KR20120119849A - Snp genotyping assay set for apoe genes and method of detecting apoe using the same - Google Patents

Abstract

PURPOSE: A single nucleotide polymorphism analyzing of genotype for ApoE gene and a distinguishing method using the same are provided to be used for preventing and treating cardiovascular disease or Alzheimer disease. CONSTITUTION: A distinguishing method of single nucleotide polymorphism using ApoE gene comprises the following steps: isolating genomic DNA from a sample of test object; detecting the polymorphic site existed in the isolated genomic DNA by using a kit including a primer set, and a probe; and comparing the existence of the detected polymorphic sites. The primer set for detecting the single nucleotide polymorphism detects SNP(single nucleotide polymorphism) inside the ApoE(apolipoprotein E) gene. The primer complementarily unites with a codon which codes the 112th or 158th amino acid inside the ApoE gene. The ApoE gene is a gene consisting of the sequence number 1(SEQ ID NO:1). [Reference numerals] (AA) Set 1; (BB) Set 2; (CC) Set 3; (DD) Set 4; (EE) Set 5; (FF) Set 6

Description

SNP genotyping assay set for ApoE genes and method of detecting ApoE using the same}

The present invention relates to a single nucleotide polymorphism genotype analysis for the ApoE gene and a method for identifying the same.

ApoE (apolipoprotein E) genes have been linked to apo C-I, C-II and LDL receptor genes, and polymorphisms have been shown to be associated with the development of several diseases such as hyperlipidemia and dementia.

Molecular mediators play a role in the synthesis of β-Amyloid protein in patients with Alzheimer's disease. ApoE e4 is thought to have strong binding mediation properties. Accelerating the formation of neuronal fiber bundles has also been reported.

The human ApoE gene is located in the long arm of chromosome 19 and there are three alleles 2, 3, and 4 isotypes E2, E3, and E4, which are distinguished by the difference between the 112th and 158th amino acids. The combination of these three alleles results in six different genotypes of the ApoE gene polymorphism of Table 1, which is associated with the risk of cardiovascular disease and Alzheimer's disease.

Among them, ApoE4 protein is known as an important risk factor for Alzheimer's disease and coronary artery disease, whereas ApoE2 protein has a prophylactic effect on Alzheimer's disease, whereas most patients with type III hypercholesterolemia have been reported as homozygotes of ApoE2. Therefore, the polymorphism of the ApoE gene has important implications for cardiovascular disease and dementia, and has been recognized as an important test not only for patients but also for healthy people.

On the other hand, SNPs for detecting single nucleotide polymorphism, which are widely used in molecular genetics, include sequencing, PCR-SSCP (Polymerase chain reaction-Single stranded conformation polymorphism), allele specific hybridization, and ollie. There are oligo-ligation, mini-sequencing and enzymatic cleavage. A method using a DNA chip has also been introduced, which is in principle no different from an allele specific hybridization, but differentiated from a stationary phase to which an oligonucleotide probe is attached.

The sequencing method includes the Macsam-Gilbert method and the Sanger method, but the latter method is mainly used. This method is not only to investigate the genetic variation of a specific position, but to identify the nucleotide sequence of all or a part of the gene, but if the nucleotide sequence is revealed, it can be used for SNP scoring because it can also identify the genetic variation of a specific position. have. However, it is inefficient in that it reads not only the mutation of the SNP to be investigated but also reads the nucleotide sequence of the surrounding which does not need to be investigated.

PCR-SSCP (Orita, M. et.al, Genomics , 1989, 5: 8874-8879) amplifies the sequences containing the SNPs to be analyzed by PCR, separates them into their respective chains, and then polyacrylamide gels. Perform electrophoresis at. Since the secondary structure of the DNA chain is changed by one base difference, the base variation is examined according to the electrophoretic movement speed difference caused by the difference.

Allele specific hybridization is a method of hybridizing sample DNA labeled with a radioisotope to a probe attached to a nylon filter or the like, and examining whether or not there is a base mutation by controlling conditions such as temperature and hybridization.

Real-Time Polymerase Chain Reaction is a type of polymerase chain reaction method that checks amplification according to the intensity of a fluorescence signal using a primer and a probe.

