-specific probe (FAM-CYP2A6-48T SEQ ID NO: 13) was used, each gene was distinguished to a significant degree. However, a significant level of qPCR signal was also observed for CYP2A7 and CYP2A6(-48G). The applicant's STexS technology was applied to completely suppress these nonspecific signals. 5x STexS-DB1 to suppress amplification of CYP2A7 despite use of specific primer (CYP2A6-SF, SEQ ID NO: 12).When Taq buffer (Genotech, Korea) was applied and the corresponding com-probe (CYP2A6 / -48G-Com-P SEQ ID NO: 14) was used to suppress nonspecific binding of the specific probe (FAM-CYP2A6-48T SEQ ID NO: 13), nonspecific signals of the CYP2A7 (SEQ ID NO: 29) and CYP2A6(-48G) (SEQ ID NO: 28) genes observed when the STexS technology was applied were completely suppressed, and there was no change in the qPCR Ct value derived from CYP2A6(-48T) (SEQ ID NO: 27).

[0114] Table 1 below shows the sequences, Tm values, and dosages of primers, probes, etc. for the qPCR format test.

[0115] qPCR formatprimer / probe / etcsequence (5' -> 3') (Tm)pmoles / 20ulformat ①CYP2A6 / 2A7-F1(SEQ ID NO: 9)(F-primer)5'-AGGTIATTATGTAATIAGCCAAAGTCC (63.7℃)10AS-R1 (SEQ ID NO: 10)(R-primer)5'-ACAGTCAGGCAGICCAGCAAGG (65.82℃)10FAM-R-CYP2A6 / CNV5.2 (SEQ ID NO: 11)(common probe)5'-FAM-CTGAGGCCAGCATGGTGGTAGTG-BHQ1 (68.3℃)10format ②CYP2A6-SF(SEQ ID NO: 12)([C]-specific primer)5'-GGAGGTGAAATGAGGTAATTATGTAGTC (64.4℃)10AS-R1 (SEQ ID NO: 10)(R-primer)5'-ACAGTCAGGCAGICCAGCAAGG (65.82℃)10FAM-CYP2A6-48T(SEQ ID NO: 13)([T]-specific probe)5'-FAM-TTCAGGCAGTATAAAGGCAAAC-BHQ1 (58.4℃)10format ③CYP2A6-SF(SEQ ID NO: 12)(F-primer)5'-GGAGGTGAAATGAGGTAATTATGTAGTC (64.4℃)10AS-R1 (SEQ ID NO: 10)(R-primer)5'-ACAGTCAGGCAGICCCAGCAAGG (65.82℃)10FAM-CYP2A6-48T(SEQ ID NO: 13)([T]-specific probe)5'-FAM-TTCAGGCAGTATAAAGGCAAAC-BHQ1 (58.4℃)10CYP2A6 / -48G-Com-P (SEQ ID NO: 14)(Com-Probe)5'-TTCAGGCAGTAGAAAGGCAAACp (60.1℃)20

[0116] *It is inosine.

[0117] Example 4: Multiplex qPCR for multiplex quantification

[0118] In order to construct a qPCR system to measure the genotype and copy number of CYP2A6*9 (-48T>G) using the qPCR format ③ established in Example 3, we first selected the RNaseP gene, a representative housekeeping gene, as a reference gene. Since this gene exists in one copy number on one strand of the human chromosome (2N, so the actual copy number is 2), to implement this, we used the pRNaseP / CYP2A6(-48T) standard plasmid, which linked the RNaseP and CYP2A6 genes to one plasmid of Example 1, as shown in Fig. 2a.

[0119] Using this as a template, qPCR was performed with the primer / probe set and conditions shown in Table 2. At this time, the Ct value of each gene was determined by adjusting the amount, position, and length of each primer and probe to achieve PCR harmony between the reference gene RNaseP and the target gene to be measured, CYP2A6 (-48T). At this time, the probe for detecting the reference gene RNaseP, IC1 (internal control 1), corresponds to the Ct value of the copy number CN = 2, and IC2 (internal control 2, IC2.1 + IC2.2) was configured to correspond to the Ct value of the copy number CN = 4, which is twice the number.

[0120] The prerequisites for PCR harmonization are to establish qPCR conditions that satisfy dCt(IC1 - IC2) = Ct of IC1(Cy5) - Ct of IC2(CFR610) = 1 ± 0.1 and dCt(TCN - AS) = Ct of TCN(FAM) - Ct of AS(JOE) = 0 ± 0.1. If qPCR satisfying these prerequisites is performed and Ct and dCt values ​​(Ct & dCt values ​​of qPCR) for each probe are obtained, a judgment can be made as mentioned below. The IC1 probe (Cy5:CN2) Ct value indicates that the copy number (CN) of RNaseP is 2, and the IC2 probe (CFR610:CN4) (IC2.1 + IC2.2) Ct value indicates that the copy number of RNaseP is 4, and the Ct value of the TCN-Probe (TCN-probe-FAM) corresponding to the target gene (CYP2A6) reflects that the copy number of CYP2A6 is 2 because they are linked to one plasmid, and the [T] probe (CYP2A6-48T-JOE) reflects that the copy number of CYP2A6 (-48T) is 2 because it reflects the T base sequence at position -48.

[0121] This provides a very important implication: it shows that copy number can be easily measured by performing qPCR in a single reaction mixture for a reference gene of known copy number (RNaseP in this example) and a target gene of unknown copy number (e.g., CYPs) that are physically linked.

[0122] Table 2 shows the primer / probe set information for the CYP2A6 (-48T) copy number test.

