KR102061317B1 - Kits for detecting hair loss disorders with Single Nucleotide Polymorphism and method for detecting hair loss disorders thereby - Google Patents

Abstract

The present invention relates to a kit for predicting hair loss produced by selecting 12 single nucleotide polymorphisms (SNPs) which affect hair loss, and a method for predicting hair loss using the kit. By using the kit of the present invention, it is possible for early prediction and early management of hair loss patients, and thus hair loss management can be easily and appropriately performed. A service for proposing products for alleviating hair loss will also be possible, and thus the marketability can be increased. In addition, it is possible to maximize a therapeutic effect by early prediction of hair loss, and the economic effect thereby of reducing a treatment cost and analysis cost can be expected.

Description

Kits for detecting hair loss disorders using single nucleotide polymorphism and methods for predicting hair loss using the same

The present invention relates to a kit for predicting hair loss, and more particularly, to a kit for predicting hair loss produced by selecting 12 single base polymorphisms (SNPs) that affect hair loss and a method for predicting hair loss using the same.

Recently, hair loss patients and the hair loss treatment industry is growing rapidly due to physical and mental stress and genetic causes, and the need for accurate identification of the hair loss phenomenon and prevention of treatment is increasing. Hair loss is a progressive disease, and hair loss in people with genetic predisposition is very slow, and the progress and progression of hair loss vary greatly.However, most hair loss patients can stop the progression of hair loss with proper drug treatment at an early stage. Therefore, treatment through early diagnosis is very important.

However, most of the hair loss patients have been diagnosed by traditional methods such as self-diagnosis, photos of missing hairs, and mineral test. Among them, the microscopic photographic examination and self-diagnosis include subjective factors, and the expensive hair mineral examination is conducted irrelevant to hair loss, so that accurate hair loss prediction is impossible.

On the other hand, domestic disease-related single nucleotide polymorphism (SNP) discovery technology is ranked as the most technologically advanced 20% in the US (Biotechnology, Biotechnology Research Institute), and the development of SNP markers essential for genetic diagnostic analysis is still at an early stage. It is necessary to secure. In Korea, Chromach has confirmed the possibility of hair loss through the quantitative detection of the male hormone 5α-reductase, which is well known in the male-type hair loss mechanism. There has been a report of no specific differences between the two (S. Redler et al. 2012), which is not an accurate predictive diagnosis of hair loss. Therefore, there is an urgent need to develop a method that can be used for treatment by accurately predicting hair loss.

Republic of Korea Patent Publication No. 10-2018-0036788 (2018.04.09), it is described with respect to the biomarker for the treatment of alopecia areata.

Most studies on alopecia patients have mainly used polymerase chain reaction-restriction enzyme length polymorphism (PCR-RFLP) to test the gene polymorphism of the androgen receptor. The use of restriction enzymes has the disadvantages of complexity, long analysis time and poor accuracy. In addition, since hair loss is known to be a disease with a strong genetic factor, the present invention intends to develop and provide a kit for predicting hair loss (Kit) capable of accurate prediction and early management.

The present invention provides a primer set for detecting hair loss predicting SNP markers, comprising primers represented by nucleic acid sequences of SEQ ID NOs: 1 to 24. In this case, the primer set for detecting the hair loss predicting SNP marker may preferably further include respective primers represented by nucleic acid sequences of SEQ ID NOs: 25 to 36.

On the other hand, the present invention is characterized in that it comprises a primer set for detecting the SNP markers comprising each primer represented by the nucleic acid sequence of SEQ ID NO: 1 to 24 and each primer represented by the nucleic acid sequence of SEQ ID NO: 25 to 36 It provides a diagnostic kit for predicting hair loss.

Meanwhile, the present invention provides a method for recovering amplification products by PCR amplifying rs6113491, rs201571, rs1998076, rs6137444, rs2180439, rs1352015, rs1160312, rs756853, rs2249817, rs7910290, rs6601957 and rs11253156 of human DNA (a); And determining the individual genotypes by analyzing single base polymorphisms of rs6113491, rs201571, rs1998076, rs6137444, rs2180439, rs1352015, rs1160312, rs756853, rs2249817, rs7910290, rs6601957 and rs11253156 in the amplification product (b); A hair loss prediction method is provided.

