KR102170423B1 - Age estimation method using body fluids - Google Patents

Abstract

The present invention relates to an age tracking method using body fluids. More specifically, the present invention relates to a method for tracking the age of a test subject by measuring the degree of DNA methylation for a specific CpG site of a target gene in a body fluid derived from a test subject, most preferably a saliva sample. will be.
In the field of forensic science and forensic medicine, tracking the age of a victim or perpetrator from evidence can be a great clue to case resolution. Until now, the most effective biomarker for age estimation is known as a DNA methylation marker, but the conventional age estimation method has a problem that it takes a lot of time and cost to track age because a number of genes must be analyzed.
The present invention relates to an age tracking method using a bodily fluid derived from a test object, most preferably a saliva sample. The age of the test subject can be accurately tracked by methylation of the CpG positions of four target genes, and the analysis method is simple. It is expected to be widely used in the field of forensic science and forensic medicine, as the cost and cost are reduced.

Description

Age estimation method using body fluids

The present invention relates to an age tracking method using body fluids. More specifically, the present invention relates to a method for tracking the age of a test subject by measuring the degree of DNA methylation for a specific CpG site of a target gene in a body fluid derived from a test subject, most preferably a saliva sample. will be.

In the field of forensic science and forensic medicine, tracking the age of a victim or perpetrator from evidence can be a great clue to case resolution. Even if the DNA genotyping result of the victim or perpetrator is obtained from evidence at the scene of the incident, the person cannot be identified unless DNA information consistent with the obtained analysis result exists in the genetic database, so the victim or perpetrator can be directly specified. It would be more important to track the age that is there. Until now, the most effective biomarker for age estimation is known as a DNA methylation marker, and it is highly effective because it can be applied regardless of ethnicity or group diversity. In this regard, Hannum G et al. known a method for predicting age by measuring the methylation of genes from blood samples (Mol Cell. 2013 Jan 24;49(2):359-367), Horvath S et al. Although the prediction method was known, there was a limitation in that methylation in more than 70 genes had to be analyzed (Genome Biol. 2013;14(10):R115). Recently, a method of tracking the age by measuring the degree of DNA methylation in saliva samples (Korean Patent No. 10-1873303) has been known, but the invention requires two PCR amplification processes, and the CpG of 7 target genes Since the location is analyzed, there is a problem that it takes a lot of time and cost to track age.

Therefore, the present invention was devised to solve the problems of the prior art as described above, by measuring the degree of DNA methylation for a specific CpG site of a target gene in a bodily fluid derived from a target, most preferably a saliva sample. It relates to a method of tracking the age of the test subject. The present invention is expected to be widely used in forensic science and forensic medicine since it is possible to accurately track the age of a test target by methylation of the CpG positions of four target genes, and because the analysis method is simple, time and cost are saved.

The present invention was conceived to solve the problems of the prior art as described above, and relates to an age tracking method using body fluids.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems that are not mentioned may be clearly understood by those of ordinary skill in the art from the following description.

Hereinafter, various embodiments described herein are described with reference to the drawings. In the following description, for a thorough understanding of the invention, various specific details, such as specific forms, compositions and processes, etc. are set forth. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well-known processes and manufacturing techniques have not been described in specific details in order not to unnecessarily obscure the present invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the invention. Thus, the context of “in one embodiment” or “an embodiment” expressed in various places throughout this specification does not necessarily represent the same embodiment of the invention. Additionally, particular features, shapes, compositions, or properties may be combined in one or more embodiments in any suitable manner.

Unless otherwise defined in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

In one embodiment of the present invention, the term "biological sample" means any sample capable of confirming the gene information of the test object, preferably blood, plasma, serum, saliva, etc., but if it is a type capable of confirming the CpG marker, Not limited.