[0123] CYP2A6Sequence (5' -> 3') (Tm)pmoles / 20ulCYP2A6 / -48T / GCYP2A6-SF(SEQ ID NO: 12)5'-GGAGGTGAAATGAGGTAATTATGTAGTC (64.4℃)10AS-R1(SEQ ID NO: 10)5'-ACAGTCAGGGCAGICCAGCAAGG (65.82℃)10CYP2A6-48T-JOE(SEQ ID NO: 15)5'-JOE-TTCAGGCAGTATAAAGGCAAAC-BHQ1 (58.4℃)20TCN-probe-FAM(SEQ ID NO: 16)5'-FAM-CCTGAGGGCCAGCATGGTGGTAGTG-BHQ1 (70.6℃)10CYP2A6 / -48G-Com-P5'-TTCAGGCAGTAGAAAGGCAAACp (60.1℃)9RNasePRNaseP_F6(SEQ ID NO: 17)5'-AGGCTTGCTGTTTGGGCTCT (60.5℃)10RNaseP-R2(SEQ ID NO: 18) 5'-TGACTTGCTTTTAACATGACCATTC (60.9 ℃)10IC1 (Cy5)(SEQ ID NO: 19)5'-Cy5-AGTGACGCCAAGGCTGCGGTG-BHQ3 (67.3 ℃)6IC2.1 (CFR610)(SEQ ID NO: 20)5'-CFR610-TGCCGAGCAGCGCTTCT-BHQ2 (57.3℃)9IC2.2 (CFR610)(SEQ ID NO: 21)5'-CFR610-TGTACTGGAAGAACTTGCTTAC-BHQ2 (58.4℃)9

[0124] Example 5: Correlation between dCt changes and copy number of reference gene (RNaseP) and target gene (CYP2A6)

[0125] Compared to the reference gene RNaseP, the amount of the target gene CYP2A6 was proportionally increased to confirm the change in the Ct value of each probe. To this end, qPCR was performed as in Example 2 using the primer and probe sets in Table 2 using independently prepared standard plasmids pRNaseP and pCYP2A6(-48T) (Table 3 and Fig. 3). As a result of the test, the dCt of IC1 and IC2 (Ct(IC1)(Cy5)- Ct(IC2)(CFR610)) showed a stable measurement value similar to dCt = 1.0 when the theoretical copy number is doubled, with an average of 0.99, and Ct(TCN)(FAM)- Ct(IC1)(Cy5) and Ct(TCN)(JOE)- Ct(IC1)(Cy5) clearly decreased in accordance with the proportional increase in pCYP2A6 reflecting the copy number. In particular, dCt(FAM - Cy5) and dCt(JOE - Cy5) according to the copy number showed similar calibration curves (y = -1.543 x ln(x) + 1.2144 [R² = 0.9881] and y = -1.517 x ln(x) + 1.101 [R² = 0.9979]) (Fig. 3 b and c).

[0126] Table 3 shows the change in dCt for the reference gene (RNaseP) according to the change in CYP2A6(-48T) copy number.

[0127] Copy Number(CN)Plasmid*Ct**dCtpRNasePpCYP2A6 (-48T)IC1(Cy5: CN2)IC2(CFR610: CN4)TCN (CYP2A6)(FAM)AS ([T])(JOE)IC1 - IC2(Cy5 - CFR610)TCN - IC1(FAM - Cy5)AS - IC1(JOE - Cy5)12 ul1 ul28.5327.5629.7129.530.97Average0.9911.181.0022 ul28.4427.5028.5928.550.940.140.1133 ul28.4227.4928.0028.000.93-0.42-0.4244 ul28.4427.4627.5727.520.98-0.87-0.9255 ul28.7927.7527.4927.361.04-1.30-1.4366 ul28.9427.8627.3427.221.08-1.60-1.72

[0128] *Each plasmid is 1 x 10 5 Tests were conducted using copies / ul.

[0129] **Ct values ​​are the average of 3 replicates and were measured at a threshold of 400.

[0130] The test result amplification curve and regression analysis curve are shown in Fig. 3.

[0131]

[0132] Example 6: Effect of Ct value changes according to copy number changes on allelic incorporation.

[0133] pRNaseP, pCYP2A6(-48T) and pCYP2A6(-48G) were cloned into standard plasmids (each 1 x 10 5copies / ul) were used. Assuming various diploid types and SNP types, each plasmid was mixed and tested. As a result, the dCt (Cy5-CFR610) value was 0.993 on average (STDEV = 0.059), satisfying the prerequisite of 1 ± 0.1 as in Example 5, showing an increase or decrease in Ct that stably reflects the copy number (Table 4 and Figure 4). These results show that genetic mutation typing and copy number measurement can be performed simultaneously even when alleles are mixed.

[0134] Table 4 shows the Ct values ​​of qPCR according to the copy number change of CYP2A6(-48T / G) (heterozygote).

[0135] NoDiplotypesCNPlasmids*Ct**dCtpRNasePpCYP2A6(-48T)pCYP2A6(-48G)IC1 (Cy5: CN2)IC2 (CFR610:CN4)TCN (CYP2A6)(FAM)AS([T])(JOE)IC1 - IC2(CY5 - CFR610)#1T*1 / G*122 ul1 ul1 ul28.4727.5628.6229.760.91Average0.993#2T*3 / G*142 ul3 ul1 ul28.5627.5927.7128.190.97#3T*4 / G*152 ul4 ul1 ul28.5727.6527.3427.800.92#4T*2 / G*242 ul2 ul2 ul28.6427.6627.6428.750.98#5T*3 / G*252 ul3 ul2 ul28.7627.7527.4828.271.01#6T*4 / G*262 ul4 ul2 ul28.6927.6527.1527.711.04#7T*1 / G*342 ul1 ul3 ul28.9027.8427.9930.111.06#8T*3 / G*362 ul3 ul3 ul28.9927.9327.3228.391.06

[0136] *Each plasmid is 1 x 10 5 Tests were conducted using copies / ul.

[0137] **Ct values ​​are the average of 3 replicates and were measured at a threshold of 400.

[0138]

[0139] Example 7: Calculation of Ct factor and Ct value using calibration curve

[0140] In order to calculate the Ct value according to a more precise copy number by linking it with the Ct of the known copy number, i.e. the reference gene (RNaseP), a correlation calibration curve between the copy number of the target gene and the dCt (FAM - Cy5) of the target gene and the reference gene was created using the results of the test in Example 5 (Fig. 5). The calculated dCt (calculated) of each copy number calculated using the formula y = -1.543 x ln(x) + 1.2144 derived from this calibration curve is shown in Table 5. Using this, the ratio of the difference in the Ct value with the reference gene of which the copy number is known was determined as the CN1 factor, CN3 factor, CN5 factor, and CN6 factor, as in the Ct coefficients in Table 5. This coefficient was calculated as a percentage of the average value (0.991) of dCt(JOE - CFR610) of IC1 and IC2 obtained in Example 5 to obtain a value linked to the difference in Ct value of CN2(Cy5) or CN4(CFR610) of reference gene (RNaseP) and target gene (CYP2A6) (Table 5). From each obtained Ct coefficient, Ct values ​​(CN Ct values) for each copy number can be calculated by adding or subtracting to the Ct value of CN2(Cy5) or CN4(CFR610) of RNaseP in the same way as the method of obtaining the Ct value in Table 5.

