KR20210122498A - Promer for real-time detection for SNP analysis of ApoE gene and detection method using the same - Google Patents

Promer for real-time detection for SNP analysis of ApoE gene and detection method using the same Download PDF

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KR20210122498A
KR20210122498A KR1020200039708A KR20200039708A KR20210122498A KR 20210122498 A KR20210122498 A KR 20210122498A KR 1020200039708 A KR1020200039708 A KR 1020200039708A KR 20200039708 A KR20200039708 A KR 20200039708A KR 20210122498 A KR20210122498 A KR 20210122498A
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남영현
김남효
김현미
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Abstract

The present invention relates to a promer for real-time detection of single nucleotide polymorphism of apolipoprotein E (ApoE) gene and a detection method using the same, and more particularly, to a method for real-time detection of single nucleotide polymorphism of ApoE gene by using a promer which has a structure of X-Y-Z and is composed of a nucleotide sequence capable of complementary binding to some or all of the nucleotide sequence of ApoE gene, and a kit therefor. The method for detecting the SNP of the ApoE gene in real time by using a promer that is cleaved only by a cleavage reagent according to the present invention has the advantage of measuring more accurately than a conventional method for detecting genetic variations using a probe. In addition, the E2/E2, E3/E3, E4/E4, E2/E3, E2/E4, and E3/E4 phenotypes of the ApoE gene can be distinguished quickly, accurately and simply, and thus can be effectively used for diagnosis of various diseases caused by the ApoE genotype, such as Alzheimer's and cardiovascular disease, selection of therapeutic agents, and prognostic diagnosis.

Description

ＡｐｏＥ 유전자의 ＳＮＰ 분석을 위한 실시간 검출용 단일핵산 및 이를 이용한 검출 방법{Promer for real-time detection for SNP analysis of ApoE gene and detection method using the same}A single nucleic acid for real-time detection and a detection method using the same for SNP analysis of ApoE gene and detection method of ApoE gene and detection method using the same

본 발명은 ApoE(apolipoprotein E) 유전자의 SNP 분석을 위한 실시간 검출용 단일핵산 및 이를 이용한 검출 방법에 관한 것이다. 보다 상세하게는, X-Y-Z의 구조를 가지며, 112 및 158번 코돈에서 단일염기다형성을 보이는 ApoE 유전자의 염기서열 일부 또는 전체에 상보적 결합이 가능한 염기서열로 구성된 단일핵산을 사용하여 ApoE 유전자의 단일염기다형성을 실시간으로 검출하는 방법 및 이를 위한 키트에 관한 것이다.The present invention relates to a single nucleic acid for real-time detection for SNP analysis of apolipoprotein E (ApoE) gene and a detection method using the same. More specifically, a single nucleotide of the ApoE gene using a single nucleic acid comprising a nucleotide sequence capable of complementary binding to some or all of the nucleotide sequence of the ApoE gene having the structure of XYZ and showing single nucleotide polymorphism at codons 112 and 158 is used. It relates to a method for detecting polymorphisms in real time and a kit therefor.

유전자 변형에 있어 단일염기다형성(single nucleotide polymorphism; SNP)에 의한 변형은 가장 일반적인 형태이며, 다양한 질병을 유발시키는 원인으로 작용한다(Barreiro LB, et al., Methods Mol. Biol., 578:255-276, 2009; Beaudet L. et al., Genome Res., 11(4):600-608, 2001). 이에 유전자 변형에 의한 여러 질병들을 조기에 진단하기 위해 SNP 검출을 통한 진단 방법은 매우 효율적이며 빠른 진단을 가능하게 한다. 따라서 SNP를 정확하게 검출하기 위한 많은 방법들이 제시되어 왔으며, 현재에도 이와 관련된 많은 연구가 진행되고 있다(Ermini ML. et al., Biosen. & Bioele., 61:28-37, 2014; K. Chang et al., Biosen. & Bioele., 66:297-307, 2015).In genetic modification, modification by single nucleotide polymorphism (SNP) is the most common form and causes various diseases (Barreiro LB, et al ., Methods Mol. Biol. , 578:255- 276, 2009; Beaudet L. et al. , Genome Res. , 11(4):600-608, 2001). Therefore, in order to diagnose various diseases caused by genetic modification at an early stage, a diagnostic method through SNP detection enables a very efficient and rapid diagnosis. Therefore, many methods for accurately detecting SNPs have been proposed, and many studies related thereto are still in progress (Ermini ML. et al. , Biosen. & Bioele. , 61:28-37, 2014; K. Chang et al. al. , Biosen. & Bioele. , 66:297-307, 2015).

구체적으로, 다수의 유전자 분석을 위한 가장 보편적으로 사용하는 방법으로 중합효소 연쇄반응(polymerase chain reaction, PCR)을 이용하는 방법, 다중 중합효소 연쇄반응(multiplex polymerase chain reaction, Multiplex PCR) 방법이 있다.Specifically, there are a method using a polymerase chain reaction (PCR) and a multiplex polymerase chain reaction (multiplex PCR) method as the most commonly used method for analyzing a plurality of genes.

상기 중합효소 연쇄반응은 템플레이트(template) DNA와 결합할 수 있으며, 형광물질과 소광물질이 결합된 프라이머 또는 프로브를 임의로 설계함으로써 검출하고자 하는 유전자의 원하는 부위만을 정확하게 증폭할 수 있다는 장점이 있다. 그러나, 통상 한번의 반응으로 하나의 관심 유전자만을 증폭 및 분석할 수 있어 증폭하고자 하는 관심 유전자가 다수일 경우에는 동일한 작업을 반복해서 수행하여야 하는 번거로움이 있다.The polymerase chain reaction can bind to template DNA, and has the advantage of being able to accurately amplify only a desired region of a gene to be detected by arbitrarily designing a primer or probe in which a fluorescent material and a quencher are bonded. However, in general, since only one gene of interest can be amplified and analyzed in one reaction, when there are a large number of genes of interest to be amplified, it is inconvenient to repeatedly perform the same operation.

상기 다중 중합효소 연쇄반응은 여러 개의 중합효소 연쇄반응을 한 튜브에서 수행함으로써 다수의 유전자 영역을 동시에 분석할 수 있다는 장점이 있다. 그러나, 여러 개의 프라이머 또는 프로브를 한 튜브에서 동시에 사용함에 따라 프라이머 또는 프라이머들 간의 교차반응이 발생하게 되기 때문에 한 번에 증폭할 수 있는 유전자 영역의 수에는 한계가 있다. 또한, 반응 조건을 찾기 위한 많은 노력과 시간을 필요로 하고 민감도 및 특이도에서 좋은 결과를 얻을 수 없다는 단점이 있다(Hardenbol P. et al., Nat. Biotechnol., 21(6):673-678, 2003).The multiple polymerase chain reaction has the advantage that multiple gene regions can be analyzed simultaneously by performing multiple polymerase chain reactions in one tube. However, there is a limit to the number of gene regions that can be amplified at once because cross-reactions between primers or primers occur when several primers or probes are used simultaneously in one tube. In addition, it requires a lot of effort and time to find the reaction conditions, and there are disadvantages in that good results cannot be obtained in sensitivity and specificity (Hardenbol P. et al. , Nat. Biotechnol. , 21(6):673-678). , 2003).

최근에는 다중 중합효소 연쇄반응을 사용하지 않고 공통 프라이머를 사용하여 다수의 유전자 영역을 동시에 증폭하여 대량 분석을 가능하게 하는 연구가 활발히 이루어지고 있으며, 대표적인 기술로는 동시에 여러 유전자 영역의 단일염기다형성(SNP)을 분석할 수 있는 SNPlex, Goldengate assay, molecular inversion probes(MIPs) 등이 있다.Recently, studies that enable mass analysis by simultaneously amplifying multiple gene regions using common primers without using multiple polymerase chain reaction have been actively conducted. There are SNPlex, Goldengate assay, and molecular inversion probes (MIPs) that can analyze SNPs.

그러나, 상기 SNPlex, Goldengate assay, molecular inversion probes(MIPs) 등과 같은 방법들은 첫 번째 튜브에서 반응시킨 생성물의 일부를 두 번째 튜브로 옮겨 반응을 수행해야 하거나, 여러 종류의 효소를 이용하기 때문에 서로 다른 샘플들 간의 오염이 발생될 수 있으며, 실험 방법이 복잡한 문제점이 있다. 또한, 형광표지가 부착된 프로브의 개수만큼의 단일염기다형성만이 검출 가능하므로 분석하고자 하는 단일염기다형성의 개수가 증가할수록 분석 비용이 높아지는 문제가 있다.However, methods such as SNPlex, Goldengate assay, molecular inversion probes (MIPs), etc. have to transfer a part of the product reacted in the first tube to the second tube to perform the reaction, or use different types of enzymes. Contamination may occur between them, and there is a problem in that the experimental method is complicated. In addition, since only as many single nucleotide polymorphisms as the number of fluorescently labeled probes can be detected, there is a problem in that the analysis cost increases as the number of single nucleotide polymorphisms to be analyzed increases.

한편, ApoE(apolipoprotein E) 유전자는 apo C-I, C-Ⅱ 및 LDL 수용체 유전자와 연계되어 있으며, 유전자 다형성은 고지혈증 및 치매 등의 여러 질병의 발생과의 연관성이 밝혀져 있다.On the other hand, the apolipoprotein E (ApoE) gene is linked to apo C-I, C-II and LDL receptor genes, and gene polymorphisms have been found to be associated with the occurrence of various diseases such as hyperlipidemia and dementia.

알츠하이머 환자의 β-아밀로이드(β-Amyloid) 단백 합성과정에 분자적 매개 역할을 하며, ApoE e4의 경우 이런 결합 매개적 특성이 강할 것으로 추측되고 있으며, 또한 ApoE e4는 결합 형태의 차이로 인산화 과정에 변화를 초래하여 신경원 섬유뭉치의 형성 속도를 빠르게 하는 것 역시 보고된 바 있다.It plays a molecular mediating role in the synthesis of β-Amyloid protein in Alzheimer's patients, and it is assumed that ApoE e4 has strong binding-mediated properties. It has also been reported to increase the rate of formation of neurofibrillary tangles by causing changes.

인체의 ApoE 유전자는 19번 염색체의 장완에 위치하며 3가지 대립유전자 2, 3, 4의 동위형 E2, E3,E4가 존재하며 이들은 제112번째 및 158번째 아미노산이 달라짐으로 인해 구별된다. 이러한 3가지 대립유전자의 조합으로 6개의 다른 유전형으로 ApoE 유전자 다형성 (표 7 참조)이 이루어지는데, 그 다형성은 심혈관계질환 및 알츠하이머병의 발생위험과 관계가 있다.The human ApoE gene is located on the long arm of chromosome 19 and there are three alleles 2, 3, and 4 isoforms E2, E3, and E4, and they are distinguished by the difference in amino acids 112 and 158. The combination of these three alleles results in a polymorphism of the ApoE gene (see Table 7) in six different genotypes, which polymorphism is associated with the risk of cardiovascular disease and Alzheimer's disease.

