KR101742458B1 - Genetic polymorphic markers for determining type of white skin and use thereof - Google Patents

Abstract

The present invention relates to a skin type whitening diagnostic marker comprising one or more single base polymorphic markers selected from single base polymorphism (SNP) markers, a composition comprising an agent capable of detecting said marker, a kit comprising said composition Or a microarray, and a method of providing information on whitening of a skin type using the marker. The single nucleotide polymorphic markers of the present invention provide accurate information on the individual's skin type and thus can develop personalized cosmetics and develop customized active ingredients.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a skin type skin polymorphism marker,

The present invention provides a skin type whitening diagnostic marker comprising one or more single base polymorphic markers selected from single base polymorphism (SNP) markers capable of judging whether a skin is whitened, a preparation capable of detecting the marker A kit, or a microarray comprising the composition, and a method for providing information on whitening of a skin type using the marker.

Human skin has different skin condition for each individual. In particular, even skin types of the same age and the same skin type can vary greatly depending on the internal and external environment of each individual. As the scientific civilization develops, there are more and more cases in which the skin is severely affected by exposure to various polluted environments, exposure to ultraviolet rays due to destruction of the ozone layer, various stresses, and the like, resulting in deterioration of skin function and even skin troubles . Accordingly, there is a growing demand for functional cosmetics that can improve or maintain the condition of the skin.

However, at present, it is difficult to obtain complete trust in the skin condition information because the judgment of the skin condition for each user is mainly about the skin condition of the user through simple skin test and simple skin test. In addition, functional cosmetics currently on the market are not aimed at individuals, but are universal. Therefore, there are many limitations in finding suitable cosmetics for users. Therefore, there is almost no system to provide customized cosmetics by precisely diagnosing skin condition by approaching scientific basis.

With the increasing demand for customized cosmetics according to skin characteristics, it is required to establish a systematic standard of skin classification and to characterize product prescription accordingly. However, the current water balance classification standard used for skin classification is not able to accurately classify the skin. In addition, in a method of providing customized cosmetics by measuring the skin condition of the prior art, in order to check the current skin condition of the user, the condition of the skin wrinkles is grasped by an image analyzer system using mainly a replica plate in case of wrinkles, In the case of skin whitening, a change in erythema or skin color is measured using a mexameter and a colorimeter. In the case of skin moisturizing, a moisturizing amount is measured mainly using a corneometer have. These measurement methods simply represent the physical state of the skin surface by numerical values and may vary depending on the program to be measured. Therefore, there is a fear that errors may occur in evaluating the degree of improvement of the subject. In addition, in the case of the visual evaluation of the tester and the evaluation of the questionnaire by the tester, the subjective aspect greatly affects and it is difficult to evaluate the subject's skin condition objectively and precisely.

Under these circumstances, the present inventors constructed a scientific skin classification standard by grasping the genetic characteristics that determine the skin characteristics of the individual, developed a personalized customized ingredient based on the classification, developed customized cosmetics according to skin characteristics through various product segmentation (SNP) markers having a significant correlation with skin whitening were selected, and a method of diagnosing whether skin type was whitening was selected to confirm the present invention.

It is an object of the present invention to provide a skin type whitening diagnosis marker comprising one or more single base polymorphism markers selected from single base polymorphism (SNP) markers capable of judging the whitening skin condition.

It is still another object of the present invention to provide a skin type whitening detection composition comprising a probe capable of detecting the skin type whitening diagnosis marker or an amplifiable preparation.

Another object of the present invention is to provide a skin type whitening diagnosis kit or microarray comprising the skin type whitening presence detection composition.

Yet another object of the present invention is to provide a method for providing skin whitening information including identifying a polymorphic site of the single nucleotide polymorphism marker.

In one aspect of the present invention, there is provided a skin type whitening diagnostic marker comprising at least one single base polymorphism marker selected from single nucleotide polymorphism (SNP) markers capable of determining whitening skin to provide.