Hair loss prediction method of the present invention, after the step (b), preferably analyzing the hair loss traits according to the individual genotype, and determining the degree of hair loss expected (c); may further comprise a.

By using the kit of the present invention, early prediction and early management of alopecia patients can be facilitated, and a proper hair loss management can be performed.

In addition, the economic effect of maximizing the treatment effect by the early prediction of hair loss, the resulting treatment cost and analysis cost reduction can be expected.

Figure 1 shows a schematic diagram of the NGS target concentration method.
Figure 2 shows a base sequence generation schematic.
3 is a schematic diagram of a method (SNaPshot assay) for confirming the accuracy of genotyping.
Figure 4 is a result of verifying the hair loss prediction through genetic analysis by performing a multiplex SNP extension assay (multiplex SNP extension assay) to be amplified at the same time by combining the 12 SBE primers for the SNPs.

More than 10 companies are currently doing genetic testing, including 23andMe and TheMakingsofMe in the United States. In particular, TheMakingsofMe offers DNA testing services using SNP markers related to hair loss, but only because it tests only four genetic markers. The prediction accuracy is low. In Korea, as the regulation of hair loss-related DTC (Direct-To-Consumer) test is lifted, domestic companies are in the process of testing. However, only four hair loss-related SNPs are allowed, which leads to low accuracy of hair loss prediction.

Therefore, the present invention finds 12 highly accurate hair loss predictive SNP markers using the NGS technique, and develops a customized hair loss predictive diagnosis kit by using the same. We anticipated to establish a scientific gene management system that can be customized for hair loss treatment.

To this end, the present invention provides a primer set for detecting hair loss predicting SNP markers, comprising primers represented by nucleic acid sequences of SEQ ID NOs: 1 to 24. In this case, the primer set for detecting the hair loss predicting SNP marker may preferably further include respective primers represented by nucleic acid sequences of SEQ ID NOs: 25 to 36. In the present invention, each of the primers represented by the nucleic acid sequences of SEQ ID NOs: 1 to 24 is a hair loss predicting SNP marker 'amplification' primer set targeted in the present invention, and each primer represented by the nucleic acid sequences of SEQ ID NOs: 25 to 36. The amplification products (SNP-containing nucleic acid sequences) amplified using a primer set for amplifying hair loss predicting SNP markers including the respective primers represented by the nucleic acid sequences of SEQ ID NOs: 1 to 24 were visually identified by dye. This is a set of 'check' single-base extension (SBE) primers.

In addition, the present invention is characterized in that it comprises a primer set for detecting the SNP markers comprising each primer represented by the nucleic acid sequence of SEQ ID NO: 1 to 24 and each primer represented by the nucleic acid sequence of SEQ ID NO: 25 to 36 To provide a diagnostic kit for predicting hair loss, the diagnostic kit for predicting hair loss of the present invention is preferably a polymerase chain reaction (PCR) kit. The diagnostic kit for predicting hair loss of the present invention includes a buffer and the like in addition to the primer set. In addition to the primers used in the PCR diagnostic kit as a secondary component is widely known, so a detailed description thereof will be omitted.

In the present invention, 12 SNPs characteristically found in the hair loss group were selected by comparing the SNPs of the hair loss group and the control group. By determining the presence of the 12 SNPs using the primer set for predicting hair loss of the present invention, it is possible to predict hair loss, and as the number of SNP markers is examined compared to the existing test, the prediction rate is also very high. There is an advantage.

Meanwhile, the present invention provides a method for recovering amplification products by PCR amplifying rs6113491, rs201571, rs1998076, rs6137444, rs2180439, rs1352015, rs1160312, rs756853, rs2249817, rs7910290, rs6601957 and rs11253156 of human DNA (a); And determining the individual genotypes by analyzing single base polymorphisms of rs6113491, rs201571, rs1998076, rs6137444, rs2180439, rs1352015, rs1160312, rs756853, rs2249817, rs7910290, rs6601957 and rs11253156 in the amplification product (b); It provides a hair loss prediction method characterized in that, the hair loss prediction method of the present invention, after the step (b), preferably analyzing the hair loss trait according to the individual genotype, and (c) determining the expected degree of hair loss; It is better to include more.