그 중, ApoE4 단백질은 알츠하이머병과 관상동맥 질환의 중요한 위험인자로 알려져 있으며, ApoE2 단백질은 반대로 알츠하이머병에 예방적인 효과가 있는 반면 제Ⅲ형 고콜레스테롤혈증 환자의 대부분은 ApoE2의 동형접합체로 ApoE 유전자의 다형성은 심혈관계질환 및 치매에 있어 중요한 의미를 가지고 있어 환자 뿐 아니라 건강한 일반인에서도 중요한 검사로 인식되고 있다.Among them, ApoE4 protein is known as an important risk factor for Alzheimer's disease and coronary artery disease. ApoE2 protein, on the other hand, has a preventive effect on Alzheimer's disease, whereas most patients with type III hypercholesterolemia are homozygous for ApoE2, Polymorphism has an important meaning in cardiovascular disease and dementia, and is recognized as an important test not only in patients but also in healthy general population.

이에, 본 발명자들은 ApoE 유전자의 SNP에 의한 유전자 변형을 실시간으로 검출하는데 있어서 낮은 민감도와 특이도를 증가시켜 ApoE 유전자 변형으로 인해 유발되는 질환을 간단하고 정확하게 진단할 수 있는 방법을 오랫동안 연구하여 오던 중, 본 발명자들의 단일핵산을 활용할 경우 높은 민감도 및 높은 특이성을 나타내어 ApoE 유전자 변형에 의한 다양한 질환, 예컨대 알츠하이머 및 심혈관계 질환 진단에 매우 유용함을 발견하고, 본 발명을 완성하게 되었다.Accordingly, the present inventors have been researching for a long time a method for diagnosing diseases caused by ApoE gene modification simply and accurately by increasing low sensitivity and specificity in real-time detection of genetic modification by SNP of the ApoE gene. , The present inventors have found that the mononucleic acid of the present inventors exhibits high sensitivity and high specificity and is very useful in diagnosing various diseases caused by ApoE gene modification, such as Alzheimer's and cardiovascular diseases, and completed the present invention.

Barreiro LB, et al., Methods Mol. Biol., 578:255-276, 2009Barreiro LB, et al., Methods Mol. Biol., 578:255-276, 2009 Beaudet L. et al., Genome Res., 11(4):600-608, 2001Beaudet L. et al., Genome Res., 11(4):600-608, 2001 Ermini ML. et al., Biosen. & Bioele., 61:28-37, 2014Ermini ML. et al., Biosen. & Bioele., 61:28-37, 2014 K. Chang et al., Biosen. & Bioele., 66:297-307, 2015K. Chang et al., Biosen. & Bioele., 66:297-307, 2015 Hardenbol P. et al., Nat. Biotechnol., 21(6):673-678, 2003Hardenbol P. et al., Nat. Biotechnol., 21(6):673-678, 2003

본 발명의 하나의 목적은 ⅰ) X-Y-Z의 구조를 가지며, ⅱ) 단일염기다형성 부위를 포함한 ApoE 유전자의 염기서열 일부 또는 전체에 상보적 결합을 하고, ⅲ) 양 말단 또는 내부에 동일하거나 적어도 2개의 상이한 탐지 가능한 마커가 부착되어 있으며, 상기 Y는 ApoE 유전자에 위치하는 1개 또는 2개의 염기서열로 구성된 RNA인 것을 특징으로 하는, ApoE 유전자의 단일염기다형성 검출용 단일핵산을 제공하는 것이다.One object of the present invention is to i) have the structure of XYZ, ii) complementary binding to some or all of the nucleotide sequence of the ApoE gene including the single nucleotide polymorphism site, iii) the same or at least two at both ends or inside To provide a single nucleic acid for detecting single nucleotide polymorphisms in the ApoE gene, characterized in that different detectable markers are attached, and Y is RNA composed of one or two nucleotide sequences located in the ApoE gene.

본 발명의 다른 하나의 목적은 ApoE 유전자의 단일염기다형성(single nucleotide polymorphism; SNP) 확인을 위해 프로브(probe)로만 사용하는 경우 상기 단일핵산은 (a) 상기 X는 4 내지 20개의 염기서열로 구성되는 DNA이며, (b) 상기 Z는 1 내지 20개의 염기서열로 구성되는 DNA인 것을 특징으로 하는, ApoE 유전자의 단일염기다형성 검출용 단일핵산을 제공하는 것이다.Another object of the present invention is that when used only as a probe to confirm single nucleotide polymorphism (SNP) of the ApoE gene, the single nucleic acid is (a) the X is composed of 4 to 20 nucleotide sequences to provide a single nucleic acid for detecting single nucleotide polymorphisms in the ApoE gene, characterized in that the DNA is a DNA that becomes

본 발명의 또 다른 하나의 목적은 상기 단일핵산을 프라이머(primer) 및 프로브(probe)로 동시에 사용하는 경우 (c) 상기 X는 10 내지 30개의 염기서열로 구성되는 DNA이며, (d) 상기 Z는 1 내지 5개의 염기서열로 구성되는 DNA인 것을 특징으로 하는, ApoE 유전자의 단일염기다형성 검출용 단일핵산을 제공하는 것이다.Another object of the present invention is when the single nucleic acid is used as a primer and a probe at the same time (c) wherein X is a DNA consisting of 10 to 30 base sequences, (d) the Z is to provide a single nucleic acid for detecting single nucleotide polymorphisms of the ApoE gene, characterized in that the DNA is composed of 1 to 5 nucleotide sequences.

본 발명의 또 다른 하나의 목적은 상기 단일핵산을 포함하는, ApoE 유전자의 단일염기다형성 실시간 검출용 키트를 제공하는 것이다.Another object of the present invention is to provide a kit for real-time detection of a single nucleotide polymorphism of an ApoE gene, including the mononucleic acid.

본 발명의 또 다른 하나의 목적은 a) 생물학적 시료로부터 검출하고자 하는 ApoE 유전자의 단일염기다형성 부위를 포함하는 표적 핵산을 수득하는 단계; b) 상기 ApoE 유전자의 단일염기다형성 검출용 단일핵산을 제조하는 단계; c) 상기 단계 a)에서 수득한 표적 핵산, 상기 단계 b)에서 제조한 1형 단일핵산, ApoE 유전자 특이적 프라이머 세트, 및 절단시약과 혼합하거나 상기 단계 a)에서 수득한 표적 핵산, 상기 단계 b)에서 제조한 2형 단일핵산, 및 절단시약과 혼합한 후 신장반응을 통해 ApoE 유전자의 단일염기다형성 부위를 포함하는 표적 핵산-단일핵산 복합체를 증폭시키는 단계; 및 d) 상기 단계 c)에서 증폭된 ApoE 유전자의 단일염기다형성 부위를 포함하는 표적 핵산-단일핵산 복합체로부터 분리된 단일핵산 단편의 양을 측정하는 단계;를 포함하는, ApoE 유전자의 단일염기다형성 검출 방법을 제공하는 것이다.Another object of the present invention is to obtain a target nucleic acid comprising a single nucleotide polymorphism site of the ApoE gene to be detected from a) a biological sample; b) preparing a single nucleic acid for detecting a single nucleotide polymorphism of the ApoE gene; c) the target nucleic acid obtained in step a), the type 1 mononucleic acid prepared in step b), the ApoE gene-specific primer set, and the target nucleic acid obtained in step a) or mixed with the cleavage reagent, step b ) amplifying the target nucleic acid-mononucleic acid complex containing the mononucleotide polymorphism site of the ApoE gene through elongation after mixing with the type 2 mononucleic acid prepared in ) and a cleavage reagent; And d) measuring the amount of a single nucleic acid fragment isolated from the target nucleic acid-mononucleic acid complex comprising the mononucleotide polymorphism site of the ApoE gene amplified in step c); to provide a way

상기 목적을 달성하기 위해, 본 발명은 ApoE 유전자의 단일염기다형성 검출용 단일핵산(promer)을 제공한다.In order to achieve the above object, the present invention provides a single nucleic acid (promer) for detecting a single nucleotide polymorphism of the ApoE gene.

상세하게는, 상기 단일핵산은 ⅰ) X-Y-Z의 구조를 가지며, ⅱ) 단일염기다형성 부위를 포함한 ApoE 유전자의 염기서열 일부 또는 전체에 상보적 결합을 하고, ⅲ) 양 말단 또는 내부에 동일하거나 적어도 2개의 상이한 탐지 가능한 마커가 부착되어 있으며, 상기 Y는 단일 표적 유전자에 위치하는 1개 또는 2개의 염기서열로 구성된 RNA로서 ApoE 유전자와 혼성화시 절단 시약에 의해 절단된다.Specifically, the mononucleic acid is i) has the structure of XYZ, ii) complementarily binds to some or all of the nucleotide sequence of the ApoE gene including the single nucleotide polymorphism site, iii) the same or at least two at both ends or inside Two different detectable markers are attached, and the Y is RNA composed of one or two base sequences located in a single target gene, and is cleaved by a cleavage reagent when hybridizing with the ApoE gene.

이때, 상기 단일핵산이 ApoE 유전자의 단일염기다형성(single nucleotide polymorphism; SNP)을 확인하기 위한 프로브(probe)로만 사용하는 경우 (a) 상기 X는 4 내지 20개의 염기서열로 구성되는 DNA이며, (b) 상기 Z는 1 내지 20개의 염기서열로 구성되는 DNA인 것을 특징으로 하고, 상기 단일핵산이 ApoE 유전자의 단일염기다형성을 확인하기 위한 프라이머(primer) 및 프로브(probe)로 동시에 사용하는 경우 (c) 상기 X는 10 내지 30개의 염기서열로 구성되는 DNA이며, (d) 상기 Z는 1 내지 5개의 염기서열로 구성되는 DNA인 것을 특징으로 한다.In this case, when the single nucleic acid is used only as a probe for confirming single nucleotide polymorphism (SNP) of the ApoE gene (a), X is a DNA consisting of 4 to 20 nucleotide sequences, ( b) wherein Z is a DNA consisting of 1 to 20 nucleotide sequences, and when the single nucleic acid is used simultaneously as a primer and a probe for confirming the single nucleotide polymorphism of the ApoE gene ( c) wherein X is DNA composed of 10 to 30 nucleotide sequences, and (d) Z is DNA composed of 1 to 5 nucleotide sequences.

또한, 상기 단일핵산은 ApoE 유전자의 단일염기다형성을 확인하기 위해 프로브로만 사용될 경우에 ApoE 유전자와 혼성화된 후 절단 시약에 의해 상기 Y가 절단되는 경우 상기 X 및 Z도 ApoE 유전자로부터 분리되어 프로브로 작동하는 것을 특징으로 하거나, ApoE 유전자의 단일염기다형성을 확인하기 위해 프라이머 및 프로브로 동시 사용될 경우 ApoE 유전자와 혼성화된 후 절단 시약에 의해 상기 Y가 절단되는 경우 상기 Z는 ApoE 유전자로부터 분리되지만 상기 X는 분리되지 않고 프라이머와 프로브로 동시 작동하는 것을 특징으로 한다.In addition, when the mononucleic acid is used only as a probe to confirm the single nucleotide polymorphism of the ApoE gene, when the Y is cleaved by a cleavage reagent after hybridization with the ApoE gene, the X and Z are also separated from the ApoE gene and act as a probe When the Y is cleaved by a cleavage reagent after hybridization with the ApoE gene when used simultaneously as a primer and a probe to confirm a single nucleotide polymorphism of the ApoE gene, the Z is separated from the ApoE gene, but the X is It is characterized in that it works simultaneously as a primer and a probe without being separated.