In one embodiment of the present invention, the term "body fluids" is a generic term for liquids that move between blood vessels or tissues in the body of an animal. It includes blood, plasma, lymph, aqueous humor, cerebrospinal fluid, pleural fluid, tear fluid (tears), urine, saliva (saliva), gallbladder, amniotic fluid, semen, tissue fluid, and the like. In the present invention, the bodily fluid preferably means saliva, but is not limited thereto.

In one embodiment of the present invention, "saliva" is a secretion fluid secreted from the salivary glands into the mouth. Saliva contains more than 99% of water (moisture), in addition to various electrolytes (sodium, potassium, calcium, chloride, magnesium, bicarbonate, phosphate) and proteins, various enzymes that play a very important role in oral health. Several types of immunoglobulins, antimicrobial factors, mucosal glycoproteins, trace amounts of albumin, several types of polypeptides and oligopeptides, etc. This includes. The amount of saliva produced by healthy adults is about 1 to 1.5 L per day, and the necessary components are extracted from the blood flowing in the blood vessels from the salivary glands and converted into saliva, and the conversion time is almost real time.

Since oral epithelial cells are present in the saliva discharged to the outside, and genotyping can be performed from the oral epithelial cells present in the saliva, the saliva sample is importantly used as a genotyping sample.

In one embodiment of the present invention, “CpG site” refers to a site in which two bases of cytosine and guanine exist side by side in a DNA complementary strand, and a methyl group (CH ₃ ) may be added.

In one embodiment of the present invention, “DNA methylation” is a very important biochemical process for the development of higher organisms, and is a mechanism for regulating gene expression that inhibits transcription. It involves adding one methyl group to position 5 of the pyrimidine ring of cytosine or to the number 6 nitrogen of the purine ring of adenine (cytosine and adenine belong to the four bases of DNA). DNA methylation is critical for general organismal evolution and cellular mutation, generally disappears during zygote production, and regenerates through subsequent cell differentiation during expression. Additionally, DNA methylation can reliably reduce the gene phenotype in cells. In particular, DNA methylation at position 5 of cytosine has a special effect of reducing genotyping, and it can be used to infer the passage of time the cell has passed. In the case of the human genome, methylated cytosine is known to exist about 3 to 4% of all cytosine, and the degree or pattern of methylation of CpG varies depending on the species of mammal and is known to appear specifically depending on the tissue.

In one embodiment of the present invention, the term "kit" refers to a device for examination that provides information necessary for predicting age by identifying a CpG marker that has a relationship with predicting the age of a test subject, and It is possible to confirm the DNA methylation ratio of the CpG marker, preferably target genes of the present invention SST (somatostatin), KLF14 (Kruppel like factor 14), TSSK6 (testis specific serine kinase 6), and SLC12A5 (solute carrier family 12 member) 5) Including a set of probes or primers having a sequence complementary to the CpG marker (cg00481951, cg14361627, cg08928145, cg07547549) within, but is not limited thereto.

In one embodiment of the present invention, the term "age prediction device" refers to the age of the subject from the measurement result of the methylation level for the CpG marker (cg00481951, cg14361627, cg08928145, cg07547549) of a gene in a bodily fluid sample obtained from a subject. As a predicting device, there is no limitation as long as it is a device capable of predicting the age of a subject by measuring the methylation level of a CpG marker in a sample derived from a subject.

In the present invention, "polymerase chain reaction (PCR)" is a simple and convenient method that can exponentially amplify the DNA sequence of a specific site with only a very small amount of DNA. It is specific to the DNA to be amplified. A specific DNA is amplified by repeatedly performing the processes of denaturation, annealing, and extension using Taq DNA polymerase, which is bonded and stable at high temperatures. The detection of a target gene using PCR is amplified and confirmed using a primer that recognizes the gene.

In the present invention, "discrimination" includes quantitative and/or qualitative analysis, and includes detection of presence and absence and concentration measurement, and such methods are known in the art, and those skilled in the art will use appropriate methods for the practice of the present application. You will be able to choose. In the present invention, "discrimination" refers to determining the predicted age of the test subject by measuring the degree of methylation of the CpG marker of the gene target in the body fluid sample derived from the test subject.