In the present invention, the term "polymorphism" refers to a case where two or more alleles exist in one locus. Of the polymorphic sites, only a single base differs from a polymorphism region to a single base polymorphism (single nucleotide polymorphism, SNP). Preferred polymorphic markers have two or more alleles exhibiting an incidence of 1% or more, more preferably 10% or 20% or more, in the selected population.

The term "allele " in the present invention refers to various types of genes that exist on the same locus of a homologous chromosome. Alleles are also used to represent polymorphisms, for example, SNPs have two kinds of bialles.

In the present invention, the term "rs_id " means rs-ID, which is an independent marker assigned to all SNPs initially registered by the NCBI that has started accumulating SNP information since 1998. [ The rs_id shown in the above table means the SNP marker which is the polymorphism marker of the present invention.

In the present invention, the term "skin type " refers to a skin type of an individual to be measured or diagnosed. The type of skin that can be measured by the SNP of the present invention is not limited but may be preferably whitening. According to one embodiment of the present invention, the present inventors classified the subject's skin type into whitened skin having less pigment or skin having more pigment and having better skin burning. Since the single nucleotide polymorphic marker of the present invention enables accurate skin type measurement, information on the changed skin type of skin contacted with the active ingredient can also be provided.

Preferably, the single nucleotide polymorphism marker may be at least one single nucleotide polymorphism marker selected from the single nucleotide polymorphism markers set forth in Table 3 and Table 4. The single nucleotide polymorphism markers shown in Table 3 and Table 4 may be used to determine whether the skin type is whitening.

Whether the single nucleotide polymorphism marker of the present invention was diagnosed with the skin type was determined by measuring the frequency of each marker. Such significance is not limited to p-values such as less than 0.05, less than 0.01, less than 0.001, less than 0.0001, less than 0.00001, less than 0.000001, less than 0.0000001, less than 0.00000001, or less than 0.000000001 p-value. Preferably, the p-value may be less than 0.01, more preferably the p-value may be less than 0.001.

The polymorphic markers of the present invention are the markers shown in Tables 3 and 4, wherein when the base of the polymorphic site of the individual polymorphic marker is a minor allele, And the frequency of the whitening skin test case is judged to be the skin of the high frequency group. When the polymorphic base is a major allele, the skin of the well-skinned control group shown in Table 3 and Table 4 This is a marker that can be judged as the skin of a group with a low frequency in the frequency of the test group which is a whitening skin. For example, when the 54189156 base of the chromosome 10 of the individual is C, the frequency of the control group is higher in Table 3, so that the skin of the individual having the minority allele C can be judged as a control group It is. If the 54189156 base of chromosome 10 of the individual is T, which is a multiple allele, the frequency of the test group is lower than that of the test group.