또한, 본 발명은 상기 단일핵산을 포함하는, ApoE 유전자의 단일염기다형성 실시간 검출용 키트를 제공한다.In addition, the present invention provides a kit for real-time detection of a single nucleotide polymorphism in the ApoE gene, including the mononucleic acid.

또한, 본 발명은 a) 생물학적 시료로부터 검출하고자 하는 ApoE 유전자의 단일염기다형성 부위를 포함하는 표적 핵산을 수득하는 단계; b) 상기 ApoE 유전자의 단일염기다형성 검출용 단일핵산을 제조하는 단계; c) 상기 단계 a)에서 수득한 표적 핵산, 상기 단계 b)에서 제조한 1형 단일핵산, ApoE 유전자 특이적 프라이머 세트, 및 절단시약과 혼합하거나 상기 단계 a)에서 수득한 표적 핵산, 상기 단계 b)에서 제조한 2형 단일핵산, 및 절단시약과 혼합한 후 신장반응을 통해 ApoE 유전자의 단일염기다형성 부위를 포함하는 표적 핵산-단일핵산 복합체를 증폭시키는 단계; 및 d) 상기 단계 c)에서 증폭된 ApoE 유전자의 단일염기다형성 부위를 포함하는 표적 핵산-단일핵산 복합체로부터 분리된 단일핵산 단편의 양을 측정하는 단계;를 포함하는, ApoE 유전자의 단일염기다형성 검출 방법을 제공한다.In addition, the present invention provides a method comprising the steps of: a) obtaining a target nucleic acid comprising a single nucleotide polymorphism site of an ApoE gene to be detected from a biological sample; b) preparing a single nucleic acid for detecting a single nucleotide polymorphism of the ApoE gene; c) the target nucleic acid obtained in step a), the type 1 mononucleic acid prepared in step b), the ApoE gene-specific primer set, and the target nucleic acid obtained in step a) or mixed with the cleavage reagent, step b ) amplifying the target nucleic acid-mononucleic acid complex containing the mononucleotide polymorphism site of the ApoE gene through elongation after mixing with the type 2 mononucleic acid prepared in ) and a cleavage reagent; And d) measuring the amount of a single nucleic acid fragment isolated from the target nucleic acid-mononucleic acid complex comprising the mononucleotide polymorphism site of the ApoE gene amplified in step c); provide a way

본 발명에 따른 단일핵산을 이용한 ApoE 유전자의 단일염기다형성을 실시간으로 검출하는 방법은 종래 qPCR을 이용한 SNP 분석 방법과 비교하여 단일공간에서 2곳의 단일염기다형성의 측정이 가능하여 보다 간단하고 정확하게 측정할 수 있는 이점이 있다. 즉, 본 발명에 따른 단일핵산은 절단시약에 의해서만 절단되고, 단일공간에서 2곳의 단일염기다형성 측정이 가능하여 종래 프로브를 이용한 ApoE 유전자의 SNP 검출 방법에 비해 보다 정확하게 측정할 수 있다.The method for detecting the single nucleotide polymorphism of the ApoE gene using a single nucleic acid according to the present invention is simpler and more accurate than the conventional SNP analysis method using qPCR because it is possible to measure two single nucleotide polymorphisms in a single space. There are advantages to being able to That is, the single nucleic acid according to the present invention is cleaved only by a cleavage reagent, and it is possible to measure single nucleotide polymorphisms in two places in a single space, so that it can be measured more accurately compared to the SNP detection method of the ApoE gene using a conventional probe.

또한, 본 발명에 따른 단일핵산을 이용하여 SNP와 같은 유전적 변이 분석시, 용융온도(melting temperature) 분석과 같은 별도의 확인 과정 필요없이 바로 분석이 가능하다.In addition, when analyzing a genetic variation such as SNP using the single nucleic acid according to the present invention, it is possible to directly analyze it without the need for a separate confirmation process such as a melting temperature analysis.

따라서, 본 발명의 단일핵산(promer) 및 이를 이용한 ApoE 유전자의 단일염기다형성 실시간 검출 방법은 ApoE 유전자의 E2/E2, E3/E3, E4/E4, E2/E3, E2/E4 및 E3/E4 표현형을 신속 정확하게 구분할 수 있어, ApoE 유전자에 의한 다양한 질환, 예컨대 알츠하이머 및 심혈관계 질환의 진단, 치료제 선택 및 예후 진단에 유용하게 이용될 수 있다.Therefore, the single nucleic acid (promer) of the present invention and the method for real-time detection of single nucleotide polymorphisms of the ApoE gene using the same are the E2/E2, E3/E3, E4/E4, E2/E3, E2/E4 and E3/E4 phenotypes of the ApoE gene. can be quickly and accurately distinguished, so it can be usefully used for diagnosing various diseases caused by the ApoE gene, such as Alzheimer's and cardiovascular diseases, selecting therapeutic agents, and prognostic diagnosis.

도 1은 본 발명의 일 실시예에 따른 단일핵산을 이용하여 ApoE 유전자 단일염기다형성을 확인한 도로, 상세하게는 ApoE 단일핵산 1형(서열번호 3 내지 6)를 이용하여 ApoE 유전자 6종의 표현형 E2/E2, E3/E3, E4/E4, E2/E3, E2/E4, E3/E4 구분이 가능함을 확인한 PCR 결과이다.
도 2는 본 발명의 일 실시예에 따른 단일핵산을 이용하여 ApoE 유전자 단일염기다형성을 확인한 도로, 상세하게는 ApoE 단일핵산 2형(서열번호 7 내지 10)을 이용하여 ApoE 유전자 6종의 표현형 E2/E2, E3/E3, E4/E4, E2/E3, E2/E4, E3/E4 구분이 가능함을 확인한 PCR 결과이다.1 is a road confirming the ApoE gene single nucleotide polymorphism using a single nucleic acid according to an embodiment of the present invention. Specifically, the phenotype E2 of six ApoE genes using ApoE mononucleotide type 1 (SEQ ID NOs: 3 to 6). This is the PCR result confirming that /E2, E3/E3, E4/E4, E2/E3, E2/E4, and E3/E4 can be distinguished.
2 is a road confirming the ApoE gene single nucleotide polymorphism using a single nucleic acid according to an embodiment of the present invention. Specifically, the phenotype E2 of six ApoE gene types using ApoE single nucleic acid type 2 (SEQ ID NOs: 7 to 10). This is the PCR result confirming that /E2, E3/E3, E4/E4, E2/E3, E2/E4, and E3/E4 can be distinguished.

본 발명은 ApoE(apolipoprotein E) 유전자의 단일염기다형성 실시간 검출용 단일핵산 및 이를 이용한 검출 방법에 관한 것이다. 보다 상세하게는, X-Y-Z의 구조를 가지며, 단일염기다형성(SNP)을 보이는 ApoE 유전자의 염기서열 일부 또는 전체에 상보적 결합이 가능한 염기서열로 구성된 단일핵산을 사용하여 ApoE 유전자의 단일염기다형성을 실시간으로 검출하는 방법 및 이를 위한 키트에 관한 것이다.The present invention relates to a single nucleic acid for real-time detection of a single nucleotide polymorphism in an apolipoprotein E (ApoE) gene and a detection method using the same. More specifically, the single nucleotide polymorphism of the ApoE gene is monitored in real time using a single nucleic acid composed of a nucleotide sequence capable of complementary binding to some or all of the nucleotide sequence of the ApoE gene having the structure of XYZ and showing single nucleotide polymorphism (SNP). It relates to a method for detecting with and a kit for the same.

하나의 양태로서, 본 발명은 ApoE 유전자의 단일염기다형성 실시간 검출용 단일핵산을 제공한다.In one aspect, the present invention provides a single nucleic acid for real-time detection of a single nucleotide polymorphism of the ApoE gene.

본 발명에 있어서, 상기 단일핵산은 ⅰ) X-Y-Z의 구조를 가지며, ⅱ) 단일염기다형성 부위를 포함한 ApoE 유전자의 염기서열 일부 또는 전체에 상보적 결합을 하고, ⅲ) 양 말단 또는 내부에 동일하거나 적어도 2개의 상이한 탐지 가능한 마커가 부착되어 있는 것을 특징으로 한다.In the present invention, the mononucleic acid is i) has the structure of XYZ, ii) complementarily binds to some or all of the nucleotide sequence of the ApoE gene including the single nucleotide polymorphism site, iii) the same or at least at both ends or inside It is characterized in that two different detectable markers are attached.

이때 부착되는 탐지 가능한 마커의 위치는 특정 부위에 국한하지 않으며, 절단 시약에 의해 Y 부위 절단 시 탐지 가능한 마커가 분리되는 위치라면 어느 곳이든 가능하다.In this case, the position of the detectable marker to be attached is not limited to a specific site, and any position at which the detectable marker is separated when the Y site is cut by a cleavage reagent may be used.

또한, 상기 단일핵산은 실시간 검출을 목적으로 하는 단일염기다형성 부위를 포함한 ApoE 유전자의 염기서열 일부 또는 전체에 상보적 결합하여 복합체를 형성, 증폭시키는 것을 특징으로 한다.In addition, the single nucleic acid is characterized in that the complex is formed and amplified by complementary binding to a part or all of the nucleotide sequence of the ApoE gene including the single nucleotide polymorphism site for the purpose of real-time detection.

본 발명에 있어서, 상기 단일핵산은 ApoE 유전자의 단일염기다형성(single nucleotide polymorphism; SNP)을 탐지하는 핵산을 의미한다. 여기서, 단일염기다형성(SNP) 탐지 시 프로브(probe)로만 사용되는 단일핵산은 "1형 단일핵산" 또는 "단일핵산 1형"으로 표현할 수 있으며, 프라이머(primer)와 프로브(probe)로 동시에 사용되는 단일핵산은 "2형 단일핵산" 또는 "단일핵산 2형"으로 표현할 수 있다. 상기 1형 단일핵산은 2형 단일핵산과 달리 프로브(probe)로 작동 가능하며, 2형 단일핵산은 1형 단일핵산과 달리 프라이머(primer)와 프로브(probe)로 동시에 사용 가능하다.In the present invention, the single nucleic acid refers to a nucleic acid for detecting single nucleotide polymorphism (SNP) of the ApoE gene. Here, a single nucleic acid used only as a probe when detecting a single nucleotide polymorphism (SNP) can be expressed as “type 1 mononucleic acid” or “single nucleic acid type 1”, and is used as a primer and a probe at the same time The mononucleic acid to be used can be expressed as "type 2 mononucleic acid" or "mononucleic acid type 2". The type 1 mononucleic acid can operate as a probe unlike the type 2 mononucleic acid, and the type 2 mononucleic acid can be used simultaneously as a primer and a probe, unlike the type 1 mononucleic acid.

구체적으로, 1형 단일핵산이 ApoE 유전자와 혼성화된 후 절단 시약에 의해 상기 Y가 절단되는 경우 상기 X 및 Z도 ApoE 유전자로부터 분리되어 프로브로 작동 가능하며, 2형 단일핵산이 ApoE 유전자와 혼성화된 후 절단 시약에 의해 상기 Y가 절단되는 경우 상기 Z는 ApoE 유전자로부터 분리되지만 상기 X는 분리되지 않고 프라이머와 프로브로 동시 작동하는 것을 특징으로 한다.Specifically, when the Y is cleaved by a cleavage reagent after the type 1 mononucleic acid is hybridized with the ApoE gene, the X and Z are also separated from the ApoE gene and can operate as a probe, and the type 2 mononucleic acid is hybridized with the ApoE gene When the Y is cleaved by a post-cleavage reagent, the Z is separated from the ApoE gene, but the X is not separated, and it is characterized in that it operates simultaneously as a primer and a probe.