In order to solve the above problem, the present inventors selected the type of specific CpG to be analyzed in the saliva sample derived from the test object, and measured the ratio of DNA methylation and non-methylation of the selected marker to minimize the error range. The prediction method was built.

An object of the present invention is a CpG marker in the SST (somatostatin), KLF14 (Kruppel like factor 14), TSSK6 (testis specific serine kinase 6), and SLC12A5 (solute carrier family 12 member 5) genes for a saliva sample derived from a test object ( cg00481951, cg14361627, cg08928145, cg07547549) by measuring the degree of DNA methylation to predict the age of a test subject or to provide a kit for predicting the age of the test subject.

The CpG marker cg00481951 is defined as the CpG position chr3:187387650 in DNA, cg14361627 as chr7:130419116, cg08928145 as chr19:19625364, and cg07547549 as chr20:44658225.

In one embodiment of the present invention, as a method for predicting age comprising a methylation assay step for a CpG marker of a gene with a sample isolated from an unknown individual, the gene is SST (somatostatin), KLF14 (Kruppel like factor) 14), TSSK6 (testis specific serine kinase 6), and SLC12A5 (solute carrier family 12 member 5) provides an age prediction method.

The CpG markers are most preferably cg00481951 for SST, cg14361627 for KLF14, cg08928145 for TSSK6, and cg07547549 for SLC12A5, but are not limited thereto.

In addition, the methylation analysis step provides an age prediction method in which the K value, which is the degree of methylation of each CpG marker, is calculated by the formula of “{methylated copy/(methylated copy + unmethylated copy)}×100”.

In addition, after the methylation analysis step, the K value, which is the degree of methylation of each CpG marker, is "-N ₁ + N ₂ × (K value of cg00481951) + N ₃ × (K value of cg14361627) + N ₄ × (cg08928145). K value) + N ₅ × (K value of cg07547549)” is an age calculation step of calculating an age, further comprising an age prediction method, wherein N ₁ is -16.2 to 3.8, and N ₂ is -3.3 To 6.7, N ₃ is -2.6 to 7.4, N ₄ is -2.7 to 3.3, and N ₅ is -2.6 to 3.4.

The sample is any one selected from the group consisting of body fluid, body tissue, and hair root, and the body fluid is blood, plasma, lymph fluid, aqueous humor, cerebrospinal fluid, pleural fluid, tear fluid (tears), urine, saliva (saliva), bile fluid, amniotic fluid, semen , And any one selected from the group consisting of tissue fluid, saliva (saliva) is most preferred, but is not limited thereto.

In another embodiment of the present invention, there is provided a kit for age prediction comprising a primer set for methylation analysis of CpG markers in a gene group including SST, KLF14, TSSK6, and SLC12A5, wherein the CpG marker is cg00481951 for SST, Provides a kit for age prediction of cg14361627 for KLF14, cg08928145 for TSSK6, and cg07547549 for SLC12A5, the primer set being SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO It provides a kit for age prediction comprising any one or more selected from the group consisting of 10, SEQ ID NO: 13, and SEQ ID NO: 14.

The kit may further include a probe set for methylation analysis of CpG markers in the gene group including SST, KLF14, TSSK6, and SLC12A5, and the probe set is SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, and sequence Number 8, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 15, and may include any one or more selected from the group consisting of SEQ ID NO: 16.

In another embodiment of the present invention, an analysis unit for measuring the methylation level of a CpG marker of a gene in a sample isolated from an unknown individual; And a calculation unit for calculating the age of the individual from the methylation level measurement result, wherein the genes are SST, KLF14, TSSK6, and SLC12A5.

The CpG markers are most preferably cg00481951 for SST, cg14361627 for KLF14, cg08928145 for TSSK6, and cg07547549 for SLC12A5, but are not limited thereto.