Recently, the applicant also filed a patent in Korean Patent No. 2011-0004724, “Method for determining the single nucleotide polymorphism of a target gene through PCR”.

An object of the present invention is to provide a single nucleotide polymorphic U-type analysis of the ApoE gene and a method of discriminating the same using the same. More specifically, it is effective and reliable against single nucleotide polymorphism (SNP) in human apoE (apolipoprotein E) gene. To provide a method for determining the single nucleotide polymorphism of the high ApoE gene.

In order to achieve the above object, the present invention provides a primer set for single nucleotide polymorphism discrimination, which can detect single nucleotide polymorphism (SNP) in human apoE (apolipoprotein E) gene.

The present invention also provides a single nucleotide polymorphism determination probe capable of detecting single nucleotide polymorphism (SNP) in human apoE (apolipoprotein E) gene.

The present invention also provides a kit for determining the single nucleotide polymorphism of the human ApoE gene, comprising the primer set for determining the ApoE single nucleotide polymorphism, the probe for determining the ApoE single nucleotide polymorphism, or a mixed construct thereof.

The present invention also provides a method for determining the single nucleotide polymorphism of the human ApoE gene using the kit for determining the single nucleotide polymorphism of the human ApoE gene.

Hereinafter, the present invention will be described in detail.

The present invention provides a primer set for single nucleotide polymorphism determination, which can detect single nucleotide polymorphism (SNP) in human apoE (apolipoprotein E) gene.

In the present invention, the ApoE gene is characterized in that the gene described in SEQ ID NO: 1.

In the present invention, the primers are characterized by binding complementary to the codon encoding the 112th or 158th amino acid in the ApoE gene.

In the present invention, the primer set is characterized in that a mixture of two or more primers selected from the primers set forth in SEQ ID NO: 2 to 15, 18 and 19.

More specifically, the forward primer of the ApoE SNP 112 allele is the nucleotide sequence of SEQ ID NO: 2 to 7, the reverse primer is SEQ ID NO: 8, the forward primer of the ApoE SNP 158 allele is SEQ ID NO: 9 to 14, the reverse primer The base sequence of SEQ ID NO: 15, the internal positive control primer (Internal Positive Control), the forward primer is SEQ ID NO: 18, the reverse primer is the base sequence described in SEQ ID NO: 19, used by mixing the forward primer and the reverse primer do.

In the present invention, “primer” refers to a single stranded oligonucleotide sequence that is complementary to the nucleic acid strand to be copied and may serve as a starting point for the synthesis of the primer extension product. The length and sequence of the primer should allow to start the synthesis of the extension product. The specific length and sequence of the primers will depend on the primer utilization conditions such as temperature and ionic strength as well as the complexity of the DNA or RNA target required.

The present invention provides a single nucleotide polymorphism determination probe capable of detecting single nucleotide polymorphism (SNP) in human apoE (apolipoprotein E) gene.

In the present invention, the probe is characterized in that at least one selected from the probes set forth in SEQ ID NO: 16, 17 and 20.

The present invention provides a kit for determining single nucleotide polymorphism of a human ApoE gene, comprising the primer set for determining ApoE single nucleotide polymorphism, a probe for determining ApoE single nucleotide polymorphism, or a mixed construct thereof.

In the present invention, the mixed construct is characterized in that SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.

In the present invention, the mixed construct is characterized in that SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.

The kit may also include instructions. The guide is a printed document that explains how to use the kit, such as how to prepare reverse transcription buffer and PCR buffer, and the reaction conditions presented. The brochure includes instructions on the surface of the package including the brochure or leaflet in the form of a brochure, a label attached to the kit, and a kit. In addition, the guide includes information that is disclosed or provided through electronic media such as the Internet.

The present invention

1) separating genomic DNA from a sample to be tested;

2) detecting a polymorphic site present on the genomic DNA isolated in step 1) using a kit comprising the primer set for determining ApoE single nucleotide polymorphism, a probe for determining ApoE single nucleotide polymorphism or a mixed construct thereof; And

3) comparing the presence or absence of the polymorphic site detected in step 2);

It provides a single nucleotide polymorphism determination method of a human ApoE gene comprising a.