하나의 구체적 예로서, 상기 1형 단일핵산은 서열번호 3, 4, 5 및 6으로 구성된 것일 수 있으며, 상기 2형 단일핵산은 서열번호 7, 8, 9 및 10으로 구성된 것일 수 있다.As a specific example, the type 1 mononucleic acid may be composed of SEQ ID NOs: 3, 4, 5 and 6, and the type 2 mononucleic acid may be composed of SEQ ID NOs: 7, 8, 9 and 10.

본 발명의 단일핵산은 X-Y-Z의 구조를 가지며, 각각의 X, Y, 및 Z는 다양한 개수의 뉴클레오티드(nucleotide)를 가질 수 있다.The mononucleic acid of the present invention has a structure of X-Y-Z, and each of X, Y, and Z may have a variable number of nucleotides.

상기 Y는 ApoE 유전자에 위치하는 1개 또는 2개의 염기서열로 구성된 RNA이며, 절단 시약에 의해 절단되는 부위이다.The Y is RNA composed of one or two nucleotide sequences located in the ApoE gene, and is a site cleaved by a cleavage reagent.

여기서, 절단 시약은 DNase, RNase, 헬리카제(helicase), 엑소뉴클리아제 및 엔도뉴클리아제 같은 효소효소를 매개로 한 절단이 이루어지는 것이 바람직하나, 기타 공지된 절단시약이 사용되어도 무방하다.Here, the cleavage reagent is preferably cleaved through an enzyme enzyme such as DNase, RNase, helicase, exonuclease and endonuclease, but other known cleavage reagents may be used.

상기 X는 1형 단일핵산의 경우 4 내지 20개, 바람직하게는 4 내지 19개, 보다 바람직하게는 4 내지 18개, 보다 더 바람직하게는 5 내지 18개, 보다 더 바람직하게는 6 내지 18개, 보다 더 바람직하게는 6 내지 17개, 보다 더 바람직하게는 6 내지 16개, 가장 바람직하게는 6 내지 15개의 염기서열로 구성되는 DNA이다.In the case of type 1 mononucleic acid, X is 4 to 20, preferably 4 to 19, more preferably 4 to 18, still more preferably 5 to 18, even more preferably 6 to 18 , even more preferably 6 to 17 nucleotides, even more preferably 6 to 16 nucleotides, and most preferably 6 to 15 nucleotide sequences.

하나의 구체적인 예로서, 112번 코돈의 SNP를 검출하기 위한 1형 단일핵산의 X는 5'-GC GCG GAC ATG GAG GAC GTG-3'로 구성된 염기서열(서열번호 27)일 수 있으며, 또한 상기 염기서열에서 3' 말단을 기준으로 4개 이상, 바람직하게는 4개 내지 20개를 포함하는 서열로서 구성될 수 있다. 보다 구체적인 예로서, 112번 코돈의 SNP를 검출하기 위한 1형 단일핵산의 X 부위는 하기 표 1의 서열을 포함할 수 있다.As a specific example, X of the type 1 mononucleic acid for detecting the SNP of codon 112 may be a nucleotide sequence (SEQ ID NO: 27) consisting of 5'-GC GCG GAC ATG GAG GAC GTG-3', and also It may be constituted as a sequence comprising 4 or more, preferably 4 to 20, based on the 3' end of the nucleotide sequence. As a more specific example, the X site of the type 1 mononucleic acid for detecting the SNP of codon 112 may include the sequence shown in Table 1 below.

서열번호SEQ ID NO: 서열order 1111 5‘-C GTG-3’5'-C GTG-3' 1212 5‘-AC GTG-3’5'-AC GTG-3' 1313 5‘-GAC GTG-3’5'-GAC GTG-3' 1414 5’-G GAC GTG-3’5’-G GAC GTG-3’ 1515 5‘-AG GAC GTG-3’5'-AG GAC GTG-3' 1616 5‘-GAG GAC GTG-3’5'-GAG GAC GTG-3' 1717 5‘-G GAG GAC GTG-3’5'-G GAG GAC GTG-3' 1818 5‘-TG GAG GAC GTG-3’5'-TG GAG GAC GTG-3' 1919 5‘-ATG GAG GAC GTG-3’5'-ATG GAG GAC GTG-3' 2020 5'-C ATG GAG GAC GTG-3'5'-C ATG GAG GAC GTG-3' 2121 5'-AC ATG GAG GAC GTG-3’5'-AC ATG GAG GAC GTG-3' 2222 5'-GAC ATG GAG GAC GTG-3’5'-GAC ATG GAG GAC GTG-3' 2323 5'-G GAC ATG GAG GAC GTG-3’5'-G GAC ATG GAG GAC GTG-3' 2424 5'-CG GAC ATG GAG GAC GTG-3’5'-CG GAC ATG GAG GAC GTG-3' 2525 5'-GCG GAC ATG GAG GAC GTG-3’5'-GCG GAC ATG GAG GAC GTG-3' 2626 5'-C GCG GAC ATG GAG GAC GTG-3’5'-C GCG GAC ATG GAG GAC GTG-3' 2727 5'-GC GCG GAC ATG GAG GAC GTG-3’5'-GC GCG GAC ATG GAG GAC GTG-3'

다른 하나의 구체적인 예로서, 158번 코돈의 SNP를 검출하기 위한 1형 단일핵산의 X는 5'-AT GCC GAT GAC CTG CAG AAG-3'로 구성된 염기서열(서열번호 54)일 수 있으며, 또한 상기 112번 코돈의 SNP를 검출하기 위한 1형 단일핵산의 X와 같이 상기 서열번호 54의 염기서열에서 3' 말단을 기준으로 4개 이상, 바람직하게는 4개 내지 20개를 포함하는 서열로서 구성될 수 있다. 보다 구체적인 예로서, 158번 코돈의 SNP를 검출하기 위한 1형 단일핵산의 X 부위는 하기 표 2의 서열을 포함할 수 있다.As another specific example, X of the type 1 mononucleic acid for detecting the SNP of codon 158 may be a nucleotide sequence (SEQ ID NO: 54) consisting of 5'-AT GCC GAT GAC CTG CAG AAG-3', and also Like X of type 1 mononucleic acid for detecting the SNP of codon 112, 4 or more, preferably 4 to 20, based on the 3' end of the nucleotide sequence of SEQ ID NO: 54. can be As a more specific example, the X site of the type 1 mononucleic acid for detecting the SNP of codon 158 may include the sequence shown in Table 2 below.

서열번호SEQ ID NO: 서열order 3838 5‘-G AAG-3’5'-G AAG-3' 3939 5‘-AG AAG-3’5'-AG AAG-3' 4040 5‘-CAG AAG-3’5'-CAG AAG-3' 4141 5’-G CAG AAG-3’5’-G CAG AAG-3’ 4242 5‘-TG CAG AAG-3’5'-TG CAG AAG-3' 4343 5‘-CTG CAG AAG-3’5'-CTG CAG AAG-3' 4444 5‘-C CTG CAG AAG-3’5'-C CTG CAG AAG-3' 4545 5‘-AC CTG CAG AAG-3’5'-AC CTG CAG AAG-3' 4646 5‘-GAC CTG CAG AAG-3’5'-GAC CTG CAG AAG-3' 4747 5'-T GAC CTG CAG AAG-3'5'-T GAC CTG CAG AAG-3' 4848 5'-AT GAC CTG CAG AAG-3’5'-AT GAC CTG CAG AAG-3' 4949 5'-GAT GAC CTG CAG AAG-3’5'-GAT GAC CTG CAG AAG-3' 5050 5'-C GAT GAC CTG CAG AAG-3’5'-C GAT GAC CTG CAG AAG-3' 5151 5'-CC GAT GAC CTG CAG AAG-3’5'-CC GAT GAC CTG CAG AAG-3' 5252 5'-GCC GAT GAC CTG CAG AAG-3’5'-GCC GAT GAC CTG CAG AAG-3' 5353 5'-T GCC GAT GAC CTG CAG AAG-3’5'-T GCC GAT GAC CTG CAG AAG-3' 5454 5'-AT GCC GAT GAC CTG CAG AAG-3’5'-AT GCC GAT GAC CTG CAG AAG-3'

또한, 상기 X는 2형 단일핵산인 경우 10개 내지 30개, 바람직하게는 11개 내지 30개, 보다 바람직하게는 12 내지 30개, 보다 더 바람직하게는 13 내지 30개, 보다 더 바람직하게는 14 내지 30개, 보다 더 바람직하게는 15 내지 30개, 보다 더 바람직하게는 16 내지 30개, 보다 더 바람직하게는 16 내지 29개, 보다 더 바람직하게는 16 내지 28개, 보다 더 바람직하게는 16 내지 27개, 보다 더 바람직하게는 16 내지 26개, 보다 더 바람직하게는 16 내지 25개, 보다 더 바람직하게는 16 내지 24개, 보다 더 바람직하게는 16 내지 23개, 보다 더 바람직하게는 16 내지 22개, 보다 더 바람직하게는 16 내지 21개, 가장 바람직하게는 16 내지 20개의 염기서열로 구성되는 DNA이다.In addition, in the case of type 2 mononucleic acid, X is 10 to 30, preferably 11 to 30, more preferably 12 to 30, even more preferably 13 to 30, even more preferably 14 to 30, even more preferably 15 to 30, even more preferably 16 to 30, even more preferably 16 to 29, even more preferably 16 to 28, even more preferably 16 to 27, even more preferably 16 to 26, even more preferably 16 to 25, even more preferably 16 to 24, even more preferably 16 to 23, even more preferably It is a DNA composed of 16 to 22, more preferably 16 to 21, and most preferably 16 to 20 nucleotide sequences.

하나의 구체적인 예로, 112번 코돈의 SNP를 검출하기 위한 2형 단일핵산의 X는 5'-GCC CGG CTG GGC GCG GAC ATG GAG GAC GTG-3'로 구성된 염기서열(서열번호 37)일 수 있으며, 또한 상기 염기서열에서 3' 말단을 기준으로 10개 이상, 바람직하게는 10개 내지 30개를 포함하는 서열로서 구성될 있다. 보다 구체적인 예로서, 112번 코돈의 SNP를 검출하기 위한 2형 단일핵산의 X 부위는 하기 표 3의 서열을 포함할 수 있다.As a specific example, X of the type 2 mononucleic acid for detecting the SNP of codon 112 may be a nucleotide sequence (SEQ ID NO: 37) consisting of 5'-GCC CGG CTG GGC GCG GAC ATG GAG GAC GTG-3', In addition, it may be configured as a sequence comprising 10 or more, preferably 10 to 30, based on the 3' end of the nucleotide sequence. As a more specific example, the X site of the type 2 mononucleic acid for detecting the SNP of codon 112 may include the sequence shown in Table 3 below.