The 'hair loss trait according to the individual genotype' described in the present invention is classified as a percentage of hair loss based on the 12 SNPs of the present invention. That is, genotypes having all 12 hair loss-related SNPs among 12 SNPs are classified as' 100% alopecia trait ', and genotypes having one hair loss-related SNP out of 12 SNPs are' 8% (1/12 ×). 100) hair loss traits. That is, the 'hair loss trait analysis according to the individual genotypes' described in the present invention is to predict the possibility of hair loss according to the number of SNPs possessed by the individual among the 12 hair loss prediction SNP markers discovered in the present invention.

For example, using the primer set for predicting hair loss of the present invention, as a result of analyzing an individual SNP, if it is confirmed that Kim has one hair loss-related SNP and aunt has five hair loss-related SNPs, hair loss of Kim hair The likelihood will be about 8%, and the likelihood of hair loss will be about 42%. As a result of the SNP analysis of the individual as described above, the larger the number of SNPs (12 hair loss prediction SNP markers discovered in the present invention) held by the individual, the more likely it is that the development of hair loss is very high.

As described above, the present invention provides a standardized test method for diagnosing hair loss by providing the hair loss prediction method. According to the present invention, by analyzing the genes related to hair loss, it is possible to early diagnosis and early treatment of hair loss, thereby reducing the economic burden.

Conventional hair loss diagnosis was a system to provide treatment only when the hair loss progressed, but the customized hair loss predictive diagnosis kit through this research and development prior to screening the root cause, hair loss severity, early hair loss, etc. Predictive diagnostics is an advantage that can be treated according to the characteristics of the individual.

Hereinafter, the configuration of the present invention will be described in detail through the following examples. However, the scope of the present invention is not limited only to the following examples, but includes modifications of equivalent technical ideas.

[ Production Example  1: Selection of single nucleotide polymorphism (SNP)]

(1) DNA extraction

DNA was extracted from the blood of 40 hair loss groups and 10 control groups.

(2) NGS library production

The extracted genomic DNA was fragmented through sonication and then subjected to the following process using SureSelectXT Human All Exon Kit. Adapter-tagged DNA library through end repairing, bead purification, 3'-end adenylation, adapter ligation ) Was prepared and amplified to prepare a DNA library. Targeted-enriched NGS libraries (indexed, target-enriched), generated by capturing the capturing library through hybridization of the DNA library and the captured library, and PCR amplification using indexing primers. NGS library). Figure 1 shows a schematic diagram of the NGS target concentration method.

(3) PCR amplification and sequencing platforms

Cluster generation of target-enriched DNA library using Illumina c-Bot instrument and flow cell in which cluster was created using Illumina HiSeq2500 instrument. Sequencing was performed at. The sequencing results analyzed by the NGS method were determined by the bioinformatics tool and the statistical method, and the alignment of the exogenous sequencing data was aligned through the integrated geographic viewer. analysis was performed. Figure 2 shows a base sequence generation schematic.

(4) Discovery of single nucleotide polymorphism markers

Variant sequences of the control group and the experimental group were analyzed to prepare a variant list, and SNPs showing differences between the control group and the experimental group were listed. In addition, gender and age corrections were made. The reported SNPs were examined in both lists, and they were sorted in descending order of significance (p-value).

(5) single nucleotide polymorphism (SNP) Marker  selection.

A literature review confirmed the alopecia of genes containing SNPs with low probability. Among these, three locus of SNPs (rs7910290, rs6601957, rs11253156) are present in one gene (TUBAL3), and the expression level of alpha-tubulin is reduced in alopecia areata (Scientific Reports, (2018) 8: 521), it is estimated that rs7910290, rs6601957, and rs11253156 of SNPs of this TUBAL3 gene are highly associated with hair loss. Therefore, SNP rs7910290, rs6601957 and rs11253156 were selected as test SNPs. In addition, in order to increase the probability of hair loss, a well-known hair loss-related SNP was selected as a test target marker. Finally, a total of 12 SNP markers capable of predicting hair loss were selected and shown in Table 1 below.