[0141] CN123456*dCt(FAM - Cy5)1.180.14-0.42-0.87-1.30-1.60**dCt(calculated)1.2140.145-0.481-0.925-1.269-1.550***CN Ct factorCN1 Ct factor(1.214 - 0.145) / 0.991 factor(-0.925 + 1.550) / 0.991 x 100 = 63.07 (%)****CN Ct valueCN1 Ct ValueCN2 Ct (Cy5) + dCt(Cy5-CFR610)x 107.87 % (CN1 Ct factor)CN3 Ct ValueCN2 Ct (Cy5) - dCt(Cy5-CFR610)x 63.17 % (CN3 Ct factor)CN5 Ct ValueCN4 Ct (CFR610) - dCt(Cy5-CFR610)x 34.71 % (CN5 Ct factor)CN6 Ct ValueCN4 Ct (CFR610) - dCt(Cy5-CFR610)x 63.07% (CN6 Ct factor)

[0142] *dCt is the result obtained from the test of Example 5.

[0143] **The calculated dCt is the value obtained by calculating using the formula y = -1.543 x ln(X) + 1.2144 in Fig. 5.

[0144] ***CN Ct factor is expressed as the difference between the Ct value of IC1 (CY5) and the Ct value of IC2 (CFR610) of the reference gene obtained in the test, and the average value (0.991) of dCt (JOE- CFR610) is expressed as a percentage of the calculated dCt value.

[0145] ****CN Ct value is a value calculated by adding or subtracting the Ct value corresponding to each copy number to the Ct value of the corresponding copy number of the reference gene (RNaseP).

[0146]

[0147] Example 8. Calculation of Ct coefficient and Ct value using calibration curve

[0148] Using the test values ​​of Example 5 (Table 3) as presented in Example 7, the Ct coefficients were obtained for each, and by applying these, the Ct values ​​of CN1, CN3, CN5, and CN6 were calculated (Table 6). These values ​​can be displayed on one axis together with the Ct test values ​​for each test area (copy number), as shown in Fig. 6b. In this Ct plot, the Ct values ​​corresponding to each CN calculated in Fig. 6a, where only the test values ​​are displayed, are additionally displayed, so that the test values ​​corresponding to each copy number can be compared very easily with the CN Ct values ​​corresponding to each copy number, thereby presenting results that are remarkably useful for determining the copy number.

[0149] DiplotypeCNCt*Ct value (calculated) **IC: CN2 (Cy5)IC : CN4 (CFR610)CYP2A6(FAM)T (JOE)Ct factors107.87%63.17%34.71%63.07%CN1CN3CN5CN6T*1128.5327.5629.7129.5329.5827.9 227.2226.95T*2228.4427.5028.5928.5529.4527.8527.1726.91T*3328.4227.4928.0028. 0029.4227.8327.1726.90T*4428.4427.4627.5727.5229.5027.8227.1226.84T*5528.7927 .7527.4927.3629.9128.1327.3927.09T*6628.9427.8627.3427.2230.1028.2627.4927.18

[0150] *This is the test value of Example 5.

[0151] **Ct value calculated according to the method presented in Table 5.

[0152]

[0153] Example 9. Comparison of experimental and theoretical values ​​of the calibration curve

[0154] The usefulness was confirmed by comparing the calibration curve (experimental value) presented in Examples 5 and 7 and the derived formula y = -1.543 x ln(X) + 1.2144 and each CN Ct coefficient (experimental value) using it with the theoretical value, that is, the theoretical calibration curve (theoretical value) using CN2 assuming 100% PCR amplification efficiency and the dCt value of each copy number and the CN Ct coefficient (theoretical value). The theoretical calibration curve is as shown in Fig. 8b, and the function of the log graph at this time is y = -1.443 x ln(x) + 1. Each value was similar to the calculated and theoretical values ​​based on the experimental values: CN1 Ct coefficient was 107.87% and 100%, CN3 Ct coefficient was 63.17% and 58.5%, CN5 Ct coefficient was 34.71% and 32.2%, and CN6 Ct coefficient was 63.07% and 58.6% (Tables 5 and 7).

[0155] Table 7 below shows the formula for the change in dCt according to the change in the theoretical copy number and the Ct coefficient calculated using this formula.

[0156] CN1234568Theoretical dCt10--1---2Calculated dCt*---0.585--1.322-1.586-CN1 Ct factordCt(CN1 - CN2) / dCt(CN2 - CN4)x 100100 (%)CN3 Ct factordCt(CN2 - CN3) / dCt(CN2 - CN4)x 10058.5 (%)CN5 Ct factordCt(CN4 - CN5) / dCt(CN2 - CN4)x 10032.2 (%)CN6 Ct factordCt(CN4 - CN6) / dCt(CN2 - CN4)x 10058.6 (%)

[0157] *This value is calculated using the formula Y = -1.443 x ln(CN) +1, and the formula is obtained by plotting the dCt correlation of each copy number (CN) with CN2 using a logarithmic function assuming 100% PCR amplification efficiency.

[0158] Example 10. Correlation between dCt changes and copy number of reference genes (RNaseP) and CYP2A7

[0159] Calculation of the exact copy number of CYP2A7, a pseudogene of CYP2A6, is required. For this purpose, simultaneous evaluation of the copy number of CYP2A7 is necessary. In order to measure the exact copy number of only CYP2A7 excluding CYP2A6, a test was performed using the primer and probe set in Table 8 including the ARMS primer (CYP2A7-F1-2 sequence number) that can amplify only CYP2A7. The qPCR performed at this time did not result in amplification of the pCYP2A6 gene, and the qPCR was performed under the prerequisite dCt(FAM - Cy5) = 0 ± 0.1 presented in Example 4 using pRNaseP / CYP2A7.