서열번호SEQ ID NO: 서열order 1717 5'-G GAG GAC GTG-3'5'-G GAG GAC GTG-3' 1818 5'-TG GAG GAC GTG-3'5'-TG GAG GAC GTG-3' 1919 5'-ATG GAG GAC GTG-3'5'-ATG GAG GAC GTG-3' 2020 5'-C ATG GAG GAC GTG-3'5'-C ATG GAG GAC GTG-3' 2121 5'-AC ATG GAG GAC GTG-3'5'-AC ATG GAG GAC GTG-3' 2222 5'-GAC ATG GAG GAC GTG-3'5'-GAC ATG GAG GAC GTG-3' 2323 5'-G GAC ATG GAG GAC GTG-3'5'-G GAC ATG GAG GAC GTG-3' 2424 5'-CG GAC ATG GAG GAC GTG-3'5'-CG GAC ATG GAG GAC GTG-3' 2525 5'-GCG GAC ATG GAG GAC GTG-3'5'-GCG GAC ATG GAG GAC GTG-3' 2626 5'-C GCG GAC ATG GAG GAC GTG-3'5'-C GCG GAC ATG GAG GAC GTG-3' 2727 5'-GC GCG GAC ATG GAG GAC GTG-3'5'-GC GCG GAC ATG GAG GAC GTG-3' 2828 5'-GGC GCG GAC ATG GAG GAC GTG-3'5'-GGC GCG GAC ATG GAG GAC GTG-3' 2929 5'-G GGC GCG GAC ATG GAG GAC GTG-3'5'-G GGC GCG GAC ATG GAG GAC GTG-3' 3030 5'-TG GGC GCG GAC ATG GAG GAC GTG-3'5'-TG GGC GCG GAC ATG GAG GAC GTG-3' 3131 5'-CTG GGC GCG GAC ATG GAG GAC GTG-3'5'-CTG GGC GCG GAC ATG GAG GAC GTG-3' 3232 5'-G CTG GGC GCG GAC ATG GAG GAC GTG-3'5'-G CTG GGC GCG GAC ATG GAG GAC GTG-3' 3333 5'-GG CTG GGC GCG GAC ATG GAG GAC GTG-3'5'-GG CTG GGC GCG GAC ATG GAG GAC GTG-3' 3434 5'-CGG CTG GGC GCG GAC ATG GAG GAC GTG-3'5'-CGG CTG GGC GCG GAC ATG GAG GAC GTG-3' 3535 5'-C CGG CTG GGC GCG GAC ATG GAG GAC GTG-3'5'-C CGG CTG GGC GCG GAC ATG GAG GAC GTG-3' 3636 5'-CC CGG CTG GGC GCG GAC ATG GAG GAC GTG-3'5'-CC CGG CTG GGC GCG GAC ATG GAG GAC GTG-3' 3737 5'-GCC CGG CTG GGC GCG GAC ATG GAG GAC GTG-3'5'-GCC CGG CTG GGC GCG GAC ATG GAG GAC GTG-3'

다른 하나의 구체적인 예로, 158번 코돈의 SNP를 검출하기 위한 2형 단일핵산의 X는 5'-CTC CTC CGC GAT GCC GAT GAC CTG CAG AAG-3'로 구성된 염기서열(서열번호 64)일 수 있으며, 또한 상기 112번 코돈의 SNP를 검출하기 위한 1형 단일핵산의 X와 같이 상기 서열번호 64의 염기서열에서 3' 말단을 기준으로 4개 이상, 바람직하게는 4개 내지 20개를 포함하는 서열로서 구성될 수 있다. 보다 구체적인 예로서, 158번 코돈의 SNP를 검출하기 위한 1형 단일핵산의 X 부위는 하기 표 4의 서열을 포함할 수 있다.As another specific example, X of the type 2 mononucleic acid for detecting the SNP of codon 158 may be a nucleotide sequence (SEQ ID NO: 64) consisting of 5'-CTC CTC CGC GAT GCC GAT GAC CTG CAG AAG-3' and , Also, a sequence comprising 4 or more, preferably 4 to 20, based on the 3' end of the nucleotide sequence of SEQ ID NO: 64, such as X of type 1 mononucleic acid for detecting the SNP of codon 112 can be configured as As a more specific example, the X site of the type 1 mononucleic acid for detecting the SNP of codon 158 may include the sequence shown in Table 4 below.

서열번호SEQ ID NO: 서열order 4444 5‘-C CTG CAG AAG-3’5'-C CTG CAG AAG-3' 4545 5‘-AC CTG CAG AAG-3’5'-AC CTG CAG AAG-3' 4646 5‘-GAC CTG CAG AAG-3’5'-GAC CTG CAG AAG-3' 4747 5'-T GAC CTG CAG AAG-3'5'-T GAC CTG CAG AAG-3' 4848 5'-AT GAC CTG CAG AAG-3’5'-AT GAC CTG CAG AAG-3' 4949 5'-GAT GAC CTG CAG AAG-3’5'-GAT GAC CTG CAG AAG-3' 5050 5'-C GAT GAC CTG CAG AAG-3’5'-C GAT GAC CTG CAG AAG-3' 5151 5'-CC GAT GAC CTG CAG AAG-3’5'-CC GAT GAC CTG CAG AAG-3' 5252 5'-GCC GAT GAC CTG CAG AAG-3’5'-GCC GAT GAC CTG CAG AAG-3' 5353 5'-T GCC GAT GAC CTG CAG AAG-3’5'-T GCC GAT GAC CTG CAG AAG-3' 5454 5'-AT GCC GAT GAC CTG CAG AAG-3’5'-AT GCC GAT GAC CTG CAG AAG-3' 5555 5'-GAT GCC GAT GAC CTG CAG AAG-3'5'-GAT GCC GAT GAC CTG CAG AAG-3' 5656 5'-C GAT GCC GAT GAC CTG CAG AAG-3'5'-C GAT GCC GAT GAC CTG CAG AAG-3' 5757 5'-GC GAT GCC GAT GAC CTG CAG AAG-3'5'-GC GAT GCC GAT GAC CTG CAG AAG-3' 5858 5'-CGC GAT GCC GAT GAC CTG CAG AAG-3'5'-CGC GAT GCC GAT GAC CTG CAG AAG-3' 5959 5'-C CGC GAT GCC GAT GAC CTG CAG AAG-3'5'-C CGC GAT GCC GAT GAC CTG CAG AAG-3' 6060 5'-TC CGC GAT GCC GAT GAC CTG CAG AAG-3'5'-TC CGC GAT GCC GAT GAC CTG CAG AAG-3' 6161 5'-CTC CGC GAT GCC GAT GAC CTG CAG AAG-3'5'-CTC CGC GAT GCC GAT GAC CTG CAG AAG-3' 6262 5'-C CTC CGC GAT GCC GAT GAC CTG CAG AAG-3'5'-C CTC CGC GAT GCC GAT GAC CTG CAG AAG-3' 6363 5'-TC CTC CGC GAT GCC GAT GAC CTG CAG AAG-3'5'-TC CTC CGC GAT GCC GAT GAC CTG CAG AAG-3' 6464 5'-CTC CTC CGC GAT GCC GAT GAC CTG CAG AAG-3'5'-CTC CTC CGC GAT GCC GAT GAC CTG CAG AAG-3'

상기 1형 단일핵산에서 Z부위는 1 내지 20개, 바람직하게는 2 내지 20개, 보다 바람직하게는 3 내지 20개, 보다 더 바람직하게는 4 내지 20개, 보다 더 바람직하게는 4 내지 19개, 보다 더 바람직하게는 4 내지 18개, 보다 더 바람직하게는 4 내지 17개, 가장 바람직하게는 4 내지 16개 등의 염기서열로 구성되는 DNA이다.In the type 1 mononucleic acid, the number of Z sites is 1 to 20, preferably 2 to 20, more preferably 3 to 20, even more preferably 4 to 20, even more preferably 4 to 19. , even more preferably from 4 to 18, even more preferably from 4 to 17, most preferably from 4 to 16, and the like.

하나의 구체적인 예로서, 112번 코돈의 SNP를 검출하기 위한 1형 단일핵산의 Z는 5‘-GCG GCC GCC TGG TGC AGT AC-3’로 구성된 염기서열(서열번호 84)일 수 있으며, 또한 상기 염기서열(서열번호 84)에서 5‘ 기준으로 4개 이상을 포함하는 서열로서 구성될 수 있다. 보다 구체적인 예로서, 112번 코돈의 SNP를 검출하기 위한 1형 단일핵산의 Z 부위는 하기 표 5의 서열을 포함할 수 있다.As a specific example, Z of the type 1 mononucleic acid for detecting the SNP of codon 112 may be a nucleotide sequence (SEQ ID NO: 84) consisting of 5'-GCG GCC GCC TGG TGC AGT AC-3', and also It may be configured as a sequence including 4 or more 5' in the base sequence (SEQ ID NO: 84). As a more specific example, the Z region of the type 1 mononucleic acid for detecting the SNP of codon 112 may include the sequence shown in Table 5 below.

서열번호SEQ ID NO: 서열order 6565 5‘-G-3’5'-G-3' 6666 5‘-GC-3’5'-GC-3' 6767 5‘-GCG-3’5'-GCG-3' 6868 5‘-GCG G-3’5'-GCG G-3' 6969 5‘-GCG GC-3’5'-GCG GC-3' 7070 5‘-GCG GCC-3’5'-GCG GCC-3' 7171 5’-GCG GCC G-3‘5'-GCG GCC G-3' 7272 5’-GCG GCC GC-3‘5'-GCG GCC GC-3' 7373 5’-GCG GCC GCC-3‘5'-GCG GCC GCC-3' 7474 5’-GCG GCC GCC T-3‘5'-GCG GCC GCC T-3' 7575 5’-GCG GCC GCC TG-3‘5'-GCG GCC GCC TG-3' 7676 5’-GCG GCC GCC TGG-3‘5'-GCG GCC GCC TGG-3' 7777 5’-GCG GCC GCC TGG T-3‘5'-GCG GCC GCC TGG T-3' 7878 5’-GCG GCC GCC TGG TG-3‘5'-GCG GCC GCC TGG TG-3' 7979 5’-GCG GCC GCC TGG TGC-3‘5'-GCG GCC GCC TGG TGC-3' 8080 5’-GCG GCC GCC TGG TGC A-3‘5'-GCG GCC GCC TGG TGC A-3' 8181 5’-GCG GCC GCC TGG TGC AG-3‘5'-GCG GCC GCC TGG TGC AG-3' 8282 5’-GCG GCC GCC TGG TGC AGT-3‘5'-GCG GCC GCC TGG TGC AGT-3' 8383 5’-GCG GCC GCC TGG TGC AGT A-3‘5'-GCG GCC GCC TGG TGC AGT A-3' 8484 5’-GCG GCC GCC TGG TGC AGT AC-3‘5'-GCG GCC GCC TGG TGC AGT AC-3'

다른 하나의 구체적인 예로서, 158번 코돈의 SNP를 검출하기 위한 1형 단일핵산의 Z는 5‘-GCC TGG CAG TGT ACC AGG CC-3’로 구성된 염기서열(서열번호 104)일 수 있으며, 또한 상기 염기서열(서열번호 104)에서 5‘ 기준으로 4개 이상을 포함하는 서열로서 구성될 수 있다. 보다 구체적인 예로서, 158번 코돈의 SNP를 검출하기 위한 1형 단일핵산의 Z 부위는 하기 표 6의 서열을 포함할 수 있다.As another specific example, Z of the type 1 mononucleic acid for detecting the SNP of codon 158 may be a nucleotide sequence (SEQ ID NO: 104) consisting of 5'-GCC TGG CAG TGT ACC AGG CC-3', and also It may be configured as a sequence including 4 or more 5' in the base sequence (SEQ ID NO: 104). As a more specific example, the Z region of the type 1 mononucleic acid for detecting the SNP of codon 158 may include the sequence of Table 6 below.

