GENETIC POLYMORPHIC MARKER FOR TYPE OF WRINKLED SKIN, AND USE THEREOF

For in one aspect the order to achieve this object, the invention relates to a predetermined region of the skin (SNP) markers selected from 1 single nucleotide sequence comprising at least one single base polymorphic marker provides a marker for the diagnosis of wrinkle skin type.

In the present invention terms, "polymorphisms (polymorphism)" RM one gene allele (locus) are printed allele (allele) in areas it is if there is a, person only single base along the others (single nucleotide polymorphism, SNP) nucleotide sequence is combined with a load. Polymorphic marker that preferably selected population 1% or more, 10% or more preferably 20% or more indicative of the frequency of occurrence of one or more alleles have.

In the present invention terms, "allele (allele)" is the same gene to chromosome homologous to bring the various types of the genes set forth above left said substrate. The wherein the polymorphisms alleles can be used to indicate, for example, is an allele of a SNP two factor (biallele).

In the present invention terms, provide the means by which information recording year 1998 "rs_id" SNP from percent of the NCBI SNP that it initially all be altered independent rs a-ID a marking becoming a big. The table of the present invention is a SNP marker big rs_id polymorphic marker.

In the present invention terms, meaning that the skin type "skin type" RM to individuals having a measuring or diagnostic unit, SNP of the present invention measuring the skin type may include an unlimited number, be a preferred embodiments. The present inventors believe that according to an exemplary embodiment of the present invention is classified into his skin or corrugated many skin wrinkles type skin portion of a subject. Single base of the present invention measure an accurate skin type liquid jetted through a polymorphic marker, information about a change in the skin type active ingredient be skin in contact with the ceramic.

Table 3 and table 4 single base preferably polymorphic marker on the display is at least one selected among single base polymorphic marker 1 be a single base polymorphic marker. The table 3 and table 4 single base polymorphic marker displayed skin type can be judges whether the wrinkles.

Single base of the present invention determines the frequency of a polymorphic marker each markers was judged by measuring a number of diagnostic skin type. The significance of the 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 less than, or 0. 000000001 p-a value of less than one not limited to such as characterized p - value. Preferably p a-value is 0. 01 can be less than, more preferably p a-value is 0. 001 can be less than disclosed.

Table 3 and table 4 polymorphic marker display as a marker of the present invention, polymorphic marker of an individual when table 3 and table 4 represent areas of minority alleles (minor allele) display (control) showed many skin is skin wrinkles is corrugated and test group (case) be dissolved at high frequency is a frequency of skin and decides which of the group consisting of, when multiple alleles (major allele) table 3 and table 4 base areas displayed on the test group showed many skin is skin wrinkles is corrugated and is dissolved at low frequency marker that can be notified to a user be frequency group of skin are disclosed. For example, when the second base is 7 times of an individual chromosome 24551208 G set forth in table 3 herein are regulated is a higher frequency of minority alleles G is a lens-capturing skin of an individual in many wrinkles troops can be judged skin are disclosed. If multiple alleles of an individual chromosome 24551208 A 7 is set forth in table 3 when once microarray device includes a test group is a multi-axis frequency lower skin wrinkles are test troops can be judged or hypermetropia.