[0160] primer / probesSequence (5'→ 3') (Tm)pmole / 20ulCYP2A7CYP2A7-F1-25'-TGTAGTTGGGAGGTGAAATAAGTTG (62.1℃)20AS primer 15'-ACAGTCAGGCAGICCAGCAAGG (65.82℃)20FAM-R-CYP2A6 / CNV5.15'-FAM-CCTGAGGCCAGCATGGTGGTAGTG-BHQ1 (70.6℃)6RNasePRnaseP_F65'-AGGCTTGCTGTTTGGGCTCT (60.5℃)10RnaseP-R25'-TGACTTGCTTTTAACATGACCATTC (60.9℃) ℃) 10Cy5-Rnase P-1.2.25'-Cy5-TGACGCCAAGGCTGCGG-BHQ3 (59.8 ℃)10g610-R-CNV25'-CFR610-GTGTACTGGAAGAACTTGCTTACCT-BHQ2 (62 ℃)10

[0161] The Ct test values ​​of each probe were obtained by adding the pCYP2A7 plasmid as a template so that the copy number was 1 to 6 compared to the reference gene RNaseP (Table 9). As a result of this test, the dCt [Ct(IC1)Cy5) - Ct(IC2)(CFR610)] of IC1 and IC2 showed an average of 1.04, which was a stable measurement value similar to dCt = 1.0 when the theoretical copy number was doubled, and Ct(TCN)(FAM) - Ct(IC1)(Cy5) clearly decreased in accordance with the proportional increase in pCYP2A6 reflecting the copy number. In particular, the dCt(FAM - Cy5) calibration curve according to the copy number (y = -1.524 x ln(x) + 1.146 [R² = 0.9943]) (b in Fig. 7) showed that it was similar to the test result of CYP2A6 in Example 5, y = -1.543 x ln(x) + 1.2144 (Fig. 5).

[0162] Table 9 shows the change in dCt for the reference gene (RNaseP) according to the change in CYP2A7 copy number.

[0163] CNPlasmid*Ct**dCtpRNasePpCYP2A7IC: CN2(Cy5)IC : CN4(CFR610)CYP2A7(FAM)Cy5 - CFR610FAM - Cy512 ul1 ul29.3528.2930.501.06Average1.041.1522 ul29.3928.3229.561.060.1833 ul29.3228.2028.691.11-0.6344 ul29.5228.4328.471.09-1.0555 ul29.3928.4328.110.96-1.2866 ul29.4928.5427.970.95-1.52

[0164] *Each plasmid is 1 x 10 5 Tests were conducted using copies / ul.

[0165] **Ct values ​​are the average of 3 replicates and were measured at a threshold of 400.

[0166]

[0167] Example 11: Comparison of copy number calculations using experimental and theoretical CN Ct coefficient values.

[0168] The CN Ct coefficients of each experimental value and theoretical value were used to calculate the number of copies and compare them to examine their usefulness. The experimental CN Ct coefficients were obtained using the tests of Examples 5 and 10. From the test of Example 5 using CYP2A6, the CYP2A6 CN Ct coefficient (FAM) (②) was obtained using the FAM signal value, and the CYP2A6 [T] Allele CN Ct coefficient (JOE) (③) was obtained using the JOE signal value. In addition, from the test of Example 10 using pCYP2A7, the CYP2A7 CN Ct coefficient (FAM) (④) (Table 10) was obtained using the same method. The CN Ct values ​​were calculated by applying the CN Ct coefficients of ②, ③, and ④ obtained in this way and the theoretical CN Ct coefficient (①) (Table 11).

[0169] Using the Ct values ​​of each CN calculated in this way, the logarithmic function (y = A x ln(x) + B) was derived, and using this, the Ct value could be accurately calculated. In this formula, y is the Ct value of the sample, A is the coefficient of the natural logarithm term, and the constant B represents the copy number, that is, the Ct value when x = 1. Based on this, the copy number of the target gene can be calculated using the Ct value of the qPCR Ct value of the target gene, that is, the FAM signal value (total copy number, TCN) or the JOE signal value ([T] allele copy number), and the copy number can be judged based on this (Table 11). The results of the calculation of the copy number and the judgment based on it were the same in all tests whether the judgment result was using the theoretical Ct coefficient or the Ct coefficient derived from the experimental value, and the judged copy number was the same as the test execution conditions and results (Table 11).

[0170] Table 10 shows the CN Ct coefficients, and Table 11 compares the copy number calculations using the CN Ct coefficients.

[0171] ① TheoreticalCN Ct factor② ExperimentalTotal CYP2A6CN Ct factor (FAM)③ ExperimentalCYP2A6 [T] AlleleCN Ct factor (JOE)④ ExperimentalTotal CYP2A7CN Ct factor (FAM)CN1 Ct factor100 %107.91 %105.60 %100.61 %CN3 Ct factor58.5 %63.17 %61.77 %58.85 %CN5 Ct factor32.2 %34.74 %34.00 %32.39 %CN6 Ct factor58.6 %63.12 %61.77 %58.85 %

[0172] Experimental Ct & Calculated CN Ct valuesCopy Number CalculationCYP2A6(-48T>G)*y= A x ln(x) + BTotal Copy Number[T] Allele Copy NumberCtfactorNo.FAMJOECN1CN2(Cy5)CN3CN4(610)CN5CN6ABCalculatedJudgementCalculatedJudgement①129.7129.5329.5028.5327.9627.5627.2526.99-1.40029.5000.86110.9791228.5928.5529.3828.4427.8927.5027.2026.95-1.35629.3801.79121.8442328.0028.0029.3528.4227.8827.4927.1926.95-1.34229.3502.73532.7353②129.71-29.5828.5327.9227.5627.2226.95-1.45829.5580.9011--228.59-29.4528.4427.8527.5027.1726.91-1.41329.4361.8202--328.00-29.4228.4227.8327.4927.1726.90-1.39829.4052.7333--③1-29.5329.5528.5327.9327.5627.2326.96-1.44129.541--1.00812-28.5529.4328.4427.8627.5027.1826.92-1.39729.420--1.86423-28.0029.4028.4227.8527.4927.1726.92-1.38229.389--2.7333CYP2A7**y= A x ln(x) + BTotal Copy Number-CtfactorNo.FAMJOECN1CN2(Cy5)CN3CN4(610)CN5CN6ABCalculatedJudgement①130.50-30.4129.3528.7328.2927.9527.67-1.53030.4100.9431229.56-30.4629.3928.7628.3227.9827.69-1.54430.4601.7912328.69-30.4429.3228.6628.2027.8427.54-1.61630.4402.9533④130.50-30.4229.3528.7328.2927.9527.67-1.53430.4150.9461229.56-30.4729.3928.7628.3227.9727.69-1.54930.4651.7942328.69-30.4529.3228.6628.2027.8427.54-1.62130.4452.9533.