More preferably, Table 3 shows a polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 54189156 base of the human chromosome 10, T or C (rs1100311), and the 54189156 base; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 82834514 base sequence of the human chromosome 5 (G) or G (A) (rs1833890), and the 82834514 base sequence; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 55831811 base of the human chromosome 1, T or C (rs7526426), and the 55831811 base; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising 191042669 base of human human chromosome 2, G or A (rs1019321), said 191042669 base; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 54169046 base of C on human chromosome 10 (rs1238575) and the base 54169046 base; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 8282972792 nucleotide of human chromosome 5 (rs2652106) with G or T; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising 43428336 base of human chromosome 13, G or A (rs7322264), and 43428336 base; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 54196212 base of the human chromosome 10 (rs1082479), which is T or C, and the 54196212 base; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 191009613 base, wherein the 191009613 base of human chromosome 2 is A or G (rs9646748); A polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 55855971 base of human chromosome 1 (T) or T (C) rs1118219, including the 55855971 base; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 134657139 base of human chromosome 3 is C or T (rs9865003), the 134657139 base; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising 54182014 base of human chromosome 10 (Tg or G) (rs1100311), including 54182014 base; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 54191096 base of human human chromosome 10, C or T (rs 1693307), and the 54191096 base; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising 4860027 base (489005785) of human chromosome 6 with A or G (rs9405785); A polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 79431934 base of human chromosome 4, G or T (rs1484344), and the 79431934 base; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 32866528 base of the human chromosome 14, G or A (rs8011225), and the 32866528 base; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 8396938686 base of human chromosome 1 (A rs3470503) and the 83969386 base; A polynucleotide consisting of 5-100 consecutive DNA sequences containing the 22067299 base of the human chromosome 16 (rs1164222) with T or G, and the 22067299 base; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 134656869 base of human chromosome 3 is C or T (rs2370246), the 134656869 base; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 191098371st base of human chromosome 2 (A rs1093145), which is A or G, and the 191098371st base; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 188206372 base of the human chromosome 1 (rs1711758), which is G or A; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 191041973 base of human human chromosome 2, T or C (rs4340549), and the 191041973 base; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 96368401 base of the human chromosome 15, T or C (rs2845633), the 96368401 base; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 54203366 base of human chromosome 10 (A) or G (rs1031101), and the 54203366 base; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 29970271 base of human human chromosome 8, G or A (rs6987138), and the 29970271 base; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 900218 base of C on human chromosome 20 (rs2223962), the 900218 base; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 3765524040 base of human 6 or G (A rs804846) comprising the 37655240 base; A polynucleotide consisting of 5-100 contiguous DNA sequences comprising the 235675961 base of human chromosome 2 (rs4277473) with A or G, and the 235675961 base; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 43418348 base of the human chromosome 13, T or C (rs9525880), and the 43418348 base; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising 58468246 bases of human chromosome 5 (Cs or T) (rs1006239) and comprising the above 58468246 bases; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 54173326 base of human chromosome 10, G or A (rs996232), and the 54173326 base; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 244670509 base of human chromosome 1 (rs2340778), which is C or T, and the 244670509 base; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 158512290 base of the human chromosome 5 (A) or G (rs6556401), and the 158512290 base; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising 17963006 base of human chromosome 8 (rs1763609) which is A or G, and 17963006 base; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 67559320 base of the human chromosome 17, T or C (rs9911193), and the 67559320 base; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 65093758 base of the human chromosome 16, T or C (rs1428106), and the 65093758 base; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising 20186505 base of the human chromosome 9 (rs1456958) with T or G, and 20186505 base; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising 20186466 base of human chromosome 9 with T or C (rs1456959), 20186466 base; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 20218652 base of the human chromosome 9 (rs321218), which is C or T; A polynucleotide consisting of 5-100 consecutive DNA sequences comprising 19969813 base of the human chromosome 9 (A rs1892011), said 19969813 base; And at least one polynucleotide selected from the group consisting of complementary polynucleotides thereof, whitening of the skin type that judges whether the skin type is a skin that is well burned because of a large number of pigment, It may be a diagnostic marker.

According to one embodiment of the present invention, the VCAN (versican) gene shows meaningful results in Table 3, and in particular, the two positions of Versican showed a significant SNP, whereas the Versican gene SNPs rs1833890 and rs2652106 are chromosome 5 Respectively, at positions 82834514 and 82829792, respectively. Versican is chondroitin sulfate proteoglycan (CSPG), one of the major components of the extracellular matrix (ECM) that provides a loose & hydrated matrix at the critical point of development and disease. Versican plays an important role in morphogenesis and maintenance by participating in cell binding, proliferation, migration, and angiogenesis. In addition, Versican is involved in atherosclerotic vascular disease, cancer, tendon remodeling, hair cycle, central nervous system damage, and neurite outgrowth. Versican consists of a core protein and a glycosaminoglycan side chain (GAG) side chain. Versican is differentially expressed by different cells in time and space, and is also expressed differently depending on the developmental pathology (Fig. 3). The Versican gene is located on chromosome 5 of the human genome and consists of 15 exons of 90 kb base. Neurons and melanocytes are originated from the neuroectoderm. They are similar in shape and have a cell body at the center and a process (neurite / dendrite) It stretches in shape. Small GTP (guanosine triphosphate) -binding proteins Rho, Rac, and cdc42 are involved in both neuronal and melanocyte expansion and extension. The effect of GTP-binding proteins (Rho, Rac1, cdc42) on the neurite outgrowth of neurons was also applied to melanocytes in the same way. Versican v1 isoform (or part of the G3 domain) Induce differentiation and promote dendritic extension (Figs. 4A and 4B). Ultimately, therefore, it is assumed that the Versican will also affect the pigmentation of the skin.