More preferably, the table 3 at second base is in human 7 chromosome 24551208 A or G (rs2711118), the second-containing 5 - 100 24551208 of continuous DNA sequences polynucleotides; second base is 5 times in human chromosome 76839279 A or G (rs4273585), the second-containing 5 - 100 76839279 of continuous DNA sequences polynucleotides; at second base is 1 in human chromosome 208672298 C or T (rs11119496), the second-containing 5 - 100 208672298 of continuous DNA sequences polynucleotides; second base is 20 times in human chromosome 52738724 T or C (rs6127255), the second-containing 5 - 100 52738724 of continuous DNA sequences polynucleotides; at second base is C or T (rs2158769) in human chromosome 27346024 7, the second-containing 5 - 100 27346024 of continuous DNA sequences polynucleotides; second base is 15 times in human chromosome 47954398 A or C (rs16963008), the second-containing 5 - 100 47954398 of continuous DNA sequences polynucleotides; second base is T or C in human chromosome 101951190 3 times (rs1048364), the second-containing 5 - 100 101951190 of continuous DNA sequences polynucleotides; second base is 3 times in human chromosome 101936968 G or A (rs931206), the second-containing 5 - 100 101936968 of continuous DNA sequences polynucleotides; second debt [...] T or C in human chromosome 106740691 13 times (rs9555341), the second-containing 5 - 100 106740691 of continuous DNA sequences polynucleotides; second base is 10 times in human chromosome 122472521 C or T (rs10886747), the second-containing 5 - 100 122472521 of continuous DNA sequences polynucleotides; second base is 4 times in human chromosome 177330499 G or T (rs4538426), the second-containing 5 - 100 177330499 of continuous DNA sequences polynucleotides; second base is 4 times in human chromosome 177322010 C or T (rs2170576), the second-containing 5 - 100 177322010 of continuous DNA sequences polynucleotides; at second base is G or A in human chromosome 51222720 12 (999 0000247999), the second-containing 5 - 100 51222720 of continuous DNA sequences polynucleotides; second base is 6 times in human chromosome 107123424 T or C (rs3747790), the second-containing 5 - 100 107123424 of continuous DNA sequences polynucleotides; at second base is T or G in 11 human chromosome 68751073 (rs7931342), the second-containing 5 - 100 68751073 of continuous DNA sequences polynucleotides; at human chromosome 199554152 C or T (rs916398) in second base is 1, the second-containing 5 - 100 199554152 of continuous DNA sequences polynucleotides; second base is 10 times in human chromosome 72820331 G or A (rs10999801), 5 - 100 continuous DNA sequences of the microarray [...] 72820331 polynucleotides; second base is 10 times in human chromosome 72805925 A or G (rs1417210), the second-containing 5 - 100 72805925 of continuous DNA sequences polynucleotides; and complementary polynucleotide sequence consisting of at least one substrate is selected from the group consisting of, there is provided a skin fold-skin type is determining whether cognitive or corrugated skin wrinkle skin type be a marker for the diagnosis.

In accordance with one embodiment of the present invention, skin wrinkles on many people and the skin wrinkles on human saliva is aimed gene of table 3 using statistically significant delay of SNP 18 during one of ABI gene family member 3 is adaptor protein family, involved in oligonucleotides (cell motility) and mobile control (migration regulation) is known. This SNP is phenomena when seen through the viewer appears much wrinkles in assays, high mobility associated with oligonucleotides and pleated egcg appear to substrate. (TRK-a fused gene) TFG does not well known, but which interact with PLSCR 1 is known. Recent studies report that it is involved in TFG NF-a kappa B path (pathway) flow tides. PLSCR 1 polymerase 1 (phospholipid scramblase 1) into phospholipid scrambling, scramble proposal TFG executed the role of indirectly can be converted. (Phospholipid translocation) to plasma membrane phospholipid scrambling sacrifice pattern to proteins that are involved, mitochondrial metabolism, lipid metabolism, thrombosis (thrombosis), and apoptosis involved in substrate. Skin wrinkles is highly interacting TFG sacrifice and scrambling up to automatically control the level of apoptosis is through a skin wrinkle when cells can affect the object mask substrate.

The present inventors believe that the SNP is confirmed through the following demonstrated that the marker for the diagnosis of skin type. Specifically, the cosmetic skin wrinkle evaluation method (reference KFDA) (visual assessment; VA) functional visual evaluation criteria based on wrinkles VA grade 8 or more of the many controls (control), VA grade 6 test group (case) was classified into small wrinkles or less. The classified from multiple individuals gDNA saliva or from blood extracts, allelotyping experiments (TM) conducting to Axiom ASI array plate (also 1). As a result, wrinkles SNP (table 3 and table 4) was significantly different skin type with classifying. The SNP is a fold-skin type classifying whether the character for the first time by the inventors are disclosed.