[0173] *The results of tests 1, 2, and 3 of Example 5 (Table 3) were used.

[0174] **Value tested using pCYP2A7.

[0175]

[0176] Example 12: Method for presenting results for SNP and CNV determination and its usefulness

[0177] The present inventors sought to propose a judgment system for determining CNV and genotype (SNP) more efficiently and visually usefully. Figure 9 is an exemplary diagram for this purpose. As shown in the figure of Figure 9, the present invention is a system that obtains each Ct value obtained through a qPCR test and the CN Ct value calculated from the value, and graphically displays them for each test area in a clear manner. The judgment result values ​​in the figure were calculated according to the method of Example 11 (Table 12). The result analysis of Figure 9 and Table 12 used the test and results of Example 5 (Table 3), the test and results of Example 6 (Table 4), and the test and results of Example 10 (Table 9). This method of result analysis, presentation of analysis results, and judgment has the advantage of clearly showing information about pseudogenes, copy number information, and genotype (SNP) information of multiple test samples.

[0178] Table 12 shows the calibration curve for calculating the number of copies and the calculation table for calculating the number of copies calculated from it.

[0179] CYP2A6 (-48T>G)y= A x ln(x) + BTotal Copy Number[T] Allele Copy NumberTest No.FAMJOECN1CN2(Cy5)CN3CN4(610)CN5CN6ABcalculatedJudgementcalculatedJudgement129.7129.5329.5028.5327.9627.5627.2526.99-1.40029.5000.86110.9791228.5928.5529.3828.4427.8927.5027.2026.95-1.35629.3801.79121.8442328.0028.0029.3528.4227.8827.4927.1926.95-1.34229.3502.73532.7353427.5727.5229.4228.4427.8727.4627.1426.89-1.41429.4203.70043.8334527.4927.3629.8328.7928.1827.7527.4227.14-1.50129.8304.75655.1865627.3427.2230.0228.9428.3127.8627.5127.23-1.55830.0205.58366.0306728.6229.7629.3828.4727.9427.5627.2727.03-1.31329.3801.78420.7491827.7128.1929.5328.5627.9927.5927.2827.02-1.40029.5303.67142.6053927.3427.8029.4928.5728.0327.6527.3527.11-1.32829.4905.05153.57241027.6428.7529.6228.6428.0727.6627.3427.09-1.41429.6204.05641.85021127.4828.2729.7728.7628.1727.7527.4227.16-1.45729.7704.81352.79931227.1527.7129.7328.6928.0827.6527.3227.04-1.50129.7305.58063.84241327.9930.1129.9628.9028.2827.8427.5027.22-1.53029.9603.62640.90711427.3228.3930.0528.9928.3727.9327.5927.31-1.53030.0505.95962.9613CYP2A7y= A x ln(x) + BTotal Copy Number-Test No.FAMJOECN1CN2(Cy5)CN3CN4(610)CN5CN6ABcalculatedJudgement1530.50-30.4129.3528.7328.2927.9527.67-1 .53030.4100.94311629.56-30.4629.3928.7628.3227.9827.69-1.54430.4601.79121728.69-30.4429.3228.6628.2 027.8427.54-1.61630.4402.95331828.47-30.6129.5228.8828.4328.0827.79-1.57330.6103.89941928.11-30.3529.3928.8328.4328.1227.87-1.38530.3505.03852027.97-30.4429.4928.9328.5428.2327.98-1.37130.4406.0616.

[0180]

[0181] Example 13: SNP and CNV analysis using human genomic DNA

[0182] In the above examples, it was confirmed that SNP typing and copy number measurement of CYP2A6 were accurately performed using a standard plasmid. The test method constructed in this way was tested using human genomic DNA as a clinical sample. Human genomic DNA was quantified and approximately 5 ng was added to each test group for testing. At this time, primers / probes and qPCR were performed in the same manner as in Examples 5 and 10. 100 human genomic DNAs were used, and the test results for 10 samples are presented as representative examples (Figs. 10, 11, 12, and Table 13). As a result of this test, the diploid genotype was clearly identified depending on the clinical sample, and the analysis results, as shown in Fig. 11b and Table 13c, showed that the results of each of the three replicate tests were similar to each other and determined to be the same CN and diploid genotype. The analysis results using the average of the results of the repeated tests were also determined to be the same diploid genotype as the results of the individual tests (c in Fig. 11 and Table 14). The diploid genotype of the target gene CYP2A6 could be clearly determined by determining the copy number of the exact target gene CYP2A6 and the allele type (SNP, etc.) and determining the presence or absence of the pseudogene (CYP2A7) and its copy number. To determine this, the CN coefficient is determined using the same method as in Examples 7, 9, and 10, and the CN Ct value is calculated and secured using this, and the calibration curve is obtained by obtaining the function of the natural logarithm function y = A x ln (x) + B, and each can be calculated and obtained as in the example of Sample 1 (a and b in Fig. 13). Let us explain this using the first test result of sample 1 (Table 13) as an example. The dCt (1.12 = 28.57 - 27.45) of CN2 (IC1, Cy5) and CN4 (IC2, CFR610) was obtained, and using this, the theoretical Ct coefficients of Example 9 (Table 7) and Example 11 (Table 10) were applied to obtain the respective CN Ct values ​​(CN1, 29.69; CN2, 28.57; CN3, 27.91; CN4, 27.45; CN5, 27.09; CN6 26.79) were obtained. Using the Ct values ​​of each CN, a calibration curve was created as shown in Fig. 13, and its regression analysis equation, y = -1.616 x ln(x) + 29.69, was obtained. From this equation, Ct for y. (FAM) or Ct (JOE) By substituting x, we can easily obtain the copy number. That is, the total number of CYP2A6 genes in sample 1 can be calculated as follows.

[0183] 28.62 (Ct [FAM] ) = -1.616 x ln (copy number of #1) + 29.69(Ct [CN1] ) From the formula, ln (CN of #1) = (29.69 - 28. 62) / 1.616 = 0.662, CN of #1 (CYP2A6) = EXP(0.662), i.e., 1.959 copy number (calculated copy number).