The present inventors confirmed that the above-mentioned SNP is a skin type diagnostic marker through the following proof. Specifically, the skin whitening status was classified according to the minimum erythema (MED), MED 47 or higher as a control group, and MED 38 or lower as a test group. GDNA was extracted from the saliva or blood from the classified individuals and genotype analysis experiments were performed with an Axiom (TM) ASI array plate (Fig. 1). As a result, SNPs having significance in whitening skin type were classified (Table 3 and Table 4). SNPs capable of classifying such skin types were first identified by the present inventors.

In another aspect, the present invention provides a skin type whitening detection composition comprising a probe capable of detecting the skin type whitening diagnostic marker or an amplifiable preparation.

In the present invention, the term "a probe capable of detecting a skin type whitening diagnostic marker" refers to a composition capable of diagnosing whether a skin type is whitening by specifically hybridizing with a polymorphic site of the above- And the specific method of such gene analysis is not particularly limited and may be by any gene detection method known in the art to which this invention belongs.

In the present invention, the term "agent capable of amplifying a skin type whitening diagnosis marker" means a composition capable of diagnosing whether a skin type is whitened by confirming a polymorphic site of the above- Preferably means a primer capable of specifically amplifying the polynucleotide of the skin-type whitening diagnostic marker.

The primers used for the polymorphic marker amplification can be amplified by PCR using appropriate conditions in suitable buffers (e.g., four different nucleoside triphosphates and polymerase such as DNA, RNA polymerase or reverse transcriptase) and template-directed DNA Refers to a single stranded oligonucleotide that can serve as a starting point for synthesis. The appropriate length of the primer may vary depending on the intended use, but is usually 15 to 30 nucleotides. Short primer molecules generally require a lower temperature to form a stable hybrid with the template. The primer sequence need not be completely complementary to the template, but should be sufficiently complementary to hybridize with the template.

The term "primer" in the present invention means a base sequence having a short free 3 'hydroxyl group and can form base pairs with a complementary template, It means a short sequence functioning as a point. The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i.e., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. PCR amplification can be performed to predict skin type through the production of desired products. The PCR conditions, the lengths of the sense and antisense primers can be modified based on what is known in the art.

The probes or primers of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, "capping ", replacement of natural nucleotides with one or more homologues, and modifications between nucleotides, such as uncharged linkers, such as methylphosphonate, Phosphoamidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).

In another aspect, the present invention provides a skin type whitening diagnosis kit comprising the skin type whitening detection composition.

The kit may be an RT-PCR kit or a DNA chip kit.