서열번호SEQ ID NO: 서열order 8585 5‘-G-3’5'-G-3' 8686 5‘-GC-3’5'-GC-3' 8787 5‘-GCC-3’5'-GCC-3' 8888 5‘-GCC T-3’5'-GCC T-3' 8989 5‘-GCC TG-3’5'-GCC TG-3' 9090 5’-GCC TGG-3‘5'-GCC TGG-3' 9191 5’-GCC TGG C-3‘5'-GCC TGG C-3' 9292 5’-GCC TGG CA-3‘5'-GCC TGG CA-3' 9393 5’-GCC TGG CAG-3‘5'-GCC TGG CAG-3' 9494 5’-GCC TGG CAG T-3‘5'-GCC TGG CAG T-3' 9595 5’-GCC TGG CAG TG-3‘5'-GCC TGG CAG TG-3' 9696 5’-GCC TGG CAG TGT-3‘5'-GCC TGG CAG TGT-3' 9797 5’-GCC TGG CAG TGT A-3‘5'-GCC TGG CAG TGT A-3' 9898 5’-GCC TGG CAG TGT AC-3‘5'-GCC TGG CAG TGT AC-3' 9999 5’-GCC TGG CAG TGT ACC-3‘5'-GCC TGG CAG TGT ACC-3' 100100 5’-GCC TGG CAG TGT ACC A-3‘5'-GCC TGG CAG TGT ACC A-3' 101101 5’-GCC TGG CAG TGT ACC AG-3‘5'-GCC TGG CAG TGT ACC AG-3' 102102 5’-GCC TGG CAG TGT ACC AGG-3‘5'-GCC TGG CAG TGT ACC AGG-3' 103103 5’-GCC TGG CAG TGT ACC AGG C-3‘5'-GCC TGG CAG TGT ACC AGG C-3' 104104 5’-GCC TGG CAG TGT ACC AGG CC-3‘5'-GCC TGG CAG TGT ACC AGG CC-3'

또한, 상기 2형 단일핵산에서 Z 부위는 1 내지 5개, 바람직하게는 1 내지 4개, 보다 바람직하게는 2 내지 4개의 염기서열로 구성되는 DNA이다.In addition, in the type 2 mononucleic acid, the Z region is a DNA composed of 1 to 5, preferably 1 to 4, more preferably 2 to 4 nucleotide sequences.

하나의 구체적인 예로서, 112번 코돈의 SNP를 검출하기 위한 2형 단일핵산의 Z는 5‘-G-3’(서열번호 65), 5’-GC-3’(서열번호 66), 5‘-GCG-3’(서열번호 67), 5‘-GCG G-3’(서열번호 68), 또는 5-GCG GC-3’(서열번호 69)일 수 있다.As a specific example, Z of the type 2 mononucleic acid for detecting the SNP of codon 112 is 5'-G-3' (SEQ ID NO: 65), 5'-GC-3' (SEQ ID NO: 66), 5' -GCG-3' (SEQ ID NO: 67), 5'-GCG G-3' (SEQ ID NO: 68), or 5-GCG GC-3' (SEQ ID NO: 69).

다른 하나의 구체적인 예로서, 158번 코돈의 SNP를 검출하기 위한 2형 단일핵산의 Z는 5‘-G-3’(서열번호 85), 5’-GC-3’(서열번호 86), 5‘-GCC-3’(서열번호 87), 5‘-GCC T-3’(서열번호 88), 또는 5-GCC TG-3’(서열번호 89)일 수 있다.As another specific example, Z of the type 2 mononucleic acid for detecting the SNP of codon 158 is 5'-G-3' (SEQ ID NO: 85), 5'-GC-3' (SEQ ID NO: 86), 5 '-GCC-3' (SEQ ID NO: 87), 5'-GCC T-3' (SEQ ID NO: 88), or 5-GCC TG-3' (SEQ ID NO: 89).

본 발명에 있어서, 상술한 단일핵산으로서 X 및 Z를 구성하는 염기 개수에 따라 이들 각각의 단일염기다형성(SNP) 검출을 특이적이며 민감하게 탐지할 수 있다.In the present invention, the detection of each of the single nucleotide polymorphisms (SNPs) can be specifically and sensitively detected according to the number of bases constituting X and Z as the above-described mononucleic acid.

본 발명의 일 실시예에 있어서, ApoE 단일핵산(서열번호 3 내지 6, 및 서열번호 7 내지 10)을 이용하여 ApoE 유전자 6종의 표현형 E2/E2, E3/E3, E4/E4, E2/E3, E2/E4, E3/E4 구분이 명확하게 가능함을 실시간 PCR을 통해 확인하였다 (도 1 및 2).In one embodiment of the present invention, phenotypes E2/E2, E3/E3, E4/E4, E2/E3 of six ApoE genes using ApoE mononucleic acids (SEQ ID NOs: 3 to 6, and SEQ ID NOs: 7 to 10) , E2/E4, and E3/E4 were clearly identified through real-time PCR ( FIGS. 1 and 2 ).

본 발명에 있어서, 상기 탐지 가능한 마커는 단일핵산에 공유 결합 또는 비공유 결합에 의해 결합하는 형광물질, 또는 형광물질과 소광물질로 이루어진 형광쌍을 사용할 수 있다.In the present invention, the detectable marker may be a fluorescent material that binds to a single nucleic acid by covalent or non-covalent bonding, or a fluorescent pair composed of a fluorescent material and a quencher material.

상기 형광물질은 반드시 이로 제한되는 것은 아니지만, 예를 들어 Cy3, Cy5, Cy5.5, Bodipy, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 594, Alexa 660, 로다민(Rhodamine), TAMRA, FAM, FITC, Fluor X, ROX, Texas Red, ORNAge green 488X, ORNAge green 514X, HEX, TET, JOE, Oyster 556, Oyster 645, Bodipy 630/650, Bodipy 650/665, Calfluor ORNAge 546, Calfluor red 610, Quasar 670 및 비오틴으로 이루어지는 군으로부터 선택되는 어느 하나일 수 있다.The fluorescent material is not necessarily limited thereto, but for example, Cy3, Cy5, Cy5.5, Bodipy, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 594, Alexa 660, Rhodamine, TAMRA, FAM, FITC, Fluor X, ROX, Texas Red, ORNAge green 488X, ORNAge green 514X, HEX, TET, JOE, Oyster 556, Oyster 645, Bodipy 630/650, Bodipy 650/665, Calfluor ORNAge 546, Calfluor red 610, It may be any one selected from the group consisting of Quasar 670 and biotin.

또한 상기 소광물질은 반드시 이로 제한되는 것은 아니지만, 예를 들어 DDQ-1, Dabcyl, Eclipase, 6-TAMRA, BHQ-1, BHQ-2, BHQ-3, lowa Black RQ-Sp, QSY-7, QSY-2 및 MGBNFQ로 이루어진 군으로부터 선택되는 어느 하나일 수 있다.In addition, the matting material is not necessarily limited thereto, for example, DDQ-1, Dabcyl, Eclipase, 6-TAMRA, BHQ-1, BHQ-2, BHQ-3, lowa Black RQ-Sp, QSY-7, QSY It may be any one selected from the group consisting of -2 and MGBNFQ.

본 발명에서 탐지 가능한 마커로 형광쌍을 사용하는 경우, 형광물질과 소광물질의 위치는 X 또는 Z에 위치한 형태이거나 Y 부위에 위치할 수 있으며, 그 어느 것에 한정되는 것은 아니다. 하나의 예로서, 형광물질은 X에 위치하고 소광물질은 Y 또는 Z에 위치할 수 있다.When a fluorescent pair is used as a detectable marker in the present invention, the positions of the fluorescent material and the quencher material may be in the form of X or Z, or may be located at the Y site, but is not limited thereto. As an example, the fluorescent material may be located at X and the quencher may be located at Y or Z.

본 발명의 단일핵산은 ApoE 유전자의 단일염기다형성을 확인하는데 있어서, ⅰ) 핵산 중 RNA로부터 cDNA를 합성하기 위한 RT 프라이머; ⅱ) 핵산(DNA 또는 RNA)으로부터 합성된 cDNA를 증폭하기 위한 정방향 프라이머; ⅲ) 핵산(DNA 또는 RNA)으로부터 합성된 cDNA를 증폭하기 위한 역방향 프라이머; ⅳ) 핵산(DNA 또는 RNA)으로부터 합성된 cDNA를 증폭하기 위한 정방향 프라이머 및 역방향 프라이머; 또는 ⅴ) 검출하고자 하는 핵산(DNA 또는 RNA)을 실시간으로 확인하기 위한 프로브로 사용될 수 있다.In confirming the single nucleotide polymorphism of the ApoE gene, the mononucleic acid of the present invention includes: i) an RT primer for synthesizing cDNA from RNA among nucleic acids; ii) a forward primer for amplifying cDNA synthesized from a nucleic acid (DNA or RNA); iii) a reverse primer for amplifying cDNA synthesized from a nucleic acid (DNA or RNA); iv) forward and reverse primers for amplifying cDNA synthesized from nucleic acids (DNA or RNA); Alternatively, v) may be used as a probe for real-time confirmation of a nucleic acid (DNA or RNA) to be detected.

특히, 본 발명의 단일핵산을 RNA(miRNA 등 포함)를 cDNA로 합성 및 증폭하기 위한 RT 프라이머; 또는 정방향 프라이머와 프로브; 또는 역방향 프라이머와 프로브로 사용하는 경우에, cDNA 합성시 RT 프라이머를 루프형태로 제작하거나 폴리 A를 형성하는 과정이 필요하지 않고 검출하고자 하는 RNA와 혼성화되어 cDNA를 합성하고 탐지하고자 하는 RNA(miRNA 등 포함)를 증폭 및 실시간 검출할 수 있다.In particular, RT primers for synthesizing and amplifying RNA (including miRNA, etc.) of the mononucleic acid of the present invention into cDNA; or forward primers and probes; Alternatively, when used as a reverse primer and probe, the RT primer is not required to form a loop or to form poly A during cDNA synthesis. ) can be amplified and detected in real time.

다른 하나의 양태로서, 본 발명은 상기 단일핵산을 포함하는, ApoE 유전자의 단일염기다형성 실시간 검출용 키트를 제공한다.In another aspect, the present invention provides a kit for real-time detection of a single nucleotide polymorphism in the ApoE gene, comprising the mononucleic acid.

본 발명의 단일핵산을 ApoE 유전자의 단일염기다형성을 확인하기 위한 키트로 사용하는 경우, 상기 키트는 본 발명의 단일핵산 이외에 단일핵산의 Y 부위를 절단할 수 있는 효소를 추가로 포함하는 것이 바람직하다.When the mononucleic acid of the present invention is used as a kit for confirming the single nucleotide polymorphism of the ApoE gene, the kit preferably further includes an enzyme capable of cleaving the Y site of the mononucleic acid in addition to the mononucleic acid of the present invention. .