In addition, the methylation level measurement provides an age prediction device that calculates the K value, which is the degree of methylation of each CpG marker, by the formula of “{methylated copy/(methylated copy + unmethylated copy)}×100”, and the In the calculation of the age of the individual, the K value, which is the degree of methylation of each CpG marker, is "-N ₁ + N ₂ × (K value of cg00481951) + N ₃ × (K value of cg14361627) + N ₄ × (K value of cg08928145). + N ₅ × (K value of cg07547549)” provides an age prediction device that is calculated by the formula, wherein N ₁ is -16.2 to 3.8, N ₂ is -3.3 to 6.7, and N ₃ is -2.6 To 7.4, and N ₄ is -2.7 to 3.3, and N ₅ is -2.6 to 3.4.

Hereinafter, the present invention is described in detail step by step.

In the field of forensic science and forensic medicine, tracking the age of a victim or perpetrator from evidence can be a great clue to case resolution. Until now, the most effective biomarker for age estimation is known as a DNA methylation marker, but the conventional age estimation method has a problem that it takes a lot of time and cost to track age because a number of genes must be analyzed.

The present invention relates to an age tracking method using a bodily fluid derived from a test object, most preferably a saliva sample. The age of the test subject can be accurately tracked by methylation of the CpG positions of four target genes, and the analysis method is simple. It is expected to be widely used in the field of forensic science and forensic medicine, as the cost and cost are reduced.

1 is a result of quantification of methylation at a CpG position in a 52-year-old saliva sample according to an embodiment of the present invention. In FIG. 1, the blue circle indicates the detected FAM copy (methylated copy), and the green circle indicates the detected HEX copy (non-methylated copy).
2 is a result of calculating the methylation percentage with a 52-year-old saliva sample according to an embodiment of the present invention. In FIG. 2, the blue square represents the detected FAM copy (methylated copy), the green square represents the detected HEX copy (unmethylated copy), and the orange diamond represents the percentage (%) of the methylated copy.
3 is a scatter graph in which the percent methylation and actual age derived from a total of 76 saliva samples of various ages are prepared as variables according to an embodiment of the present invention.
4 is a scatter graph in which predicted age and actual age derived from a total of 76 saliva samples of various ages are prepared as variables according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention more specifically, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example 1: DNA extraction and sulfite conversion from saliva

DNA was extracted from 400 μl of saliva sample using QIAamp DNA Mini Kit (QIAGEN, Germany), and the extracted saliva DNA was used in D.W. It was eluted with 20 µl and converted to sulfite with an EZ DNA methylation Kit (Zymo Research, USA). All experimental procedures using the kit were performed according to the protocol provided by the manufacturer.

Example 2: DNA amplification using digital PCR

The DNA of Example 1 was amplified by digital PCR. For PCR, a QX200™ Droplet Digital™ PCR System (Bio-rad, USA) was used, and a 4-well digital PCR amplification was set to be performed for one sample analysis. Information on the sequence of the four primers/probes included in each of the 4 wells is shown in Table 1 below. Each primer/probe set was prepared for methylation quantification of four CpG sites, and FAM fluorescence was applied to 5'of the probe for quantifying methylated copies, and the probe for quantifying unmethylated copies (Unmethyl probe). 5'was labeled with HEX fluorescence, and 3'of all probes labeled BHQ1. That is, the FAM fluorescent probe was composed of a sequence complementary to the methylated copy, and the HEX fluorescent probe was composed of a sequence complementary to the unmethylated copy.