The kit of the present invention can diagnose the skin type by confirming the SNP polymorphism marker which is a skin type diagnostic marker through amplification or by confirming the expression level of SNP polymorphism marker with the mRNA expression level. As a specific example, in the present invention, the kit for measuring the mRNA expression level of the skin type diagnostic marker may be a kit containing essential elements necessary for performing RT-PCR. In addition to the individual primer pairs specific for the genes of the skin type diagnostic marker, the RT-PCR kit also includes a test tube or other appropriate container, reaction buffer (pH and magnesium concentrations vary), deoxynucleotides (dNTPs) , Enzymes such as Taq polymerase and reverse transcriptase, DNase, RNAse inhibitors, DEPC-water, sterile water, and the like. It may also contain a primer pair specific for the gene used as a quantitative control. Also preferably, the kit of the present invention may be a skin type diagnostic kit containing essential elements necessary for carrying out a DNA chip. DNA chip kits are those in which nucleic acid species are attached in a gridded array on a generally flat solid support plate, typically a glass surface not larger than a slide for a microscope, and nucleic acids are uniformly arranged on the chip surface, Hybridization reaction occurs between the nucleic acid on the surface and the complementary nucleic acid contained in the solution treated on the surface of the chip to enable a mass parallel analysis.

In another aspect, the present invention provides a skin-type whitening-detecting microarray comprising a polynucleotide of the skin-type whitening diagnostic marker.

The microarray may comprise DNA or RNA polynucleotides. The microarray comprises a conventional microarray except that the polynucleotide of the present invention is contained in the probe polynucleotide.

Methods for producing microarrays by immobilizing probe polynucleotides on a substrate are well known in the art. The probe polynucleotide means a polynucleotide capable of hybridizing, and means an oligonucleotide capable of binding to the complementary strand of the nucleic acid in a sequence-specific manner. The probe of the present invention is an allele-specific probe in which a polymorphic site exists in a nucleic acid fragment derived from two members of the same species and hybridizes to a DNA fragment derived from one member but does not hybridize to a fragment derived from another member . In this case, the hybridization conditions show a significant difference in the intensity of hybridization between alleles, and should be sufficiently stringent to hybridize to only one of the alleles. This can lead to good hybridization differences between different allelic forms. The probe of the present invention can detect an allele and can be used for diagnosis of skin type and the like. The diagnostic methods include detection methods based on hybridization of nucleic acids such as Southern blots, and may be provided in a form pre-bonded to a substrate of a DNA chip in a method using a DNA chip. The hybridization can usually be performed under stringent conditions, for example, a salt concentration of 1 M or less and a temperature of 25 ° C or higher. For example, conditions of 5x SSPE (750 mM NaCl, 50 mM Na Phosphate, 5 mM EDTA, pH 7.4) and 2530 [deg.] C may be suitable for allele-specific probe hybridization.

Immobilization on the substrate of the probe polynucleotide associated with the skin diagnosis of the present invention can also be easily made using this conventional technique. In addition, hybridization of nucleic acids on a microarray and detection of hybridization results are well known in the art. The detection can be accomplished, for example, by labeling the nucleic acid sample with a labeling substance capable of generating a detectable signal comprising a fluorescent material, such as Cy3 and Cy5, and then hybridizing on the microarray and generating The hybridization result can be detected.

In another aspect, the present invention provides a method for producing a DNA comprising: (a) obtaining DNA from a sample isolated from an individual; (b) amplifying the polymorphic site of the single nucleotide polymorphic marker from the DNA obtained in step (a) or hybridizing with the probe; And (c) identifying the base of the amplified or hybridized polymorphic site of step (b).

The term "individual" of the present invention means a subject for diagnosing whether or not the skin type is whitening. DNA can be obtained from the sample, such as hair, urine, blood, various body fluids, isolated tissues, isolated cells or saliva, but is not limited thereto.

The method of obtaining the genomic DNA of step (a) may be any method known to those skilled in the art.

The amplification of the polymorphic site of the single nucleotide polymorphic marker from the DNA obtained in step (a) of step (b) or hybridization with the probe may be performed by any method known to those skilled in the art. For example, the target nucleic acid can be obtained by PCR amplification and purification thereof. Other ligase chain reaction (LCR) (Wu and Wallace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077 (1988)), transcription amplification (Kwoh et al., Proc. Natl. Acad. Sequence amplification based on nucleic acids (NASBA) can be used as well as self-sustaining sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA 87, 1874 (1990)).