As a method for separating total DNA from a sample of the present invention, a method known in the art may be used, for example, a phenol extraction method may be used. Using the isolated whole DNA as a template, a target sequence may be amplified by real-time polymerase chain reaction using a primer set / probe according to an embodiment of the present invention. Such PCR methods are well known in the art, and commercially available kits may be used.

The real time polymerase chain reaction monitors the result of the reaction in real time by using the probe of the present invention in which the primer and the fluorescent material according to the present invention are chemically bonded. During the polymerase chain reaction, the probe binds to the complementary sequence in the nucleic acid of the sample, such as two forward primers and two reverse primers complementary to ApoE SNP 112 and ApoE SNP 158, and the position is slightly separated from the primer.

The probe of the present invention has a structure in which both a reporter and a quencher are attached to both ends, and when the reporter and the quencher are in close proximity, the probes cancel each other and the fluorescence of the reporter is not detected, but the amplification proceeds. Therefore, when the reporter is separated from the quencher, the reporter's fluorescence is detected. Thus, the intensity of fluorescence increases gradually as the amplification cycle increases. The difference between the detected values Ct of each increased fluorescence (ΔCt) distinguishes homo-type and hetero-type conjugates.

The primer / probe set for determining the single nucleotide polymorphism of the human ApoE gene according to the present invention can efficiently and accurately detect the single nucleotide polymorphism site of the human ApoE gene in a short time, thereby greatly contributing to the study of cardiovascular disease or Alzheimer's disease.

1 shows the nucleotide sequence of the ApoE gene according to the present invention,
2 is a result of performing a polymerase chain reaction of the DNA template of Example 1 using an ExiGenotyperTM 96 Real-Time Quantitative Thermal Block device using a single nucleotide polymorphism determination kit of a human ApoE gene according to the present invention.
3 is a result of performing a real-time polymerization chain reaction using a kit for determining a single nucleotide polymorphism of a human ApoE gene according to the present invention for a clinical sample (E2 / E4 type) in which a single nucleotide polymorphism is verified by sequencing. ego,
FIG. 4 is a graph showing 44 conditions of ApoE clinical specimens verified by sequencing method, and the detection conditions of homozygotes and heterozygotes of ApoE SNP 112 and ApoE SNP 158 to obtain optimal sensitivity and specificity.
5 is a single nucleotide polymorphism determination result for the ApoE gene using the single nucleotide polymorphism determination method of the human ApoE gene according to the present invention,
(A: Polymerization chain reaction result using ExiGenotyperTM 96 Real-Time Quantitative Thermal block device, B: Electrophoresis result, C: Discrimination result)
6 is a program flow diagram for automatically determining ApoE genotyping,
7 is a test result for ApoE type specimens verified by sequencing,
(A: E2 / E2, B: E2 / E3)
8 is a test result for ApoE type specimens verified by sequencing.
(A: E2 / E4, B: E3 / E3)
9 is a test result for ApoE-type specimens verified by sequencing.
(A: E3 / E4, B: E4 / E4)

The present invention will be described in more detail with reference to the following examples. However, the following examples are provided to aid understanding of the present invention, and the scope of the present invention is not limited by these examples in any sense.

Hereinafter, the technical and scientific terms used herein will be understood by those skilled in the art without departing from the scope of the present invention. Descriptions of known functions and configurations that may be unnecessarily blurred are omitted.

Example 1 Template DNA Preparation

In order to perform real-time polymerase chain reaction, template DNA was first prepared. Specifically, ApoE SNP 112 and ApoE SNP 158 single, codons encoding the 318-933 bp (630 bp) portion of human ApoE ((NC_000019.9: SEQ ID NO: 1), specifically the 112th or 158th amino acid in the ApoE gene. Template DNA and internal positive control (IPC) (NM_002046.3) 659 to 1019 bp (361 bp) portions, including base polymorphism, each having a single nucleotide polymorphism were synthesized by the method of gene synthesis (NBiochem. Biophys. Res. Commun. 1998). , 248, 200-203) and some of them were cloned into pGEM-T-Easy Vector (Cat: A1360, Promega, USA), specifically 5 μl of 2 × rapid ligation buffer (Promega, USA). 1 μl of T-Easy Vector (Promega, USA), 1 μl of T4 DNA ligase (manufactured by Promega, USA), 3 μl of synthesized gene (8 ng) were mixed in the same tube, and then allowed to stand at 37 ° C. for 1 hour.