[0184] Therefore, the total copy number of the CYP2A6 gene in sample number 1 can be determined to be 2. This was determined to be the same as the results of the repeated test (Table 13) and the average test (Table 14).

[0185] Applying the same method to Ct (JOE) [T] Allele copy number (Tables 13 and 14) and Ct of CYP2A7 test using (FAM) Using this, the total copy number of CYP2A7 (Table 14) could also be precisely measured. Using the total copy number of CYP2A6 and the copy number of [T] allele obtained in this way, the diploid genotype of CYP2A6 can be accurately determined, and the number of genes of CYP2A7, a pseudogene of CYP2A6, can also be clearly determined in the sample. Taking sample 1 as an example, the total CYP2A6 copy number is 2, the [T] allele copy number is also 2, which is the diploid genotype Tx2, and the total copy number of CYP2A7 can be determined to be 2.

[0186] Table 13 shows the calibration curve for calculating the copy number and the copy number calculation table calculated from it (Human Genome Test - Repeat Test).

[0187] CYP2A6 -48T>Gy= A x ln(x) + BTotal Copy Number[T] Allele Copy NumberSample No.FAMJOECN1CN2(Cy5)CN3CN4(610)CN5CN6ABCalculatedJudgementCalculatedJudgement128.6228.5229.6928.5727.9127.4527.0926.79-1.61629.6901.93922.063228.4928.3629.7328.5827.9127.4327.0626.76-1.65929.7302.11122.283228.4328.2929.4328.3327.6927.2326.8826.59-1.58729.4301.87822.0512230.1830.0130.1429.1128.5128.0827.7527.48-1.48630.1400.97411.092130.0129.7630.1229.0728.4628.0227.6827.40-1.51530.1201.07511.268130.1629.929.9728.9728.3927.9727.6527.38-1.44329.9700.87711.0501329.4529.2830.4529.3528.7128.2527.9027.61-1.58730.4501.87822.090229.3929.2930.4929.3928.7528.2927.9427.65-1.58730.4902.00022.130229.629.4530.5329.4028.7428.2727.9127.61-1.63130.5301.76921.9402429.4429.3130.3129.2028.5528.0927.7327.44-1.60230.3101.72221.867229.2929.1530.1829.1228.5028.0627.7227.44-1.53030.1801.79021.961229.629.530.4429.3228.6628.2027.8427.54-1.61630.4401.68221.7892528.8828.7230.4229.2828.6128.1427.7727.47-1.64530.4202.55032.811328.6428.4730.4929.3528.6828.2127.8427.54-1.64530.4903.07933.415328.5528.4830.1029.0628.4528.0227.6927.41-1.50130.1002.80932.9433629.1629.0830.2529.1928.5728.1327.7927.51-1.53030.2502.04022.149228.9328.8529.9328.9628.3927.9927.6827.42-1.40029.9302.04322.163229.0929.1230.0129.0228.4428.0327.7127.45-1.42930.0101.90421.8652727.627.5329.1428.1127.5127.0826.7526.48-1.48629.1402.81932.955327.5927.629.1128.1027.5127.0926.7626.50-1.45729.1102.83832.818327.7127.6829.1728.1227.5127.0726.7326.45-1.51529.1702.62132.6743829.8329.6429.9828.9428.3327.9027.5727.29-1.50129.9801.10511.254129.9529.6930.0228.9728.3627.9227.5827.30-1.51530.0201.04711.243130.1930.130.1229.0428.4127.9627.6127.33-1.55830.1200.95611.0131929.0329.7629.9028.7728.1127.6427.2826.98-1.63129.9001.70521.090128.7129.6329.7828.6728.0227.5627.2026.91-1.60229.7801.95121.098128.6129.5729.5728.4927.8627.4127.0626.78-1.55829.5701.85221.00011028.2829.1729.3228.2527.6227.1826.8426.55-1.54429.3201.96121.102128.1829.229.0128.0127.4327.0126.6926.42-1.44329.0101.77820.877128.129.1328.9627.9827.4127.0026.6826.43-1.41428.9601.83720.8871.

[0188]

[0189] Table 14 shows the calibration curve for calculating the copy number and the copy number calculation table calculated from it (Human Genome Test - Average).

[0190] CYP2A6 (-48T>G)y= A x ln(x) + BTotal Copy Number[T] Allele Copy NumberSampleNo.FAMJOECN1CN2(Cy5)CN3CN4(610)CN5CN6ABCalculatedJudgementCalculatedJudgement128.5128.3929.6128.4927.8327.3727.0126.71-1.61629.6101.97522.1282230.1229.8930.0829.0528.4528.0227.6927.42-1.48630.0800.97411.1371329.4829.3430.4929.3828.7328.2727.9127.62-1.60230.4901.87922.0502429.4429.3230.3029.2128.5728.1227.7727.48-1.57330.3001.72821.8652528.6928.5630.3429.2328.5828.1227.7627.47-1.60230.3402.80233.0393629.0629.0230.0729.0628.4728.0527.7227.46-1.45730.0702.00022.0562727.6327.629.1428.1127.5127.0826.7526.48-1.48629.1402.76232.8193829.9929.8130.0328.9828.3727.9327.5927.31-1.51530.0301.02711.1561928.7829.6529.7428.6428.0027.5427.1926.90-1.58729.7401.83121.05811028.1929.1729.1028.0827.4827.0626.7326.46-1.47229.1001.85620.9541CYP2A7y= A x ln(x) + BTotal Copy Number-SampleNo.FAMJOECN1CN2(Cy5)CN3CN4(610)CN5CN6ABECalculatedJudgement128.89-29.8728.8928.3227.9127.5927.34-1.41429.8702.0142229.74-30.7429.7429.1628.7428.4228.15-1.44330.7402.0992330.04-30.9930.0029.4229.0128.6928.43-1.42930.9901.9452429.78-30.7829.7829.2028.7828.4628.19-1.44330.7801.9592529.76-30.7629.7629.1828.7628.4428.17-1.44330.7601.0351629.94-30.9229.9429.3728.9628.6428.39-1.41430. 9202.1012728.99-30.0128.9928.3927.9727.6427.37-1.47230.0101.1231829.59-30.6429.5928.9828.5428.2027.92-1.51530.6402.0672929.25-30.2629.2528.6628.2427.9127.65-1.45730.2602.02821028.98-29.9828.9828.4027.9827.6627.39-1.44329.9802.0282.