Determination of bases in the polymorphic site of step (c) of the above method can be performed by sequencing analysis, hybridization by microarray, allele specific PCR, dynamic allele-specifichybridization, DASH), PCR extension analysis, SSCP, PCR-RFLP analysis or TaqMan technique, SNPlex platform (Applied Biosystems), mass spectrometry (e.g., Sequenom's MassARRAY system), mini-sequencing method, Bio- But are not limited to, the BioRad system, the CEQ and SNPstream system (Beckman), the Molecular Inversion Probe array technology (e.g., Affymetrix GeneChip), and BeadArray Technologies (e.g. Illumina GoldenGate and Infinium assay). One or more alleles in a polymorphic marker, including microsatellite, SNP or other types of polymorphic markers, can be identified by such methods or by other methods available to those skilled in the art to which this invention pertains. The base of such a polymorphic site can be determined preferably through a SNP chip.

The term "SNP chip" in the present invention means one of DNA microarrays capable of confirming each base of several hundred thousand SNPs at a time.

The TaqMan method comprises the steps of: (1) designing and constructing a primer and a TaqMan probe to amplify a desired DNA fragment; (2) labeling probes of different alleles with FAM dyes and VIC dyes (Applied Biosystems); (3) performing PCR using the DNA as a template and using the primer and the probe; (4) after completion of the PCR reaction, analyzing and confirming the TaqMan assay plate with a nucleic acid analyzer; And (5) determining the genotype of the polynucleotides of step (1) from the analysis results.

In the above, the sequencing analysis can be performed using a conventional method for determining the nucleotide sequence, and can be performed using an automated gene analyzer. The allele-specific PCR means a PCR method in which a DNA fragment in which the SNP is located is amplified with a primer set including a primer designed with the base at the 3 'end at which the SNP is located. The principle of the above method is that, for example, when a specific base is substituted by A to G, an opposite primer capable of amplifying a primer containing the A as a 3 'terminal base and a DNA fragment of an appropriate size is designed, In the case where the base at the SNP position is A, the amplification reaction is normally performed and a band at a desired position is observed. When the base is substituted with G, the primer can be complementarily bound to the template DNA, And the amplification reaction is not performed properly due to the inability of complementary binding at the terminal. DASH can be performed by a conventional method, preferably by a method such as Prince et al.

On the other hand, in the PCR extension analysis, first, a DNA fragment containing a base in which a single base polymorphism is located is amplified by a pair of primers, and all nucleotides added to the reaction are deactivated by dephosphorylation, and SNP- specific extension primers, a dNTP mixture, a digoxin nucleotide, a reaction buffer, and a DNA polymerase to perform a primer extension reaction. At this time, the extension primer has a base immediately adjacent to the 5 'direction of the base in which the SNP is located at the 3' terminus, and the nucleic acid having the same base as the dodecoxynucleotide is excluded in the dNTP mixture, and the dodecoxynucleotide indicates the SNP Base type. For example, when dGTP, dCTP and TTP mixture and ddATP are added to the reaction in the presence of substitution from A to G, the primer is extended by the DNA polymerase in the base in which the substitution has occurred, The primer extension reaction is terminated by ddATP at the position where the base first appears. If the substitution has not occurred, the extension reaction is terminated at the position, so that it is possible to discriminate the type of the base representing the SNP by comparing the lengths of the extended primers.

As the detection method, when the extension primer or the dioxynucleotide is fluorescently labeled, the SNP is detected by detecting fluorescence using a gene analyzer (for example, Model 3700 of ABI Co., Ltd.) used for general nucleotide sequence determination And when the unlabeled extension primer and the didyxin nucleotide are used, the SNP can be detected by measuring the molecular weight using MALDI-TOF (matrix assisted laser desorption ionization-time of flight) technique.