Subsequently, 5 μl of the reaction solution placed at 50 ° C. in 50 μl of E. coli pluripotent cells (competent cells) was placed on ice for 40 minutes, incubated at 42 ° C. for 40 seconds, and then placed on ice for 2 minutes. The reaction solution was inoculated on an LB plate containing ampicillin, IPTG, and X-Gal and cultured at 37 DEG C for 16 hours. Color colonies were taken, incubated for 16 hours in LB liquid medium, centrifuged, and the supernatant was discarded. Plasmid DNA was extracted from the pellets using Accuprep plasmid DNA prep kit (Bioneer, Korea). Plasmid DNA was measured and measured using a UV spectrometer (Shimazu, Japan) and then confirmed that the purity is between 1.8 and 2.0.

6.02 × 10 ²³ × concentration (concentration g / ml measured by UV spectrometer) / (3015 + 630) × 660 [3015 bp: size of T-easy vector, 630 bp: size of Apolipoprotein E template DNA] Copy number of template DNA Calculate and dilute with 1 × TE buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) using ApoE SNP 112/158 template 4 × 10 ⁵ copy / reaction and IPC (GAPDH) 4 × 10 ⁵ copy / reaction, respectively. ApoE SNP 112 template and IPC template, ApoE SNP 158 template and IPC template were mixed at 1/2 and stored at -20 ° C until use.

[ Example  2] primer  And probe design

The bases of the human ApoE (NC_000019.9) of SEQ ID NO: 1 between 318 bp and 933 bp each contain a single base polymorphism, and the corresponding length is 20 to 22 mer, Tm value of 60 ℃ to 65 ℃ optionally base Sequences were selected to be forward and reverse primers.

In addition, the length was 23 mer, the Tm value was selected as a probe at random between 67 and 72 ℃ forward and reverse primers, and the Tm value was checked using the Primer3Plus program.

All primers / probes were prepared as in Table 2 below.

All the primers / probes described above were subjected to real-time polymerase chain reaction using ExiGenotyper ™ Quantitative Thermal Block (Bionia, Korea) by combining primer / probe sets having good PCR efficiency (Table 3, Table 4).

Specifically, 20 pmol of the forward primer and the reverse primer, 30 pmol of the probe, 5 μl of 10 × PCR Buffer, Taq polymerase 20 U, dNTP 20 mM, and DEPC (Diethylpyrocarbonate) were mixed and mixed to make 40 μl per well. Dispense into well plates. The template RNA used here was diluted in Example 1 and 5 μl of each ApoE SNP 112/158 template mixed with the IPC template was used so that the final total dose was 50 μl.

In addition, the ApoE SNP 112/158 probe was used for FAM fluorescence at the 5 ′ region and BHQ1 attached to the 3 ′ region, and the IPC probe was custom-made (Bionia Co., Ltd.) to request TAMRA-BHQ1.

After denaturation at 95 ° C. for 10 minutes, 45 cycles were reacted at 95 ° C. for 30 seconds and at 65 ° C. for 30 seconds. Specifically, a synthetic template and a clinical sample (E2 / E4 type), which were synthesized in Example 1 and mixed with ApoE SNP 112/158 template and IPC template, respectively, were selected for each ExiGenotyperTM Quantitative Thermal Block (FIGS. 2 and 3). ), One optimal ApoE SNP 112 set (Table 3, set 3) and one ApoE SNP 158 set (Table 4, Set 4) were selected for the optimal primer / probe set.

[ Example  3] Diagnostic test

The sensitivity and specificity of 44 clinical specimens were examined using the synthetic template and the optimal primer / probe (set 3, set 4) selected in Examples 1 and 2 as an ExiGenotyperTM Quantitative Thermal Block device. A reference value for 100% discrimination between the conjugate and the heterozygote was statistically obtained (FIG. 4), and based on this, an ApoE genotyping program was developed.