Primer/probe set Name Sequence No. Seq (5’ to 3’) cg00481951
(SST) SST Forward primer SEQ ID NO: 1 AAG AGG TAG TAG TTT TAG AGG TTT SST Reverse primer SEQ ID NO: 2 CAC AAT TCA AAT AAA TTC TTA CTT AAC AT SST Methyl probe SEQ ID NO: 3 AGG GAT AGA CGT TAT TGT YGT SST Unmethyl probe SEQ ID NO: 4 TTA GGG ATA GAT GTT ATT GTY GTT T cg14361627
(KLF14) KLF14 Forward primer SEQ ID NO: 5 GGT TAA GTT ATG TTT AAT AGT TTT AGA AAT T KLF14 Reverse primer SEQ ID NO: 6 AAC TAC TAC AAC CCA AAA ATT CC KLF14 Methyl probe SEQ ID NO: 7 TTT TGT TTT CGY GTT TTT TTT TTT G KLF14 Unmethyl probe SEQ ID NO: 8 TTA TTT TGT TTT TGY GTT TTT TTT TTT G cg08928145
(TSSK6) TSSK6 Forward primer SEQ ID NO: 9 GGG AAG AAA GGG AGG GA TSSK6 Reverse primer SEQ ID NO: 10 AAT AAT TCC AAC CAT TCC TAC AAC TSSK6 Methyl probe SEQ ID NO: 11 TGY GTC GTT TTG GTT TAG TG TSSK6 Unmethyl probe SEQ ID NO: 12 TGY GTT GTT TTG GTT TAG TGT cg07547549
(SLC12A5) SLC12A5 Forward primer SEQ ID NO: 13 TGG AGT TTA GTT TTA TTG GAA TGT SLC12A5 Reverse primer SEQ ID NO: 14 ATT TAA CTA ACC CAA ATC AAC CC SLC12A5 Methyl probe SEQ ID NO: 15 TTA AGG TGT TGG AAT GCG TY SLC12A5 Unmethyl probe SEQ ID NO: 16 TTA AGG TGT TGG AAT GTG TYG

The composition of the droplet digital PCR mixture per well is shown in Table 2 below.

Name Quantity Primer/probe set cg00481951
(SST) cg14361627
(KLF14) cg08928145
(TSSK6) cg07547549
(SLC12A5) ddPCR Supermix for Probes 10 μl Forward primer 20 pmole Reverse primer 20 pmole Methyl probe 5 pmole Unmethyl probe 5 pmole Template DNA (Bisulfite treated) 2 μl D.W. Up to 20 μl Total 20 μl

The droplet digital PCR amplification protocol is shown in Table 3 below.

Name Temp, °C Time Cycles Initial denaturation 95 10 min - Denaturation 95 30 sec 55 Annealing/Extension 56 2 min

In addition, all droplet digital PCR processes were performed in the same manner as the manufacturer's provided protocol.

Example 3: Droplet reading

Fig. 1 shows the results of droplet reading performed in Examples 1 and 2 with a 52-year-old saliva sample, and Fig. 2 shows the result of calculating the methylated copy percentage according to the formula in Table 4 below. It is shown in 5.

Percentage of methylated copies (%) = {FAM copy / (FAM copy + HEX copy)} * 100

Chr Age cg00481951(SST) cg14361627 (KLF14) cg08928145 (TSSK6) cg07547549 (SLC12A5) 52 12.00 5.00 44.00 22.00

Example 4: Regression Analysis Model Design

A total of 76 saliva samples (20 for 20, 18 for 30, 19 for 40, 19 for 50) were used in the same manner as above to perform methylation copy percentage for 4 CpG positions, and the results are as follows. It is described in Table 6.