본 발명에 있어서, 상기 단일핵산의 Y 부위를 절단할 수 있는 효소는 단일핵산의 Y 부위를 특이적으로 절단할 수 있는 것이라면 어느 것이든 사용 가능하다. 예를 들어 Y 부위가 DNA인 경우 DNA 뉴클라아제(DNA nuclease, DNase), 구체적으로 DNase Ⅰ, DNase Ⅱ, S1 핵산 가수분해효소, 핵산가수분해효소 P1, AP 핵산내부가수분해효소, 또는 UvrABSC 핵산가수분해효소 등을 사용하는 것이 바람직하며, Y 부위가 RNA인 경우 RNA 가수분해효소(ribonuclease, RNase), 구체적으로 RNase Ⅱ, RNase Ⅲ, RNase Ⅳ, RNaseH, 또는 RNase T2 등을 사용하는 것이 바람직하다.In the present invention, any enzyme capable of cleaving the Y site of the mononucleic acid may be used as long as it can specifically cut the Y site of the mononucleic acid. For example, when the Y site is DNA, a DNA nuclease (DNase), specifically DNase I, DNase II, S1 nuclease, nuclease P1, AP nuclease, or UvrABSC nucleic acid It is preferable to use a hydrolase, etc., and when the Y site is RNA, it is preferable to use an RNA hydrolase (ribonuclease, RNase), specifically RNase II, RNase III, RNase IV, RNaseH, or RNase T2. .

본 발명의 단일핵산을 ApoE 유전자의 단일염기다형성을 확인하기 위한 키트로 사용하는 경우, 상기 키트는 본 발명의 단일핵산 및 단일핵산의 Y 부위를 절단할 수 있는 효소 이외에 DNA의 증폭반응에 필요한 시약을 추가로 포함할 수 있다.When the mononucleic acid of the present invention is used as a kit for confirming the single nucleotide polymorphism of the ApoE gene, the kit is a reagent necessary for the DNA amplification reaction in addition to the mononucleic acid of the present invention and an enzyme capable of cleaving the Y site of the mononucleic acid. may further include.

상기 증폭반응에 필요한 시약은 예를 들어, 적당량의 DNA 중합효소(예를 들어, Thermusaquatiucs(Taq), Thermusthermophilus(Tth), Thermusfiliformis, Thermis flavus, Thermococcusliteralis 또는 Phyrococcusfuriosis(Pfu)로부터 얻은 열 안정성 DNA 중합효소), DNA 중합효소 조인자(Mg2+), 완충용액, dNTPs(dATP, dCTP, dGTP 및 dTTP) 및 물(dH2O)을 들 수 있다. 또한, 상기 완충용액은 이에 제한되지는 않으나 적당량의 트리톤 X-100(Triton X-100), 디메틸설폭사이드(dimethylsufoxide, DMSO), Tween20, nonidet P40, PEG 6000, 포름아마이드 및 소혈청 알부민(BSA) 등이 있다.Reagents necessary for the amplification reaction include, for example, an appropriate amount of a DNA polymerase (eg, a thermostable DNA polymerase obtained from Thermusaquatiucs (Taq), Thermusthermophilus (Tth), Thermusfiliformis, Thermis flavus, Thermococcusliteralis or Phyrococcusfuriosis (Pfu)). , DNA polymerase cofactor (Mg 2+ ), buffer solution, dNTPs (dATP, dCTP, dGTP and dTTP) and water (dH 2 O). In addition, the buffer solution is not limited thereto, but an appropriate amount of Triton X-100 (Triton X-100), dimethylsufoxide (DMSO), Tween20, nonidet P40, PEG 6000, formamide and bovine serum albumin (BSA) etc.

또 다른 하나의 양태로서, 본 발명은 a) 생물학적 시료로부터 검출하고자 하는 ApoE 유전자의 단일염기다형성 부위를 포함하는 표적 핵산을 수득하는 단계; b) 상술한 단일핵산을 제조하는 단계; c) 상기 단계 a)에서 수득한 표적 핵산, 상기 단계 b)에서 제조한 1형 단일핵산, ApoE 유전자 특이적 프라이머 세트, 및 절단시약과 혼합하거나 상기 단계 a)에서 수득한 표적 핵산, 상기 단계 b)에서 제조한 2형 단일핵산, 및 절단시약과 혼합한 후 신장반응을 통해 ApoE 유전자의 단일염기다형성 부위를 포함하는 표적 핵산-단일핵산 복합체를 증폭시키는 단계; 및 d) 상기 단계 c)에서 증폭된 ApoE 유전자의 단일염기다형성 부위를 포함하는 표적 핵산-단일핵산 복합체로부터 분리된 단일핵산 단편의 양을 측정하는 단계;를 포함하는, ApoE 유전자의 단일염기다형성 검출 방법을 제공한다.In yet another aspect, the present invention provides a method comprising: a) obtaining a target nucleic acid comprising a single nucleotide polymorphism site of an ApoE gene to be detected from a biological sample; b) preparing the above-described mononucleic acid; c) the target nucleic acid obtained in step a), the type 1 mononucleic acid prepared in step b), the ApoE gene-specific primer set, and the target nucleic acid obtained in step a) or mixed with the cleavage reagent, step b ) amplifying the target nucleic acid-mononucleic acid complex containing the mononucleotide polymorphism site of the ApoE gene through elongation after mixing with the type 2 mononucleic acid prepared in ) and a cleavage reagent; And d) measuring the amount of a single nucleic acid fragment isolated from the target nucleic acid-mononucleic acid complex comprising the mononucleotide polymorphism site of the ApoE gene amplified in step c); provide a way

본 발명에 따른 ApoE 유전자의 단일염기다형성을 실시간으로 검출하는 방법을 각 단계에 따라 구체적으로 설명하면 다음과 같다.The method for detecting the single nucleotide polymorphism of the ApoE gene according to the present invention in real time will be described in detail according to each step as follows.

a) 생물학적 시료로부터 검출하고자 하는 ApoE 유전자의 단일염기다형성 부위를 포함하는 표적 핵산을 수득하는 단계이다.a) obtaining a target nucleic acid including a single nucleotide polymorphism site of the ApoE gene to be detected from a biological sample.

본 발명에 있어서, 상기 ApoE 유전자의 단일염기다형성 부위를 포함하는 표적 핵산은 시료로부터 검출하고자 하는 RNA 또는 DNA이거나, 상기 RNA를 역전사 중합효소로 증폭하여 얻은 cDNA일 수 있다.In the present invention, the target nucleic acid including the single nucleotide polymorphism site of the ApoE gene may be RNA or DNA to be detected from a sample, or cDNA obtained by amplifying the RNA with a reverse transcription polymerase.

상기 시료는 생물학적 시료이거나, 생물학적 시료로부터 분리된 RNA, DNA 또는 이들의 단편일 수 있다. 구체적으로, 상기 시료는 혈액, 타액, 뇨, 분변, 조직, 세포 및 생검 표본으로 이루어진 군으로부터 선택된 어느 하나 이상일 수 있으며, 또는 보관된 생물학적 시료로부터 분리된 RNA, DNA 또는 이들의 단편일 수 있다. 그러나, 반드시 이로 한정되는 것은 아니다.The sample may be a biological sample or RNA, DNA, or fragments thereof isolated from the biological sample. Specifically, the sample may be any one or more selected from the group consisting of blood, saliva, urine, feces, tissue, cell, and biopsy samples, or may be RNA, DNA, or fragments thereof isolated from stored biological samples. However, it is not necessarily limited thereto.

상기 보관된 생물학적 시료는 당업계에 통상적으로 알려진 보관방법으로 1주일 이상, 1년 이상, 예를 들면 1년 내지 10년 동안 보관되거나, 냉동 보관, 또는 포르말린으로 고정된 조직을 상온에서 보관한 조직으로부터 유래된 것일 수 있다.The stored biological sample is stored for 1 week or more, 1 year or more, for example, 1 to 10 years by a storage method conventionally known in the art, or stored frozen, or tissue stored at room temperature in formalin-fixed tissue may be derived from

본 발명에 있어서, 시료로부터 RNA 또는 DNA의 추출은 당업계에 공지된 다양한 방법을 이용할 수 있다.In the present invention, extraction of RNA or DNA from a sample may use various methods known in the art.

b) 상기 단일핵산을 제조하는 단계이다.b) preparing the mononucleic acid.

본 발명에 있어서, 단일핵산은 전술한 바와 같으며, 상세하게는 ⅰ) X-Y-Z의 구조를 가지며, ⅱ) 단일염기다형성 부위를 포함한 ApoE 유전자의 염기서열 일부 또는 전체에 상보적 결합을 하고, ⅲ) 양 말단 또는 내부에 동일하거나 적어도 2개의 상이한 탐지 가능한 마커가 부착되어 있는 것을 특징으로 한다.In the present invention, the mononucleic acid is as described above, and specifically i) has the structure of XYZ, ii) complementarily binds to some or all of the nucleotide sequence of the ApoE gene including the single nucleotide polymorphism site, iii) It is characterized in that the same or at least two different detectable markers are attached to both ends or inside.

본 발명에 있어서, 상기 탐지 가능한 마커는 단일핵산에 공유 결합 또는 비공유 결합에 의해 결합하는 형광물질, 또는 형광물질과 소광물질로 이루어진 형광쌍을 사용할 수 있다.In the present invention, the detectable marker may be a fluorescent material that binds to a single nucleic acid by covalent or non-covalent bonding, or a fluorescent pair composed of a fluorescent material and a quencher material.

상기 형광물질은 반드시 이로 제한되는 것은 아니지만, 예를 들어 Cy3, Cy5, Cy5.5, Bodipy, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 594, Alexa 660, 로다민(Rhodamine), TAMRA, FAM, FITC, Fluor X, ROX, Texas Red, ORNAge green 488X, ORNAge green 514X, HEX, TET, JOE, Oyster 556, Oyster 645, Bodipy 630/650, Bodipy 650/665, Calfluor ORNAge 546, Calfluor red 610, Quasar 670 및 비오틴으로 이루어지는 군으로부터 선택되는 어느 하나일 수 있다. 또한 상기 소광물질은 반드시 이로 제한되는 것은 아니지만, 예를 들어 DDQ-1, Dabcyl, Eclipase, 6-TAMRA, BHQ-1, BHQ-2, BHQ-3, lowa Black RQ-Sp, QSY-7, QSY-2 및 MGBNFQ로 이루어진 군으로부터 선택되는 어느 하나일 수 있다.The fluorescent material is not necessarily limited thereto, but for example, Cy3, Cy5, Cy5.5, Bodipy, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 594, Alexa 660, Rhodamine, TAMRA, FAM, FITC, Fluor X, ROX, Texas Red, ORNAge green 488X, ORNAge green 514X, HEX, TET, JOE, Oyster 556, Oyster 645, Bodipy 630/650, Bodipy 650/665, Calfluor ORNAge 546, Calfluor red 610, It may be any one selected from the group consisting of Quasar 670 and biotin. In addition, the matting material is not necessarily limited thereto, for example, DDQ-1, Dabcyl, Eclipase, 6-TAMRA, BHQ-1, BHQ-2, BHQ-3, lowa Black RQ-Sp, QSY-7, QSY It may be any one selected from the group consisting of -2 and MGBNFQ.

c) ApoE 유전자의 단일염기다형성 부위를 포함하는 표적 핵산-단일핵산 복합체를 증폭시키는 단계이다.c) amplifying the target nucleic acid-mononucleic acid complex including the mononucleotide polymorphism site of the ApoE gene.