The results were compared with the existing commercially available ApoE genotyping products (Seegene, Korea), and the results were compared with those of the ApoE genotyping program developed by Bioneer.

As a result, as can be seen in Figure 5, the primer / probe set of Example 2 was able to efficiently detect the senile dementia-related single nucleotide polymorphism of the ApoE gene without limitation, it was confirmed that the reliability is also excellent.

[ Example  4] clinical specimen testing

A single base polymorphism of ApoE SNP 112 and ApoE SNP 158 was determined by sequencing using the primer / probe set (set 3, set 4) finally selected in Table 3 of Example 2, and 6 (E2) of ApoE / E2, E2 / E3, E3 / E3, E3 / E4, E4 / E4) were determined.

As a result of the test, homozygotes and heterozygotes were determined using the difference in cycle number (Ct) of fluorescence. Specifically, it was based on ΔCt ± 2. If the difference in the number of cycles of fluorescence is greater than ΔCt ± 2, the homozygosity is determined. If the difference in the number of cycles of fluorescence is less than ΔCt ± 2, the heterozygosity is determined. The homozygosity of the fluorescence detection signal that satisfies a value greater than Ct ± 2 and has a small number of cycles (Ct) of fluorescence was determined.

As can be seen from the results of FIG. 7, it was confirmed that the primer / probe set for determining the single nucleotide polymorphism of the human ApoE gene according to the present invention can efficiently and accurately detect the single nucleotide polymorphism site of the human ApoE gene in a short time. This is a result confirming that it can be useful for cardiovascular disease or Alzheimer's disease research.