In another aspect, the present invention relates to detect such markers for the diagnosis of the wrinkles of skin type can be a probe or amplifying comprising an, compositions for the diagnosis of the wrinkles of skin type.

In the present invention terms, "detect such markers for the diagnosis of the wrinkles of skin type probe" be the genetic areas specifically hybridization reactions by selecting a skin type means a composition for diagnosing whether wrinkles and, the genetic analysis of the specific method involves the special spraying is, known to the invention is provided to all gene detection method can be.

In the present invention terms, "marker for the diagnosis of the wrinkles of skin type capable of amplifying" areas such as gene amplification is the skin type by selecting a diagnosis whether wrinkles which means a composition, preferably the skin type which enables to specifically amplify a polynucleotide of marker for the diagnosis of the wrinkles of big.

The antibody amplification the polymorphic marker, of suitable buffer under suitable conditions (for example, 4 different nucleoside triphosphate and DNA, RNA polymerase or reverse transcriptase such as a first) and suitable temperature and mold - DNA synthesis of single stranded oligonucleotides can act starting point indicating said substrate. The primers suitable length depending on the intended use but, typically 15 to 30 nucleotide sequence disclosed. The mold and the short primer molecules generally require lower temperatures in order to form a stable hybrid. The mold and the primer sequence is completely complementary but are not necessarily, the mold and the complementary hybridization enough to S7 substrate.

In the present invention terms, "primer" have short free 3 'end hydroxyl radicals (free 3' hydroxyl group) having complementary base pair sequence template (template) (base pair) to form nascent strand radiation can be short sequences by big starting point function. Appropriate buffer solution at temperature and polymerization primer (i.e., DNA polymer produced or reverse transcriptase) 4 for DNA synthesis in the presence of nucleoside triphosphate is different reagents and initiate disclosed. PCR amplification is performed through skin type can be predicting whether generation of desired product. PCR conditions, based on the length of the antisense primer into a sense can be known.

gun [lu[lu] Oh this [thu[thu] which it gets torn to quickly reduce the probes or primers of the present invention solid support method, or other methods can be chemically synthesized using known. Such nucleic acid sequences also relates to transformed capable of many known means. These modified non - limiting examples include methylation, "cap and", to one or more natural nucleotide analogs substituted, modified between and nucleotide, for example, charged connecting body (example: methyl phosphonate, phosphate gun tree ester, esters capable of inhibiting the formation, such as carbamate) or charged connecting body (example: phosphorothioate, such as phosphor with D thio polycarbonate) changing to 2000.

In another aspect, the invention relates to a composition for the diagnosis of the wrinkles of skin type provides a kit for the diagnosis of the wrinkles of skin type.

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

A kit of the invention is for diagnosing skin type SNP marker for identifying or through amplification polymorphic marker, SNP polymorphic marker identifying the expression level of the mRNA expression levels of diagnosis of skin type can be. Specific as an example, mRNA expression levels of marker for diagnosing skin type in the present invention for measuring kit for performing RT-a PCR kit comprising the required essential element can be. RT-a PCR kit, each specific gene marker for the diagnosis of skin type in addition RT-a PCR test tubes or other suitable primer kit container, reaction buffer (pH various and magnesium concentration), the jade city new the [ley[ley] five tie [tu[tu] which will grow (dNTPs), such as Taq - obesity and reverse it buys the enzyme and enzyme, DNase, RNAse inhibitors, DEPC - be (DEPC-a water), sterilization can be like. Primer pairs specific for quantitative controls also can be used. Also preferably, a kit of the invention the DNA chip to perform requisite element be a kit for diagnosing skin type. DNA chip kit, generally flat solid support plate, typically a microscope slide is not greater than a nucleic acid species lattice-like arrangement (gridded array) that is attached to a glass surface, a nucleic acid chip surface arranged constant, DNA chip on the chip surface treated nucleic acid complementary nucleic acids contained in multiple hybridization (hybridization) tank prior to massively parallel analysis tool are disclosed.