No. Chr Age cg00481951(SST) cg14361627 (KLF14) cg08928145 (TSSK6) cg07547549 (SLC12A5) One 23 4.8 1.6 39.1 17.7 2 24 7 2.1 53 12.2 3 25 5.4 3.5 50.3 13.7 4 25 7 2 38.8 14.4 5 25 6.32 1.76 49.8 11.42 6 25 5.9 1.57 52.4 12 7 27 4 2.3 75 11 8 27 6.9 2.9 39.3 13.4 9 27 8 2.43 47.7 13.4 10 27 7.4 1.5 49.5 18.3 11 27 7.3 3.1 59.1 17 12 27 6.6 2.4 38.3 14.7 13 27 6.8 3.1 46.4 12.5 14 27 8.1 3.6 38.3 20.6 15 28 6.6 2.1 51.1 15.7 16 28 6.9 1.8 49.2 13.1 17 28 7.3 1.6 44.9 13.9 18 28 7.1 1.8 55.1 13.1 19 28 6.3 3.2 45.3 11.7 20 28 6.7 2.9 49.9 12.9 21 30 8.5 2.6 42.2 13.7 22 31 8.4 4.1 36.5 25 23 32 7.6 4.8 50.4 13.1 24 32 6.97 2.96 62 10.6 25 33 8.9 2.7 35.3 17.1 26 34 10.8 2.7 56.9 14.2 27 34 11 2.6 38 23.1 28 34 6.8 3.12 41.9 13.5 29 35 8.3 2.7 52.7 11.4 30 36 8.2 2.6 48.6 14.5 31 36 7.8 3.7 43 17.6 32 36 9.27 3.85 41.9 14.1 33 37 8 5.4 43 24 34 37 8.9 3.54 58.4 17.3 35 37 7.7 4.3 40.1 19 36 37 9.9 3.9 39.3 17.9 37 37 7.7 4.6 36 24.9 38 38 8.7 3.48 54.7 16.5 39 40 10.6 5.1 45.9 20.2 40 40 8.4 3.7 39.7 12.5 41 40 10.4 4.3 46.9 17.3 42 41 9.6 5.6 43 25.9 43 41 8.9 3.3 47.1 14.2 44 42 9.6 4.6 51.1 18.5 45 42 9.2 4 54.7 17.2 46 42 11.3 5.7 49.4 18.2 47 42 7.6 6.2 43.4 23.3 48 43 10.4 5.8 52.3 20.9 49 43 11.2 5.3 51.4 18.7 50 44 7.7 4 40.7 24 51 44 11.1 5.8 47.7 19 52 44 9.9 3.68 54.9 15.6 53 45 8.4 4.4 69.9 15.7 54 46 6.9 6.2 58 13.2 55 48 11.6 6.2 59 23.1 56 49 11.3 5.2 60.6 24.2 57 49 10.3 5.98 58.5 19.7 58 50 10.9 3.85 62.5 19.4 59 50 11.4 6.3 42.7 33.9 60 50 11.3 4.6 71.2 19.9 61 51 11.4 4.48 66 20 62 52 12 5 44 22 63 53 10.5 7 53 18 64 54 11 3.3 63 23 65 54 10.5 7.2 57.2 27.2 66 54 9.6 4.5 60.4 23.3 67 54 10.6 7.1 68.3 21.1 68 54 11 6.8 66 18.2 69 55 14.8 6 60.8 20.2 70 55 16 6.9 65 21.5 71 57 19.5 1.7 63 17.6 72 57 10.4 5.88 73.2 17.6 73 58 12.7 7.5 50.9 22.5 74 58 15.2 5 74.6 22.4 75 58 17.5 7.2 65.6 18.9 76 58 12.2 6.9 47.8 24.8

As a result of the above, in order to confirm whether regression analysis is possible, a scatter graph composed of “methylation percentage X actual age” for each CpG location was created, and is shown in FIG. 3. As a result, it was confirmed that the percent methylation at the four CpG sites had a positive correlation with the actual age.

A multivariate regression model was designed using the regression analysis results of the analyzed four CpG locations. Using the designed results, a first-order equation in Table 7 was established, and the age of each salivary DNA was predicted based on this and described in Table 8.