SNP Markers for Hair Loss Prediction No. SNP Polymorphism Gene Location One rs6113491 C / A 20p11 Chr. 20: 22076777 2 rs201571 C / T 20p11 Chr. 20: 22032876 3 rs1998076 G / A 20p11 Chr. 20: 21899407 4 rs6137444 C / T 20p11 Chr. 20: 21805001 5 rs2180439 C / T 20p11 Chr. 20: 21872462 6 rs1352015 G / A EDA2R Chr.X: 66624001 7 rs1160312 G / A LINC01432 Chr. 20: 22069865 8 rs756853 G / A HDAC9 Chr.7: 18850377 9 rs2249817 G / A HDAC9 Chr. 7: 18856388 10 rs7910290 G / A TUBAL3 Chr. 10: 5394297 11 rs6601957 G / A TUBAL3 Chr. 10: 5394524 12 rs11253156 G / A TUBAL3 Chr. 10: 5395402

[ Example  1: for hair loss prediction Kit (multiplex SNaPshot Kit) Development of

(1) hair loss prediction Of kit  Configuration

The kit for predicting hair loss of the present invention was constructed by using a PCR method and a single base extension (SBE) method.

A primer set for amplifying the 12 SNP marker sites selected in Preparation Example 1 and a SBE (single base extension) primer set for identifying each SNP marker were developed and shown in Tables 2 and 3 below.

Primer set for SNP marker amplification SNP Primer persons Sequence (5 '→ 3') Concentration Used to Make Mix SEQ ID NO: rs2180439
B1_rs2180439_F1u GGTTGGATGGATGCTGGATG 10 One B1_rs2180439_R2u CTGTAATGGTGGGCACAAGTCA 10 2 rs1998076 B2_rs1998076_F2 GGACTGAAAAACCCACCAGG 5 3 B2_rs1998076_R1 CGGTTGCCCAAATCAAGCTA 5 4 rs6137444 B3_rs6137444_F2m GACTTTTGGCTCCACCCCAG 5 5 B3_rs6137444_R3 CACTCCAGGGACAACAACAGG 5 6 rs6113491 B4_rs6113491_F3u GCTTCCCTTTGGACACTCTT 10 7 B4_rs6113491_R4u GTGAAGTGGTCAGAATAGGTAAATCC 10 8 rs201571 B5_rs201571_F5m AGTCCGAGCCACTATCCTTTG 2 9 B5_rs201571_R9 GGCAGTCAGAGAAACCATGTAA 2 10 rs1160312 B6_rs1160312_F1m AGGTAGCCAGGAGGTAGAAC 5 11 B6_rs1160312_R1u CTGTGTGGCAGGTTAATTGAGA 5 12 rs1352015 B7_rs1352015_F3 ATTCCTATCCCCGTTTCCAG 2 13 B7_rs1352015_R1 GCCAGGCACTATTGTAAGCAC 2 14 rs756853 B8_rs756853_F3 ACGATCTGAATGTGGAGTGTGC 2 15 B8_rs756853_R9 TCCTCCCCTACGCTTTCAT 2 16 rs2249817 B9_rs2249817_F9m GCATCATAGGTTCACGCTAAGG 20 17 B9_rs2249817_R2 AGGGGCAATTTCTATGCAGC 20 18 rs7910290 Bw8_rs7910290_F2 TGAAGGTACAGTCCGTGTGC 2 19 Bw8_rs7910290_R5 GGCACTGGTTCAGGGTTTACG 2 20 rs6601957 Bw9_rs6601957_F1 TCCCTGAAGTCCACCACACT 2 21 Bw9_rs6601957_R1u GCTCTGAAGTGCTTTGCCTACTC 2 22 rs11253156 Bw10_rs11253156_F1u CAGGGAGAGACGGTTGGTG 2 23 Bw10_rs11253156_R4u CAGCACCGTTCACTCTTCCA 2 24