In another aspect, the invention relates to a marker for the diagnosis of the wrinkles of skin type comprising a polynucleotide of the wrinkles of skin type provides a microarray for the diagnosis.

DNA or RNA polynucleotide can be a micro-array. The microarray probe polynucleotides having comprising a polynucleotide of the present invention except except consisting of conventional microarray.

The method of making the probe polynucleotide immobilized on a microarray substrate contrast well known. The probe hybridization to a polynucleotide containing a polynucleotide includes, complementary strand nucleic acid capable of binding to sequence-specific oligonucleotide big. The probes of the allele specific probes, such as species derived from nucleic acid fragment areas with the presence of two members, one derived from DNA fragments is to modulate the hybridization, hybridization is not only derived from other members. In this case hybridization condition allows show significant difference in hybridization intensity between alleles, alleles sufficiently stringent hybridization only one of accomplishing. The difference between induced by other alleles can form good hybridization. Probe of the present invention can be used to detect alleles that diagnostic method skin type. The diagnostic process unit such as nucleic acid hybridization [pul[pul] line based on detection included, DNA chip in one method DNA chip which was previously provided in the form of the substrate may be filled. The hybrid microbial rigorous conditions, for example 1M salt concentrations of less than 25 °C and usually can be performed under a temperature of at least. For example, 5x SSPE (750 mm NaCl, 50 mm Na Phosphate, 5 mm EDTA, pH 7. 4) and 2530 °C allele specific probe hybridization condition can be suitable.

For immobilizing polynucleotide probe associated with skin diagnosis of the present invention immobilized on a substrate using such known also process can be readily prepared. Also, the detection of nucleic acid hybridization and contrast on microarray hybridization result well known. Said detection comprises for example, nucleic acid sample of the phosphor for example Cy3 and Cy5 detectable signal such as material capable of generating an indicator material then labeling, microarray hybridization on and the marker material from hybridization by a detection can detect the signals.

In another aspect, the invention relates to (a) obtain an isolated DNA from multiple individuals from a sample; (b) in the (a) amplifying DNA obtained from the hybridization probe and the polymorphic marker or single base areas; and (c) the step (b) the amplified or hybridize confirming base polymorphic site comprising, there is a method of providing information of the wrinkles of skin type.

Terms of the present invention, provide the means by which "individual" fold-skin type whether big subject for diagnosis. The kit for a hair in, urine, blood, various body fluids, isolated tissue, such as from a DNA sample to obtain isolated cells or with saliva but, are not limited to are not correct.

(A) a method for obtaining the DNA genome of step known to one skilled in certain soil or rock is available disclosed.

(B) obtained in step (a) the DNA amplification or hybridization probe areas from the single base polymorphic marker includes any method known to one skilled depending on available disclosed. For example, target nucleic acids can be achieved by amplifying purifying through PCR. Other ligase chain reactions (LCR) (Wu and Wallace, Genomics 4, 560 (1989), such as Landegren, Science 241, 1077 (1988)), transcription amplification (transcription amplification) (such as Kwoh, Proc. Natl. Acad. Sci. USA 86, 1173 (1989)) and self maintaining sequence replication (like Guatelli, Proc. Natl. Acad. Sci. USA 87, 1874 (1990)) based on sequence amplification (NASBA) nucleic acids can be used.