Predicted Age
= -6.2+1.7*cg00481951+2.4*cg14361627+0.3*cg08928145+0.4*cg07547549

No. Chr Age Predicted Age Chr-Pre No. Chr Age Predicted Age Chr-Pre One 23 23.4 -0.4 39 40 43.2 -3.2 2 24 29.7 -5.7 40 40 31.8 8.2 3 25 29.1 -4.1 41 40 40.4 -0.4 4 25 26.7 -1.7 42 41 43.7 -2.7 5 25 26.7 -1.7 43 41 34.7 6.3 6 25 26.5 -1.5 44 42 41.1 0.9 7 27 29.8 -2.8 45 42 39.7 2.3 8 27 27.9 -0.9 46 42 45.6 -3.6 9 27 31.3 -4.3 47 42 40.1 1.9 10 27 31 -4 48 43 46 -3 11 27 35.6 -8.6 49 43 45.5 -2.5 12 27 26.7 0.3 50 44 36 8 13 27 29.5 -2.5 51 44 45.3 -1.3 14 27 34 -7 52 44 39.8 4.2 15 28 29.9 -1.9 53 45 42.3 2.7 16 28 28.4 -0.4 54 46 38.5 7.5 17 28 28 0 55 48 51.6 -3.6 18 28 30.3 -2.3 56 49 50.1 -1.1 19 28 28.2 -0.2 57 49 47.2 1.8 20 28 30.1 -2.1 58 50 45.5 4.5 21 30 31.2 -1.2 59 50 51.5 -1.5 22 31 36.8 -5.8 60 50 50.1 -0.1 23 32 35.3 -3.3 61 51 48.7 2.3 24 32 32.9 -0.9 62 52 45.8 6.2 25 33 31.7 1.3 63 53 47.3 5.7 26 34 39.7 -5.7 64 54 46.3 7.7 27 34 38.6 -4.6 65 54 52.5 1.5 28 34 28.8 5.2 66 54 45.3 8.7 29 35 32.8 2.2 67 54 52.9 1.1 30 36 32.6 3.4 68 54 51.3 2.7 31 36 33.6 2.4 69 55 56.5 -1.5 32 36 34.9 1.1 70 55 61.9 -6.9 33 37 39.6 -2.6 71 57 57.2 -0.2 34 37 39.3 -2.3 72 57 50.2 6.8 35 37 34.3 2.7 73 58 53.6 4.4 36 37 37.1 -0.1 74 58 60 -2 37 37 36.2 0.8 75 58 64.4 -6.4 38 38 37.5 0.5 76 58 51.7 6.3

As a result of Table 8, a scatter graph using predicted age and actual age as variables was created and shown in FIG. 4. As a result of the analysis, the correlation coefficient (Spearman's rho) is 0.932, the absolute mean deviation (MAD from chronological age) from the actual age (Chr Age) is derived as 3.04, the age prediction using the equation (Table 7) of the present invention is actually It was confirmed that the error with age was small and was measured accurately.

In the present invention, as shown in Table 7 above, the age prediction model was constructed by measuring the degree of methylation as a target of four markers less than the CpG marker types used in the existing age prediction method. When the age prediction method according to the present invention is used, analysis cost and time can be reduced compared to the conventional age prediction method, and the error range is improved, so that the age of the test object can be accurately predicted.

All references, articles, publications, and patents and patent applications cited in this specification are incorporated herein by reference in their entirety. Accordingly, the spirit and scope of the following claims should not be limited to the description of the preferred embodiments described above.

<110> BIOCORE CO., LTD. REPUBLIC O F KOREA (FORENSIC SERVICE DIRECTOR MINISTRY OF GOVERNMENT ADMINISTRATION AND HOME AFFAIRS) <120> Age estimation method using body fluids <130> PDPB192030 <160> 16 <170> KoPatentIn 3.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SST Forward primer <400> 1 aagaggtagt agttttagag gttt 24 <210> 2 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> SST Reverse primer <400> 2 cacaattcaa ataaattctt acttaacat 29 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> SST Methyl probe <400> 3 agggatagac gttattgtyg t 21 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> SST Unmethyl probe <400> 4 ttagggatag atgttattgt ygttt 25 <210> 5 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> KLF14 Forward primer <400> 5 ggttaagtta tgtttaatag ttttagaaat t 31 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> KLF14 Reverse primer <400> 6 aactactaca acccaaaaat tcc 23 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> KLF14 Methyl probe <400> 7 ttttgttttc gygttttttt ttttg 25 <210> 8 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> KLF14 Unmethyl probe <400> 8 ttattttgtt tttgygtttt tttttttg 28 <210> 9 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> TSSK6 Forward primer <400> 9 gggaagaaag ggaggga 17 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> TSSK6 Reverse primer <400> 10 aataattcca accattccta caac 24 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TSSK6 Methyl probe <400> 11 tgygtcgttt tggtttagtg 20 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TSSK6 Unmethyl probe <400> 12 tgygttgttt tggtttagtg t 21 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SLC12A5 Forward primer <400> 13 tggagtttag ttttattgga atgt 24 <210> 14 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> SLC12A5 Reverse primer <400> 14 atttaactaa cccaaatcaa ccc 23 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SLC12A5 Methyl probe <400> 15 ttaaggtgtt ggaatgcgty 20 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> SLC12A5 Unmethyl probe <400> 16 ttaaggtgtt ggaatgtgty g 21