SBE Primer Set for SNP Marker Identification SNP Sequence (5 '→ 3') Mix production concentration (Pmole) SEQ ID NO: rs6113491 TTGGGAGAAATAGGGAGCAATT 1.0 25 rs201571 TTGGAACCAGAAAAACAATGTCTATTTAG 0.8 26 rs1998076 TTTTTTTTAATTCTCATCTGAGTTAACAGATTTGG 0.4 27 rs6137444 TTTTTTTTTTTTTTTTGGATTAGACTGCTAACTTTTTAAA 0.6 28 rs2180439 TTTTTTTTTTTTTTTTTTTTTAATCTAGCTGCCGTTTTGTGTTAT 0.6 29 rs1352015 TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATGCTATGAGGCCATGGTAC 0.6 30 rs1160312 TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAGTGTGGGGTCAGGACTC 1.0 31 rs756853 TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTACCTTGTACTTCTTGTTCGAGGA 1.0 32 rs2249817 TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCCTCTGTTCATTATAGTGACCAAAA 1.2 33 rs7910290 TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAGACAGAGTTATAAGGCTCTACCAC 0.2 34 rs6601957 TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTACCAGAAGCAGTGAATTGGAC 0.3 35 rs11253156 TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCGGCCTCGTGCGTAATTGTT 0.3 36

(2) hair loss prediction Kit (multiplex SNaPshot  Kit condition development

There are two types of reagents for analysis: a set for amplification of SNPs and a set for identification of SNPs. Reagents for amplification of SNPs consisted of DOW Hair-Loss QuickFinder HLQF Taq, 10X HLQF Taq Buffer and primer set prepared above (Table 2), and the reagent for identifying SNPs after amplification was DOW Hair-Loss QuickFinder After the SNaPshot mix (fluorescent labeled ddNTPs, buffer) and the SBE primer set (Table 3) prepared above, the reaction conditions were optimized as follows.

In the conventional sequencing analysis method for SNP analysis, sequencing was possible in samples of 10 ng or more, but the reaction conditions were optimized to enable analysis in samples of 1 ng or more in gene analysis using the kit for predicting hair loss according to the present invention.

If there is a difference in the degree of amplification for each SNP marker, the SNP genotype reading may not be accurate, so that the peak height range is within the range of 100 to 4000 RFU amplification so that a stable genotype reading can be confirmed. Optimized.

In addition, SBE primer combination conditions for 12 SNPs predicting hair loss were established and developed to identify each SNP marker. The optimized reaction conditions are as follows.

First, PCR composition for amplification of SNPs was composed of 1 ~ 60ng of genomic DNA, 0.4µl of DOW Hair-Loss QuickFinder HLQF Taq, 2µl of 10X HLQF Taq Buffer, and 5µl of SNP marker amplification primer set. Table 2), the total dose was set to 20 μl.

PCR was carried out with the PCR composition as described above. The temperature, time and cycle are 5 minutes at 95 ° C. (1 cycle, ie one repetition), followed by 37 cycles (ie 37 seconds at 20 ° C. at 95 ° C., 30 seconds at 58 ° C., 30 seconds at 72 ° C.). Cycles), and finally one cycle (once) at 72 ° C. for 5 minutes.

Residual dNTPs and primers contained in the PCR product inhibit the subsequent process, so the PCR product, 5 μl and ExoSAP-IT (Thermo Fisher Scientific), 1 μl are mixed and reacted for 45 minutes at 37 ° C. and 15 minutes at 80 ° C. dNTP and primers were removed.

The composition for performing the single-nucleotide extension (SBE) process was set to 4 μl of reaction product, 5 μl of DOW Hair-Loss QuickFinder SNaPshot mix, and 1 μl of SBE primer set (Table 3).

The SBE procedure was carried out in 25 cycles (ie 25 repetitions) under conditions of 10 seconds at 95 ° C, 10 seconds at 50 ° C, and 30 seconds at 60 ° C.

Then, to remove ddNTP that inhibits the subsequent process, 1 μl of SAP (Thermo Fisher Scientific), 1.2 μl of 10X SAP Buffer (Thermo Fisher Scientific) was added to the SBE product and reacted at 37 ° C. for 60 minutes. The reaction was carried out at 65 ° C. for 15 minutes.