Step (c) comprises determining the base density and areas of sequencing analysis, microarray (microarray) by hybridization, allele specific PCR (allele specific PCR), hybridization techniques (dynamic allele-a specifichybridization, DASH) dynamic alleles, extending PCR analysis, SSCP, TaqMan PCR-a RFLP analysis or techniques, SNPlex platform (Applied Biosystems), mass spectrometry (e.g., of Sequenom MassARRAY system), mini - sequencing (mini-a sequencing) method, system (BioRad) Bio-a Plex, CEQ and SNPstream system (Beckman), Molecular Inversion Probe array technology (for example, Affymetrix GeneChip), and BeadArray Technologies (for example, Illumina GoldenGate and Infinium assays) include, not limited. The method or this invention is in the field by to other methods available to those skilled in the, a sequence consisting, including SNP or other polymorphic marker, one or more polymorphic marker alleles can be identified. The base areas determining is preferably via SNP chip can be.

In the present invention terms, "SNP chip" is a fixing device capable of confirmation once each SNP base DNA microarray one big.

TaqMan method (1) desired DNA fragments to the amplification primers and TaqMan probe design and fabrication; (2) different alleles (Applied Biosystems) labeled with dye VIC dye and FAM probes according steps; (3) the DNA and IIS, performing PCR using the above primers and probe; (4) of the PCR reaction is completed after, nucleic acid analyzer analysis and verifying the TaqMan analysis plate; and (5) the analysis result from step (1) poly new the [ley[ley] five mote which will grow determining genotype of comprising the following steps.

In formula, sequencing analysis using base sequencing can be conventional, automated gene can be performed using a fluorometer. Also, SNP allele specific PCR is located on base 3' distally generate smoke primer DNA fragments to the primer set PCR method for amplifying an SNP located in big. The method principles of, e.g., when a specific base is substituted in G A, the A a 3 'end primers and of the relevant size DNA fragments comprising a base in opposite directions towards the heat pipes when the reaction takes place in the PCR amplification primer, the strong base is normally performed when the SNP position A is amplification reactions of band is desired location can be observed, but if the base is substituted with G DNA primer complementary mold capable of binding, 3' end towards complementary binding is not inputting using the fact by amplification reactions are disclosed. Performs in a manner that detects a variation of DASH can be, preferably [phu[phu] phosphorus [su[su] etc. can be performed by method.

On the other hand, PCR DNA-containing single base polymorphisms located extending analysis first fragment amplification primer pairs then, all nucleotide added to the deprotection reaction with inactivated by phosphorylation, specific SNP herein extending primer, dNTP mixture, FDD oxy nucleotide, DNA polymerase reaction buffer solution and added to a primer extension reaction by performing combustion chamber. The, base extending primer comprises a SNP located 5 'direction immediately adjacent the base 3' and three of the tail end, and having the same nucleic acid base except dNTP mixture contains error oxy polynucleotides and polypeptides, polynucleotides may be selected in one base representing the FDD oxy SNP types. For example, when in G to A replacement, dGTP, dCTP and TTP ddATP when added to the reaction mixture, the base being extended by DNA polymerase in primer comprises a substituted occurs and, after several base is shown in position by primer extension reaction becomes ddATP A base end through the body. If the replacement does not occur, the location extending reaction terminates, the extended primer length of SNP by comparing representing base for identifying the type is equal to or higher.

The, general methods for detecting fluorescent labeling polynucleotide primer or extending a base sequence used in a determination if FDD oxy gene analyzer (e.g., such as ABI yarn Model 3700) by detecting the SNP using fluorescence detection can be, - free labeled extension primers and FDD oxy polynucleotide is fixed (matrix assisted laser desorption ionization-a time of flight) MALDI-a TOF estimating method by measuring the molecular weight can be detecting the SNP.

Preferably, the method for providing an information (d) polymorphic site amplified or hybridize when table 3 and table 4 represent minority alleles (minor allele) is corrugated and is displayed on the test group showed many skin wrinkles skin is skin be dissolved at high frequency and decides which of the group consisting of frequency, amplified or hybridize polymorphic alleles (major allele) table 3 and table 4 multiple base sites when the tube is corrugated and display many skin is skin wrinkles is a test group showed low frequency be in water and further comprising determining a frequency group of skin may be disclosed.