Claims (15)

(a) performing a methylation assay for the CpG marker of the gene with a sample isolated from an unknown individual;
(b) substituting the result of (a) into Equation 1 to calculate a K value, which is the degree of methylation of the CpG marker of each gene; And
(c) substituting the K value of step (b) into Equation 2; as an age prediction method comprising,
The CpG marker of the gene is cg00481951 for SST (somatostatin), cg14361627 for KLF14 (Kruppel like factor 14), cg08928145 for testis specific serine kinase 6 (TSSK6), and cg07547549 for solvent carrier family 12 member 5 (SLC12A5). ego,
Formula 1 is “{methylated copy/(methylated copy + unmethylated copy)}×100”,
Formula 2 is "-N ₁ + N ₂ × (K value of cg00481951) + N ₃ × (K value of cg14361627) + N ₄ × (K value of cg08928145) + N ₅ × (K value of cg07547549)" as ,
N ₁ in Formula 2 is -16.2 to 3.8, N ₂ is -3.3 to 6.7, N ₃ is -2.6 to 7.4, N ₄ is -2.7 to 3.3, and N ₅ is -2.6 to 3.4 Phosphorus, age prediction method.
delete delete delete delete The method of claim 1,
The sample is any one selected from the group consisting of body fluids, body tissues, and hair roots, age prediction method.
The method of claim 6,
The body fluid is any one selected from the group consisting of blood, plasma, lymph fluid, aqueous humor, cerebrospinal fluid, pleural fluid, tear fluid (tears), urine, saliva (saliva), bile fluid, amniotic fluid, semen, and tissue fluid.
delete delete delete delete delete An analysis unit for analyzing a K value, which is a degree of methylation of a CpG marker of a gene in a sample isolated from an unknown individual; And
As an age prediction device comprising a; calculating unit for calculating the age of the individual from the K value,
The CpG markers of this gene are cg00481951 for SST, cg14361627 for KLF14, cg08928145 for TSSK6, and cg07547549 for SLC12A5,
The K value in the analysis unit is calculated by Equation 1,
Formula 1 is “{methylated copy/(methylated copy + unmethylated copy)}×100”,
The age calculation of the individual is calculated by Equation 2,
Equation 2 is “-N ₁ + N ₂ × (K value of cg00481951) + N ₃ × (K value of cg14361627) + N ₄ × (K value of cg08928145) + N ₅ × (K value of cg07547549)” as ,
N ₁ in Formula 2 is -16.2 to 3.8, N ₂ is -3.3 to 6.7, N ₃ is -2.6 to 7.4, N ₄ is -2.7 to 3.3, and N ₅ is -2.6 to 3.4 Phosphorus, age prediction device.
The method of claim 13,
The sample is any one selected from the group consisting of body fluids, body tissues, and hair roots, age prediction device.
The method of claim 14,
The body fluid is any one selected from the group consisting of blood, plasma, lymph fluid, aqueous humor, cerebrospinal fluid, pleural fluid, tear fluid (tears), urine, saliva (saliva), bile fluid, amniotic fluid, semen, and tissue fluid.

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