(3) Confirm the accuracy of genotype readings

After purification of each SBE amplification product obtained in Example 1, capillary electrophoresis was performed on the fluorescently labeled SBE amplification products using a genetic analyzer. SNP genotyping analysis was performed via GeneMapper software (FIG. 3, FIG. 4). Figure 3 is a schematic diagram of a method (SNaPshot assay) to confirm the accuracy of genotype reading, Figure 4 is a multiplex SNP extension assay (multiplex SNP extension assay) to be amplified simultaneously by combining the 12 SBE primers for SNPs This is a result of verifying hair loss prediction through genetic analysis.

As a result, the primer set developed in the present invention was able to effectively amplify 12 SNPs at the same time.

To summarize the present invention, in order to predict hair loss, DNA was extracted from samples of the hair loss group and the control group, and PCR was performed using primers capable of predicting hair loss. Subsequently, a multiplex SNP extension assay is performed to combine 12 SBE primers with the PCR product to be amplified at the same time, perform genetic analysis with SNP genome products, and predict hair loss after analysis. Will be done.

<110> Dowgene <120> Kits for detecting hair loss disorders with Single Nucleotide          Polymorphism and method for detecting hair loss disorders thereby <130> YP-18-195 <160> 36 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> This is forward primer for amplification of rs2180439. The name          of this primer is B1_rs2180439_F1u. <400> 1 ggttggatgg atgctggatg 20 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> This is reverse primer for amplification of rs2180439. The name          of this primer is B1_rs2180439_R2u. <400> 2 ctgtaatggt gggcacaagt ca 22 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> This is forward primer for amplification of rs1998076. The name          of this primer is B2_rs1998076_F2. <400> 3 ggactgaaaa acccaccagg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> This is reverse primer for amplification of rs1998076. The name          of this primer is B2_rs1998076_R1. <400> 4 cggttgccca aatcaagcta 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> This is forward primer for amplification of rs6137444. The name          of this primer is B3_rs6137444_F2m. <400> 5 gacttttggc tccaccccag 20 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> This is reverse primer for amplification of rs6137444. The name          of this primer is B3_rs6137444_R3. <400> 6 cactccaggg acaacaacag g 21 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> This is forward primer for amplification of rs6113491. The name          of this primer is B4_rs6113491_F3u. <400> 7 gcttcccttt ggacactctt 20 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> This is reverse primer for amplification of rs6113491. The name          of this primer is B4_rs6113491_R4u. <400> 8 gtgaagtggt cagaataggt aaatcc 26 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> This is forward primer for amplification of rs201571. The name of          this primer is B5_rs201571_F5m. <400> 9 agtccgagcc actatccttt g 21 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> This is reverse primer for amplification of rs201571. The name of          this primer is B5_rs201571_R9. <400> 10 ggcagtcaga gaaaccatgt aa 22 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> This is forward primer for amplification of rs1160312. The name          of this primer is B6_rs1160312_F1m. <400> 11 aggtagccag gaggtagaac 20 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> This is reverse primer for amplification of rs1160312. The name          of this primer is B6_rs1160312_R1u. <400> 12 ctgtgtggca ggttaattga ga 22 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> This is forward primer for amplification of rs135 2015. The name          of this primer is B7_rs1352015_F3. <400> 13 attcctatcc ccgtttccag 20 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> This is reverse primer for amplification of rs135 2015. The name          of this primer is B7_rs1352015_R1. <400> 14 gccaggcact attgtaagca c 21 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> This is forward primer for amplification of rs756853. The name of          this primer is B8_rs756853_F3. <400> 15 acgatctgaa tgtggagtgt gc 22 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> This is reverse primer for amplification of rs756853. The name of          this primer is B8_rs756853_R9. <400> 16 tcctccccta cgctttcat 19 <210> 17 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> This is forward primer for amplification of rs2249817. The name          of this primer is B9_rs2249817_F9m. <400> 17 gcatcatagg ttcacgctaa gg 22 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> This is reverse primer for amplification of rs2249817. The name          of this primer is B9_rs2249817_R2. <400> 18 aggggcaatt tctatgcagc 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> This is forward primer for amplification of rs7910290. The name          of this primer is Bw8_rs7910290_F2. <400> 19 tgaaggtaca gtccgtgtgc 20 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> This is reverse primer for amplification of rs7910290. The name          of this primer is Bw8_rs7910290_R5. <400> 20 ggcactggtt cagggtttac g 21 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> This is forward primer for amplification of rs6601957. The name          of this primer is Bw9_rs6601957_F1. <400> 21 tccctgaagt ccaccacact 20 <210> 22 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> This is reverse primer for amplification of rs6601957. The name          of this primer is Bw9_rs6601957_R1u. <400> 22 gctctgaagt gctttgccta ctc 23 <210> 23 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> This is forward primer for amplification of rs11253156. The name          of this primer is Bw10_rs11253156_F1u. <400> 23 cagggagaga cggttggtg 19 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> This is reverse primer for amplification of rs11253156. The name          of this primer is Bw10_rs11253156_R4u. <400> 24 cagcaccgtt cactcttcca 20 <210> 25 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> This is SBE primer for confirmation of rs6113491. <400> 25 ttgggagaaa tagggagcaa tt 22 <210> 26 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> This is SBE primer for confirmation of rs201571. <400> 26 ttggaaccag aaaaacaatg tctatttag 29 <210> 27 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> This is SBE primer for confirmation of rs1998076. <400> 27 ttttttttaa ttctcatctg agttaacaga tttgg 35 <210> 28 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> This is SBE primer for confirmation of rs6137444. <400> 28 tttttttttt ttttttggat tagactgcta actttttaaa 40 <210> 29 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> This is SBE primer for confirmation of rs2180439. <400> 29 tttttttttt tttttttttt taatctagct gccgttttgt gttat 45 <210> 30 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> This is SBE primer for confirmation of rs135 2015. <400> 30 tttttttttt tttttttttt tttttttttt ttatgctatg aggccatggt ac 52 <210> 31 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> This is SBE primer for confirmation of rs1160312. <400> 31 tttttttttt tttttttttt tttttttttt tttttttttt agtgtggggt caggactc 58 <210> 32 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> This is SBE primer for confirmation of rs756853. <400> 32 tttttttttt tttttttttt tttttttttt tttttttttt accttgtact tcttgttcga 60 gga 63 <210> 33 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> This is SBE primer for confirmation of rs2249817. <400> 33 tttttttttt tttttttttt tttttttttt tttttttttt tttcctctgt tcattatagt 60 gaccaaaa 68 <210> 34 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> This is SBE primer for confirmation of rs7910290. <400> 34 tttttttttt tttttttttt tttttttttt tttttttttt ttttttttag acagagttat 60 aaggctctac cac 73 <210> 35 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> This is SBE primer for confirmation of rs6601957. <400> 35 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttacc 60 agaagcagtg aattggac 78 <210> 36 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> This is SBE primer for confirmation of rs11253156. <400> 36 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 60 tttcggcctc gtgcgtaatt gtt 83