Preferably, the method of providing information of the present invention comprises the steps of: (d) when the base of the amplified or hybridized polymorphic site is a minor allele, The frequency of the frequency of the group is judged to be the skin of the higher group. If the base of the amplified or hybridized polymorphic region is a major allele, the control group and the whitened skin shown in Table 3 and Table 4 And judging the skin of the group having the lower frequency frequency frequency of the test group as the skin of the lower frequency group.

The single nucleotide polymorphic marker of the present invention is a marker capable of diagnosing skin characteristics for each individual, and can give information on the accurate skin type to an individual, thus providing a customized cosmetic according to the skin characteristics. In addition, by classifying the skin characteristics of cosmetics consumers scientifically, it is possible to develop tailor-made effective ingredients according to individuals and to develop customized cosmetics according to skin characteristics through various product segmentation.

1 is a schematic diagram of gene analysis using the Axiom (TM) ASI array plate of the present invention.
FIG. 2 is a graph showing the log value of the p-value with respect to the frequency of each SNP marker for whitening and the chromosomal location of the SNP.
FIG. 3 is a diagram showing the structure of Versican and the relationship between neighboring molecules. FIG.
Figures 4a and 4b show the mechanism and pathway of vesican.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and the scope of the present invention is not construed as being limited by these embodiments.

Example 1: Characterization of Skin and Gene Collection

In order to establish skin classification standards for gene types and to develop a personalized active ingredient, the inventors of the present invention conducted experiments to evaluate skin whitening, which is an important factor of skin physiological factors, in order to classify the skin characteristics of healthy female subjects of 20 to 50 years old. 81 subjects who met the subject selection criteria of the present invention and 20 subjects who completed whitening skin classification in the dermatology department of Gyeongsang National University were selected from the basic data of the subjects possessed by the pros and then the skin characteristics were classified and samples were collected ). The sample means saliva or blood. For example, the saliva was collected twice, and the first saliva was collected after subjects were fasted one hour before collection, and the second saliva was obtained immediately after the morning wake.

The classification was carried out according to the following criteria.

○ Skin color (whitening) classification: Skin color was classified according to the minimum erythema (MED) as follows: MED 47 or higher was used as a control group, and MED 38 or lower was used as a test group.

Classification The control (control) The test case Skin color (whitening) MED 47 or higher MED 38 or less

If you are pregnant or breastfeeding, or if you plan to become pregnant within 6 months, (2) you have used steroid-containing skins for at least one month to treat skin conditions, (3) you have not taken more than six months ⑤ If you have sensitive or irritable skin, ⑤ If you have skin abnormalities such as dots, acne, erythema and capillary dilatation on the test site. ⑥ You can not apply the same or similar cosmetics or medicines ⑦ If you have a chronic wasting disease (asthma, diabetes, hypertension, etc.), ⑨ If you have atopic dermatitis, ⑩ Other cases In cases where it was judged by the examiner that the examination was difficult, the subjects were excluded.

Example 2: Analysis of genotypes according to skin characteristics

Genotyping of the skin type samples was performed using the Affymetrix Axiom (TM) ASI array plate.

In order to determine whether the skin type was whitened according to the criteria of Example 1, the skin type consisted of 40 persons in each of 20 test groups and 20 control groups, and 50% of the test group or control group was 40 And the remaining 50% was obtained from the blood of women in their twenties.

The saliva or blood of women in each group was extracted with QIAmp mini gDNA prep kit (QIAGEN, Korea) and OD260 / 280> 1.7, more than 10ng / ㎕, 1xTAE 1% agarose gel, After confirming the band, the genotype analysis experiment was carried out. Genomic assays using the Axiom ASI plate were performed in accordance with the manual. The reagents contained in the Axiom ASI plate kit were used for amplification of gDNA, DNA fragmentation, quality control, hybridization, staining, and scanning. The image of the chip was scanned using the GeneTitan MC (Affymetrix) system and the CeneChip Command Console Software (AGCC), and the scanned image dat file was automatically converted into a cel file by the AGCC. The cel file for each chip was subjected to data quality control and genotype call using genotyping console 4 program. A general schematic diagram of a method for gene analysis using the Affymetrix Axiom (TM) ASI array plate is shown in FIG.