[0191]

[0192] Example 14: Analysis results of CYP2A6 (-48T>G) and CYP2A7 in 100 Korean samples

[0193] In order to confirm the usefulness of the genotype determination and copy number determination method of the present invention, 100 Korean clinical genomic DNA samples provided by the Daejeon Biomedical Regulatory Free Zone Human Origin Bank were tested as in Example 13, and the results are shown in Table 15. As a result of the test, there were 32 copy number mutations in CYP2A6 in total (32%), of which 19 individuals (19%) had a decrease in copy number and 13 individuals (13%) had an increase in copy number. Based on this, the total number of alleles calculated was 194 (111 + 8 + 40 + 35 = 194), and the ratio of allele [T] was 0.778 = 151 / 194, and the ratio of allele [G] was 0.221 = 43 / 194. In particular, since CYP2A7 is all T allele (T = 186), when this is reflected, the T frequency is calculated as 0.887 (151 + 186 / 380), and the allele [G] frequency is 0.113 (43 / 380), which appears to have a very similar frequency to the NIH dbSNP results for Asians (T = 0.863, G = 0.137) and East Asians (T = 0.864, G = 0.136) (https: / www.ncbi.nlm.nih.gov / snp / rs28399433). It is judged that the existing report did not exclude the gene copy number of CYP2A7, so it shows a slight difference from the results of this example (Table 16).

[0194] In addition, the results of CYP2A7 analysis of genomic DNA confirmed that the copy number of this pseudogene was highly related to CYP2A6 (-48T>G) diplotypes (Table 17). In samples with decreased or increased copy number of the entire CYP2A6, there were 16 cases with a mutation in the copy number of the entire CYP2A6, and 7 out of 8 cases (88%) of the Tx3 Type (excluding #93) and all 3 cases (100%) of the Tx2 / Gx1 Type were determined to have a mutation with 1 copy number of CYP2A7 (Table 17). This is an analysis result that clearly reflects the crossover between CYP2A6 and CYP2A7 with similar sequences, which proves that the usefulness of the analysis method of the present invention is very high.

[0195] Table 15 shows the copy number frequency of the CYP2A6 -48T>G variant (Koreans).

[0196] T homoDiplotype(Total Copy Number)Tx1 homo(1)Tx2 homo(2)Tx3 homo(3)Tx4 homo(4)58(T:111)Korean,n153481G homoDiplotype(Total Copy Number)Gx1 homo(1)Gx2 homo(2)-6(G:8)Korean,n42HeteroDiplotype(Total Copy Number)Tx1 / Gx1(2)Tx2 / Gx1(3)Tx3 / Gx1(4)ND36(T:40, G:35)Korean,n31311Total100(T:151, G:43)

[0197] Table 16 compares the copy number frequency of each CYP2A6(-48T>G) variant (Koreans).

[0198] PopulationSample SizeRef Allele frequencyAlt Allele frequencyData fromKorean100T = 0.778G = 0.221This WorkAsian204T = 0.863G = 0.137NIH dbSNP*East Asian154T = 0.864G = 0.136Total40856T = 0.94716G = 0.05284

[0199] *https: / / www.ncbi.nlm.nih.gov / snp / rs28399433; release version 20201027095038

[0200] Table 17 shows the correlation between CYP2A7 copy number variation and CYP2A6 copy number.

[0201] Sample No.571627333436466667718796102108113CYP2A6 -48T>G DiplotypeTx3Tx3Tx3Tx2Tx3Tx2Gx1Tx2Tx3Tx2Gx1Tx1Tx3Tx2Tx3Tx2Gx1Tx2Tx1CYP2A7 Copy Number1111113111111111-. Population: Korean-. Sample Size: 100-. CYP2A7 Variants: 16

[0202]

[0203] The method and kit for simultaneous determination of the copy number and genetic variant of a gene of the present invention can be widely used for genetic testing of agricultural, aquatic, and livestock products, as well as for diagnosis in the medical field of cancer and genetic diseases, as it is easy to determine the copy number of a target gene while simultaneously determining the variant or allelic genotype of a gene from a small amount of biological sample by applying real-time polymerase chain reaction.

[0204]

[0205] Electronic file attached

Claims

1. a) A step of simultaneously adding a) a target gene-specific primer, a target gene-specific probe, a probe capable of distinguishing alleles of the target gene, and b) a reference gene-specific primer and probe capable of amplifying and identifying a reference gene into one reaction chamber, and synchronizing the amplification of the gene to obtain an amplification experimental value (Ct value) by qPCR; b) a step of determining the copy number Ct factor (CN Ct factor) from the amplification experimental value and / or theoretical amplification value; d) a step of determining the Ct values of target gene copy numbers 1, 3, 5 and / or 6 from the amplification experimental values of reference gene copy numbers 2 and 4 using the determined copy number Ct coefficients; and A method for simultaneously determining the copy number and genotype of a gene, comprising: a) comparing the amplification experimental value of the target gene with the test copy number of the reference gene and the Ct values of the copy numbers 1, 3, 5 and / or 6 of the target gene determined in step c) above to determine the total copy number of the target gene and the genotype of the allele; 2. In claim 1, A method for simultaneous determination of the copy number and genotype of a gene, wherein primers and probes capable of specifically amplifying the pseudogene of the target gene are added to a) in step a).

3. In claim 1 or claim 2, A method for simultaneous determination of the copy number and genotype of a gene, wherein at least one of the target gene-specific primer and the pseudogene-specific primer is an AS primer (Allele specific primer) or an ARMS primer (Amplification Refractory Mutation System primer).

4. In any one of claims 1 to 3, In the qPCR step of the above (a) To enhance the discrimination of specific primer amplification, a discrimination-enhancing oligonucleotide that is not complementary to the primer and template, can reversibly bind to a DNA polymerase for qPCR, and forms a double strand within a single strand or partially or completely with another strand; and A method for simultaneous determination of the copy number and genotype of a gene, which additionally introduces a non-specific signal suppression probe having a structure that is complementary to the target base sequence of a non-mutated target gene in order to increase the distinguishability of a specific probe, binds preferentially to the target base sequence of a non-mutated gene over the target base sequence of a mutated target gene, and has a 3' end that is not extended by a nucleic acid polymerase.