Claims (5)

A primer set for detecting alopecia predicting SNP markers, comprising primers represented by nucleic acid sequences of SEQ ID NOs: 1 to 24.
The method of claim 1,
The primer set for detecting the hair loss prediction SNP marker,
A primer set for detecting hair loss predicting SNP markers, further comprising respective primers represented by nucleic acid sequences of SEQ ID NOs: 25 to 36.
Diagnosis for predicting hair loss comprising a primer set for detecting SNP markers comprising respective primers represented by the nucleic acid sequence of SEQ ID NO: 1 to 24 and each primer represented by the nucleic acid sequence of SEQ ID NO: 25 to 36 Kit.
Recovering the amplification product by PCR amplification to include rs6113491, rs201571, rs1998076, rs6137444, rs2180439, rs1352015, rs1160312, rs756853, rs2249817, rs7910290, rs6601957 and rs11253156 of human DNA (a); And,
(B) analyzing single base polymorphisms of rs6113491, rs201571, rs1998076, rs6137444, rs2180439, rs1352015, rs1160312, rs756853, rs2249817, rs7910290, rs6601957, and rs11253156 in the amplification product (b); Hair loss prediction method.
The method of claim 4, wherein
After step (b),
(C) analyzing the hair loss trait according to the individual genotype and determining the expected degree of hair loss.

Images