A total of 40 genotyping chip tests were performed, and one sample in the test group failed to pass QC, resulting in no genotyping results. QC standards are passed if the DISHQC value of the Axiom Chip is greater than 0,82 and the genotype call rate is more than 97%.

Example 3: Investigation of genetic polymorphisms of skin whitening skin using SNP genotype analysis and screening of significant SNPs

In order to detect genotypes associated with skin type, association analysis using SNP markers of test group / control group was performed. For this, we used the PLINK 1.07 program and set MAF (Minor Allele Frequency)> 0.1 and Hardy-Weinberg Equilibrium (HWE)> 0.001 as QC parameters for each marker.

The test group / control group were compared, and the number of data used in the comparison was as follows (Table 2). The final major SNP markers predicted to be associated with the skin type selected only the Affymetrix SNP annotation associated with the skin type, with a significance level of the chi-square test for each allele of p <0.0001 or less, or p <0.01.

The test case The control (control) Number of SNPs Whitening 19 20 40

In addition, a significant level of chi-square test for whitening was selected to be p < 0.01.

In the association analysis for whitening, the logarithm of the p-value with respect to the frequency of each marker and the chromosomal location of the SNP are shown in FIG. As a result, SNP markers showing high significance existed on chromosomes 1, 2, 5 and 10, and in particular, it was confirmed that SNPs were present at a certain consecutive position on chromosome 10. A list of key SNP markers significantly associated with such sensitivity is shown in Table 3 below.

A total of seven SNPs showed high significance in the vicinity of chromosome 10, 54169046 ~ 54203366, mostly SNP markers existing between DKK1 and MLB2 genes. Interestingly, minor alleles were present at a high frequency in the control group, while they were detected at a relatively low frequency in the test group (Table 3) (Table 3) . Therefore, minor alleles of these SNP markers were predicted to be highly related to pigmentation.

Among them, the Versican (VCAN) gene showed the most significant results, especially Versican two SNPs. Versican gene SNPs rs1833890 and rs2652106 are located on chromosome 5 at 82834514 and 82829792, respectively.

The SNP marker list with p <0.01 significantly associated with whitening is shown in Table 4 below.

Claims (5)

A skin type whitening diagnosis composition comprising a probe capable of detecting a single nucleotide polymorphism (SNP) marker for skin type whitening diagnosis or an agent capable of amplifying,
The single nucleotide polymorphism marker for skin type whitening diagnosis is a polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 900218 base of human chromosome 20, C or T (rs2223962) Or a complementary polynucleotide thereof.
A skin type whitening diagnosis kit comprising the composition of claim 1.
A skin-type whitening diagnosis microarray comprising a single nucleotide polymorphism (SNP) marker for skin type whitening diagnosis,
The single nucleotide polymorphism marker for skin type whitening diagnosis is a polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 900218 base of human chromosome 20, C or T (rs2223962) Or a complementary polynucleotide of the same or a complementary polynucleotide of the skin type.
(a) obtaining DNA from a sample isolated from an individual;
(b) amplifying or hybridizing a polymorphic site of a single nucleotide polymorphism (SNP) marker for skin type whitening diagnosis from the DNA obtained in step (a)
The single nucleotide polymorphism marker for skin type whitening diagnosis is a polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 900218 base of human chromosome 20, C or T (rs2223962) Or a complementary polynucleotide thereof; And
(c) identifying the base of the amplified or hybridized polymorphic site of step (b).
The method according to claim 4, wherein (d) the base of the amplified or hybridized polymorphic site is T, and the base of the amplified or hybridized polymorphism region is C, Wherein the skin type is a skin type.

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