5. In claim 1, A method for simultaneously determining the copy number and genotype of a gene, wherein qPCR is performed using fluorescent probes of different wavelengths for the target gene-specific probe and the allele-specific probe to confirm the copy number and allele copy number of the target gene in the qPCR step of the above a).

6. In claim 1, A method for simultaneous determination of the copy number and genotype of a gene, which performs qPCR using fluorescent probes of different wavelengths to obtain amplification experimental values of copy number 2 and copy number 4 of the reference gene in the qPCR step of the above a).

7. In claim 1, A method for determining the copy number Ct coefficient from the amplification experimental value of step b) above is a method for simultaneously determining the copy number and genotype of a gene, wherein a calibration curve is derived using the experimental Ct values of a standard gene to determine the copy number Ct coefficients of copy numbers 1, 3, 5 and / or 6 of the target gene.

8. In claim 1, A method for simultaneously determining the copy number and genotype of a gene, which is performed by determining the copy number Ct coefficient from the theoretical amplification value of the above step b) using the mathematical formula y = -1.443 x ln(x) + 1 (wherein y is the Ct value of the target gene and x is the copy number of the target gene).

9. In claim 1, A method for determining the Ct value of the copy number of a target gene by using the copy number Ct factor of the step D) is a method for simultaneously determining the copy number and genotype of a gene, wherein the difference (dCt(CN2-CN4)) between the Ct of the copy number 2 of the reference gene and the Ct of the copy number 4 obtained from the amplification experiment values of the target gene and the reference gene of which the copy numbers are known is calculated, and the Ct of the copy numbers 1, 3, 5 and / or 6 of the target gene is determined by applying the copy number Ct factor obtained in claim 6 or claim 7.

10. In claim 9, A method for comparing an amplification experimental value of a target gene to a Ct value of the copy number of the target gene is provided by applying the Ct values of copy numbers 1, 3, 5, and / or 6 of the target gene and the CN2 Ct value and the CN4 Ct value of the target gene to obtain a standard quantitative curve equation, y = A x ln(x) + B (wherein y is the Ct value of the target gene and x is the copy number of the target gene), and using this equation to obtain the copy numbers of the target gene and alleles. A method for simultaneous determination of the copy number and genotype of a gene.

11. In claim 1, A method for simultaneous determination of the copy number and genotype of a gene by performing qPCR by positioning the target gene and reference gene on a single plasmid, wherein the amplification of the target gene and reference gene is synchronized.

12. In claim 1, A method for simultaneously determining the copy number and genotype of a gene by performing qPCR by synchronizing the amplification of a target gene and a reference gene, wherein the qPCR conditions are adjusted so that the Ct value deviations dCt(TCN - IC1) and dCt(AS - IC1) satisfy ±0.1 at the same two gene copy numbers in the qPCR reactions of the reference gene and the target gene, respectively.

13. In claim 1, A method for simultaneously determining the copy number and genotype of a gene by performing qPCR so that the copy number of the reference gene is twice the copy number of the target gene.

14. In claim 13, A method for simultaneous determination of gene copy number and genotype by adjusting qPCR conditions so that dCt(CN2;IC1- CN4;IC2) satisfies 1.0 ± 0.1 when performing qPCR so that the copy number of the reference gene is twice the copy number of the target gene.

15. In claim 6 or claim 14, A method for simultaneous determination of copy number and genotype of a gene, which uses two probes with different sequences of the same fluorescence simultaneously to obtain an amplification experimental value of copy number 4 in the qPCR step.

16. In claim 1, A method for simultaneously determining the copy number and genotype of a gene, wherein a Ct value that is 1.0 less than the Ct value of the copy number of the target gene 2 is arbitrarily substituted to obtain an amplification experimental value of the copy number of the target gene 4, and the Ct value is calculated as the Ct value of the copy number 4.

17. A method for creating a simultaneous gene copy number and genotype determination table that displays the qPCR Ct result and copy number Ct value on one axis and the target gene sample to be analyzed on the other axis so that the genotype and copy number of one or more analysis samples can be visually confirmed.

18. In claim 17, A method for creating a simultaneous determination table of gene copy number and genotype, wherein the determination table includes Ct value, Ct value by copy number, copy number of diploid genotype, and allele type.

19. In claim 18, A method for preparing a simultaneous gene copy number and genotype determination table, wherein the above determination table further includes the copy number of a pseudogene.

20. In any one of claims 17 to 19, A method for creating a simultaneous determination table of gene copy number and genotype, wherein the determination table reflects the result value and / or calculation value obtained from the method of any one of claims 1 to 16.

21. A simultaneous genotype and copy number determination table that allows visual confirmation of the genotype and copy number of one or more analysis samples by displaying the qPCR Ct result and copy number Ct value on one axis and the target gene sample to be analyzed on the other axis.

22. In claim 21, The above judgment table is a simultaneous judgment table of gene copy number and genotype, including Ct value, Ct value by copy number, copy number of diplotype and allele type.

23. In claim 22, The above table is a table for simultaneous determination of gene copy number and genotype, which additionally includes the copy number of the pseudogene. 24.a) Target gene-specific primers and target gene-specific probes; a) A specific probe capable of detecting and distinguishing the alleles of the target gene; c) Reference gene-specific primers and reference gene-specific probes capable of amplifying and identifying the reference gene; and a) A qPCR kit for simultaneous determination of copy number and genotype of a target gene, comprising DNA polymerase; 25. In claim 24, A qPCR kit for simultaneous determination of the copy number and genotype of a target gene, further comprising primers and probes capable of specifically amplifying a pseudogene of the target gene.

26. In claim 24 or claim 25, b) To enhance the discrimination of specific primer amplification, a discrimination-enhancing oligonucleotide that is not complementary to the primer and the target gene template, can reversibly bind to a DNA polymerase for qPCR, and forms a double strand within a single strand or partially or completely with another strand; and A qPCR kit for simultaneous determination of copy number and genotype of a target gene, further comprising a non-specific signal suppression probe having a structure in which the probe is complementary to the target base sequence of a non-mutant target gene to increase the distinguishability of the target gene-specific probe, preferentially binds to the target base sequence of the non-mutant target gene over the target base sequence of the mutated target gene, and the 3' end is not extended by a nucleic acid polymerase.

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