<110> BIONEER CORPORATION <120> SNP genotyping assay set for ApoE genes and method of detecting          ApoE using the same <160> 20 <170> Kopatentin 1.71 <210> 1 <211> 3612 <212> DNA <213> Homo sapiens <220> <221> gene (222) (1) .. (3612) <223> ApoE gene <400> 1 gggatccttg agtcctactc agccccagcg gaggtgaagg acgtccttcc ccaggagccg 60 gtgagaagcg cagtcggggg cacggggatg agctcagggg cctctagaaa gagctgggac 120 cctgggaacc cctggcctcc aggtagtctc aggagagcta ctcggggtcg ggcttgggga 180 gaggaggagc gggggtgagg caagcagcag gggactggac ctgggaaggg ctgggcagca 240 gagacgaccc gacccgctag aaggtggggt ggggagagca gctggactgg gatgtaagcc 300 atagcaggac tccacgagtt gtcactatca tttatcgagc acctactggg tgtccccagt 360 gtcctcagat ctccataact ggggagccag gggcagcgac acggtagcta gccgtcgatt 420 ggagaacttt aaaatgagga ctgaattagc tcataaatgg aacacggcgc ttaactgtga 480 ggttggagct tagaatgtga agggagaatg aggaatgcga gactgggact gagatggaac 540 cggcggtggg gagggggtgg ggggatggaa tttgaacccc gggagaggaa gatggaattt 600 tctatggagg ccgacctggg gatggggaga taagagaaga ccaggaggga gttaaatagg 660 gaatgggttg ggggcggctt ggtaaatgtg ctgggattag gctgttgcag ataatgcaac 720 aaggcttgga aggctaacct ggggtgaggc cgggttgggg ccgggctggg ggtgggagga 780 gtcctcactg gcggttgatt gacagtttct ccttccccag actggccaat cacaggcagg 840 aagatgaagg ttctgtgggc tgcgttgctg gtcacattcc tggcaggtat gggggcgggg 900 cttgctcggt tccccccgct cctccccctc tcatcctcac ctcaacctcc tggccccatt 960 caggcagacc ctgggccccc tcttctgagg cttctgtgct gcttcctggc tctgaacagc 1020 gatttgacgc tctctgggcc tcggtttccc ccatccttga gataggagtt agaagttgtt 1080 ttgttgttgt tgtttgttgt tgttgttttg tttttttgag atgaagtctc gctctgtcgc 1140 ccaggctgga gtgcagtggc gggatctcgg ctcactgcaa gctccgcctc ccaggtccac 1200 gccattctcc tgcctcagcc tcccaagtag ctgggactac aggcacatgc caccacaccc 1260 gactaacttt tttgtatttt cagtagagac ggggtttcac catgttggcc aggctggtct 1320 ggaactcctg acctcaggtg atctgcccgt ttcgatctcc caaagtgctg ggattacagg 1380 cgtgagccac cgcacctggc tgggagttag aggtttctaa tgcattgcag gcagatagtg 1440 aataccagac acggggcagc tgtgatcttt attctccatc acccccacac agccctgcct 1500 ggggcacaca aggacactca atacatgctt ttccgctggg cgcggtggct cacccctgta 1560 atcccagcac tttgggaggc caaggtggga ggatcacttg agcccaggag ttcaacacca 1620 gcctgggcaa catagtgaga ccctgtctct actaaaaata caaaaattag ccaggcatgg 1680 tgccacacac ctgtgctctc agctactcag gaggctgagg caggaggatc gcttgagccc 1740 agaaggtcaa ggttgcagtg aaccatgttc aggccgctgc actccagcct gggtgacaga 1800 gcaagaccct gtttataaat acataatgct ttccaagtga ttaaaccgac tcccccctca 1860 ccctgcccac catggctcca aagaagcatt tgtggagcac cttctgtgtg cccctaggta 1920 ctagatgcct ggacggggtc agaaggaccc tgacccacct tgaacttgtt ccacacagga 1980 tgccaggcca aggtggagca agcggtggag acagagccgg agcccgagct gcgccagcag 2040 accgagtggc agagcggcca gcgctgggaa ctggcactgg gtcgcttttg ggattacctg 2100 cgctgggtgc agacactgtc tgagcaggtg caggaggagc tgctcagctc ccaggtcacc 2160 caggaactga ggtgagtgtc cccatcctgg cccttgaccc tcctggtggg cggctatacc 2220 tccccaggtc caggtttcat tctgcccctg tcgctaagtc ttggggggcc tgggtctctg 2280 ctggttctag cttcctcttc ccatttctga ctcctggctt tagctctctg gaattctctc 2340 tctcagcttt gtctctctct cttcccttct gactcagtct ctcacactcg tcctggctct 2400 gtctctgtcc ttccctagct cttttatata gagacagaga gatggggtct cactgtgttg 2460 cccaggctgg tcttgaactt ctgggctcaa gcgatcctcc cgcctcggcc tcccaaagtg 2520 ctgggattag aggcatgagc caccttgccc ggcctcctag ctccttcttc gtctctgcct 2580 ctgccctctg catctgctct ctgcatctgt ctctgtctcc ttctctcggc ctctgccccg 2640 ttccttctct ccctcttggg tctctctggc tcatccccat ctcgcccgcc ccatcccagc 2700 ccttctcccc gcctcccact gtgcgacacc ctcccgccct ctcggccgca gggcgctgat 2760 ggacgagacc atgaaggagt tgaaggccta caaatcggaa ctggaggaac aactgacccc 2820 ggtggcggag gagacgcggg cacggctgtc caaggagctg caggcggcgc aggcccggct 2880 gggcgcggac atggaggacg tgtgcggccg cctggtgcag taccgcggcg aggtgcaggc 2940 catgctcggc cagagcaccg aggagctgcg ggtgcgcctc gcctcccacc tgcgcaagct 3000 gcgtaagcgg ctcctccgcg atgccgatga cctgcagaag cgcctggcag tgtaccaggc 3060 cggggcccgc gagggcgccg agcgcggcct cagcgccatc cgcgagcgcc tggggcccct 3120 ggtggaacag ggccgcgtgc gggccgccac tgtgggctcc ctggccggcc agccgctaca 3180 ggagcgggcc caggcctggg gcgagcggct gcgcgcgcgg atggaggaga tgggcagccg 3240 gacccgcgac cgcctggacg aggtgaagga gcaggtggcg gaggtgcgcg ccaagctgga 3300 ggagcaggcc cagcagatac gcctgcaggc cgaggccttc caggcccgcc tcaagagctg 3360 gttcgagccc ctggtggaag acatgcagcg ccagtgggcc gggctggtgg agaaggtgca 3420 ggctgccgtg ggcaccagcg ccgcccctgt gcccagcgac aatcactgaa cgccgaagcc 3480 tgcagccatg cgaccccacg ccaccccgtg cctcctgcct ccgcgcagcc tgcagcggga 3540 gaccctgtcc ccgccccagc cgtcctcctg gggtggaccc tagtttaata aagattcacc 3600 aagtttcacg ca 3612 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ApoE 112C (112FCW-1) <400> 2 cgcggacatg gaggacgagc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ApoE 112C (112FCW-2) <400> 3 cgcggacatg gaggacgttc 20 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ApoE 112C (112FCW-3) <400> 4 ggcgcggaca tggaggacgt tc 22 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ApoE 112T (112FTW-1) <400> 5 atgccgatga cctgcagaat t 21 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ApoE 112T (112FTW-2) <400> 6 ggcgcggaca tggaggacgt tt 22 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ApoE 112T (112FTW-3) <400> 7 ggcgcggaca tggaggacgt tt 22 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ApoE 112 R (112 R) <400> 8 cagcttgcgc aggtgggag 19 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> ApoE 158C (158FCW-1) <400> 9 cgatgccgat gacctgcaga tgc 23 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ApoE 158C (158FCW-2) <400> 10 atgccgatga cctgcagaat c 21 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> ApoE 158C (158FCW-3) <400> 11 gcgatgccga tgacctgcag aatc 24 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> ApoE 158T (158FTW-1) <400> 12 cgatgccgat gacctgcaga tgt 23 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ApoE 158T (158FTW-2) <400> 13 atgccgatga cctgcagaat t 21 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> ApoE 158T (158FTW-3) <400> 14 gcgatgccga tgacctgcag aatt 24 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ApoE 158R (158R) <400> 15 cggctgccca tctcctccat 20 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> ApoE 112 probe (112Pr) <400> 16 ctggtgcagt accgcggcga ggt 23 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> ApoE 158 probe (158 Pr) <400> 17 cccgcacgcg gccctgttcc acc 23 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IPC (GF) <400> 18 tggcatggcc ttccgtgtcc 20 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> IPC (GR) <400> 19 cgacgcctgc ttcaccacct tc 22 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> IPC (GPr) <400> 20 cagcttgcgc aggtgggag 19

Claims (10)

A primer set for single nucleotide polymorphism discrimination which can detect single nucleotide polymorphism (SNP) in human apoE (apolipoprotein E) gene. The method of claim 1,
The primer is characterized in that complementary binding to the codon encoding the 112th or 158th amino acid in the ApoE gene, primer set for determining the single nucleotide polymorphism. 3. The method according to claim 1 or 2,
The ApoE gene is a set of primers for discriminating single nucleotide polymorphism, characterized in that the gene consisting of SEQ ID NO: 1. 3. The method according to claim 1 or 2,
The primer set is a primer set for determining a single nucleotide polymorphism, characterized in that a mixture of two or more primers selected from the primers set forth in SEQ ID NO: 2 to 15, 18 and 19. A single nucleotide polymorphism detection probe capable of detecting single nucleotide polymorphism (SNP) in human ApoE (apolipoprotein E) gene. 6. The method of claim 5,
The probe is a single nucleotide polymorphism determination probe, characterized in that at least one selected from the probes set forth in SEQ ID NO: 16, 17 and 20. A kit for determining a single nucleotide polymorphism of a human ApoE gene comprising a primer set according to claim 1, a probe according to claim 5, or a mixed construct thereof. 8. The method of claim 7,
Said mixed construct is SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 for determining the single nucleotide polymorphism of the human ApoE gene. 8. The method of claim 7,
Said mixed construct is SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 for determining the single nucleotide polymorphism of the human ApoE gene. 1) separating genomic DNA from a sample to be tested;
2) detecting a polymorphic site present on the genomic DNA isolated in step 1) using a kit comprising a primer set according to claim 1, a probe according to claim 4, or a mixed construct thereof; And
3) comparing the presence or absence of the polymorphic site detected in step 2);
Including, single nucleotide polymorphism determination method of human ApoE gene.

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