본 발명에 있어서, 상기 ApoE 유전자의 단일염기다형성 부위를 포함하는 표적 핵산-단일핵산 복합체의 증폭은 상기 수득한 표적 핵산, 상기 제조한 1형 단일핵산, ApoE 유전자 특이적 프라이머 세트, 및 절단시약과 혼합하거나 상기 단계 a)에서 수득한 표적 핵산, 상기 단계 b)에서 제조한 2형 단일핵산, 및 절단시약과 혼합한 후 신장반응을 통해 이루어질 수 있다.In the present invention, the amplification of the target nucleic acid-mononucleic acid complex including the single nucleotide polymorphism site of the ApoE gene is performed with the obtained target nucleic acid, the prepared type 1 mononucleic acid, ApoE gene-specific primer set, and a cleavage reagent It may be mixed or mixed with the target nucleic acid obtained in step a), the type 2 mononucleic acid prepared in step b), and a cleavage reagent, and then, through an elongation reaction.

본 발명에 있어서, 상기 ApoE 유전자 특이적 프라이머 세트는 서열번호 1 및 2로 구성된 것일 수 있으나, ApoE 유전자의 단일염기다형성 부위를 포함하는 표적 핵산과 상보적인 염기서열을 가지는 프라이머 세트라면 특별히 제한되지는 않는다.In the present invention, the ApoE gene-specific primer set may be composed of SEQ ID NOs: 1 and 2, but if it is a primer set having a nucleotide sequence complementary to a target nucleic acid including a single nucleotide polymorphism site of the ApoE gene, it is not particularly limited does not

본 발명에 있어서, 상기 절단시약은 효소를 매개로 한 절단이 이루어지는 것이 바람직하나, 기타 공지된 절단시약이 사용되어도 무방하다. 이때, DNase, RNase, 헬리카제(helicase), 엑소뉴클리아제 및 엔도뉴클리아제 같은 효소들에 의해 촉매되는 RNA 또는 DNA의 절단을 표시하기 위하여, "효소매개 절단(enzyme-mediated cleavage)"이라는 용어를 사용한다. 본 발명의 바람직한 실시예에 있어서, 혼성화된 프로브의 닉(nick) 생성 및 절단은 엔도뉴클레아제 또는 엑소뉴클레아제인 리보뉴클리아제에 의해서 수행되는 것이 보다 바람직하다. 리보뉴클리아제는 이중 가닥 DNA-RNA 혼성화 가닥으로부터 리보 핵산(ribonucleic acid)에서 닉을 생성하고 절단시키는 이중 가닥 리보뉴클리아제인 것이 더욱 바람직하다.In the present invention, the cleavage reagent is preferably cleaved through an enzyme, but other known cleavage reagents may be used. At this time, to indicate the cleavage of RNA or DNA catalyzed by enzymes such as DNase, RNase, helicase, exonuclease and endonuclease, "enzyme-mediated cleavage" is called use the term In a preferred embodiment of the present invention, nick generation and cleavage of the hybridized probe is more preferably performed by ribonuclease, which is an endonuclease or an exonuclease. More preferably, the ribonuclease is a double-stranded ribonuclease that generates and cleaves a nick in ribonucleic acid from a double-stranded DNA-RNA hybridizing strand.

본 발명에 있어서, 상기 절단시약은 이에 제한되지는 않으나, RNaseH, RNase Ⅱ, RNase Ⅲ, RNase Ⅳ 또는 RNase T2의 RNA 가수분해효소(ribonuclease, RNase)일 수 있다.In the present invention, the cleavage reagent is not limited thereto, but may be an RNA hydrolase (ribonuclease, RNase) of RNaseH, RNase II, RNase III, RNase IV, or RNase T2.

d) 상기 증폭된 ApoE 유전자의 단일염기다형성 부위를 포함하는 표적 핵산-단일핵산 복합체로부터 분리된 단일핵산 단편의 양을 측정하는 단계이다.d) measuring the amount of a single nucleic acid fragment isolated from the target nucleic acid-mononucleic acid complex including the mononucleotide polymorphism site of the amplified ApoE gene.

본 발명에 있어서, 단일핵산 단편의 양 측정은 다양한 검출방법을 사용하여 수행될 수 있다. 구체적으로, 본 발명에 따라 분리된 단일핵산의 단편은 실시간 또는 반응이 종료된 후에 측정되는 것이 바람직하며, 형광광도의 변화 또는 화학발광의 측정으로 이루어질 수 있다.In the present invention, the amount of the mononucleic acid fragment can be measured using various detection methods. Specifically, the fragment of the mononucleic acid isolated according to the present invention is preferably measured in real time or after the reaction is finished, and can be made by changing the fluorescence intensity or measuring chemiluminescence.

상기 형광광도의 변화 또는 화학발광의 측정은 당업계에 공지된 형광 표지자 검출이 가능한 모든 측정 장치를 이용할 수 있으며, 예를 들어 트라이애드 멀티모드 디텍터(TRIAD Multimode Detector), 활락/빅터 형광(Wallac/Victor fluorescence) 또는 퍼킨-엘머 LB50B 형광분광광도계(Perkin-Elmer LB50B luminescence spectrometer), LightCycler96, Applied Biosystems 7500, 또는 Biorad CFX96 real-time PCR thermocycler 등을 이용하여 이루어질 수 있으며, 이에 한정하지 않는다.The change in fluorescence intensity or the measurement of chemiluminescence may be performed using any measuring device capable of detecting a fluorescent marker known in the art, for example, a triad multimode detector (TRIAD Multimode Detector), Victor fluorescence) or a Perkin-Elmer LB50B luminescence spectrometer, LightCycler96, Applied Biosystems 7500, or Biorad CFX96 real-time PCR thermocycler, but is not limited thereto.

본 발명에 따라 절단된 단일핵산 단편의 양 측정과 검출방법은 단일핵산 또는 반응액 내로 유입된 표지 또는 탐지 가능한 마커의 종류에 따라 달라질 수 있다.The method for measuring and detecting the amount of the cleaved mononucleic acid fragment according to the present invention may vary depending on the type of the mononucleic acid or the label or detectable marker introduced into the reaction solution.

본 발명의 단일핵산은 Y 부위가 절단된 후 증폭하는 단계에 의해 Y 부위의 유전적 변이 구별을 용이하게 하므로, 이후의 핵산 증폭 반응을 통해 돌연변이 확인을 가능하게 한다. 즉, 본 발명의 단일핵산에서 Y 부위와 표적 핵산의 단일염기다형성 부위에 확인하고자 하는 유전자형의 단일핵산이 혼성화를 형성하도록 하였을 경우, Y 부위가 ApoE 유전자의 단일염기다형성 부위와 정확하게 상보적인 결합을 한 경우에만 절단되고 이후에 증폭 반응이 수행되기 때문에 ApoE 유전자의 단일염기다형성을 명확하게 확인할 수 있다. 구체적으로, 본 발명의 단일핵산에서 Y 부위와 표적 핵산의 단일염기다형성 부위가 혼성화를 형성하도록 하였음에도 Y 부위가 상보적인 결합을 하지 않은 경우라면 Y 부위가 절단되지 않으므로 증폭 반응이 일어나지 않으며, 이는 표적 핵산에 측정하고자 하는 ApoE 유전자의 단일염기다형성 부위가 확인하고자 하는 유전자형이 아님을 의미하게 된다. Since the mononucleic acid of the present invention facilitates the discrimination of genetic mutations in the Y region by amplifying the Y region after cleavage, it is possible to identify the mutation through a subsequent nucleic acid amplification reaction. That is, when the single nucleic acid of the genotype to be confirmed forms a hybridization between the Y site and the single nucleotide polymorphism site of the target nucleic acid in the single nucleic acid of the present invention, the Y site accurately complements the single nucleotide polymorphism site of the ApoE gene. Since it is cleaved only in one case and an amplification reaction is performed thereafter, the single nucleotide polymorphism of the ApoE gene can be clearly identified. Specifically, if the Y site and the single nucleotide polymorphic site of the target nucleic acid in the mononucleic acid of the present invention form a hybridization, if the Y site does not form a complementary bond, the Y site is not cleaved, so the amplification reaction does not occur, which is the target nucleic acid. This means that the single nucleotide polymorphism site of the ApoE gene to be measured in the nucleic acid is not the genotype to be confirmed.

본 발명에 쉽게 이용할 수 있는 신장반응, 즉 핵산 증폭반응은 본 발명이 속하는 기술분야의 통상의 지식을 가진 자에게 공지되어 있다. 즉, 상기 표적 핵산의 증폭은 중합효소 연쇄반응(polymerase chain reaction; PCR), 회전환 증폭(rolling circle amplification; RCA), 핵산가닥 전치 증폭(strand displacement amplification; SDA) 또는 핵산서열기반 증폭(nucleic acid sequence based amplification; NASBA)을 포함하지만 상기 방법에 국한되지 아니한다. 상기 핵산 증폭 산물은 DNA 또는 RNA이다.An extension reaction, that is, a nucleic acid amplification reaction, which can be easily used in the present invention is known to those of ordinary skill in the art to which the present invention pertains. That is, the amplification of the target nucleic acid is a polymerase chain reaction (PCR), rolling circle amplification (RCA), strand displacement amplification (SDA), or nucleic acid sequence-based amplification (nucleic acid sequence). sequence based amplification (NASBA), but is not limited to the above methods. The nucleic acid amplification product is DNA or RNA.

일반적으로, 표적 핵산의 증폭 및 상기에서 기술한 단일핵산의 절단에 의한 탐지를 동시에 가능하도록, 반응 혼합액에 표적 핵산, 단일핵산, 핵산 증폭 반응의 구성물 및 절단 효소가 포함된다. 각 증폭반응은 완충액 조건, 프라이머, 반응온도 및 단일핵산 절단 조건 등을 각각 개별적으로 최적화시키는 것이 필요하다. 핵산 증폭반응과 연계하여 본 발명의 검출방법을 사용하면 표적 핵산을 탐지하는 민감도와 속도가 현저하게 개선될 것이다.In general, a target nucleic acid, a mononucleic acid, a constituent of a nucleic acid amplification reaction, and a cleaving enzyme are included in the reaction mixture to simultaneously enable amplification of the target nucleic acid and detection by cleavage of the mononucleic acid described above. For each amplification reaction, it is necessary to individually optimize buffer conditions, primers, reaction temperature, and mononucleic acid cleavage conditions. When the detection method of the present invention is used in conjunction with a nucleic acid amplification reaction, the sensitivity and speed of detecting a target nucleic acid will be remarkably improved.

한편, 본 발명의 ApoE 유전자의 단일염기다형성 실시간 검출용 단일핵산이 알츠하이머 및 심혈관계 질환 등과 관련된 ApoE 유전자의 단일염기다형성을 신속 정확하게 검출 및 탐지할 수 있음을 확인한 바, 상기 단일핵산은 ApoE 유전자형에 의한 다양한 질환, 예컨대 알츠하이머 및 심혈관계 질환을 진단하기 위한 키트 또는 조성물에 적용가능하며, 실시간으로 ApoE 유전자형에 의한 관련 질환 진단을 수행하여 알츠하이머 등과 같은 관련 질환 발생 여부에 대한 정보를 제공하는데 유용하게 이용될 수 있다.On the other hand, as it was confirmed that the single nucleic acid for real-time detection of the single nucleotide polymorphism of the ApoE gene of the present invention can rapidly and accurately detect and accurately detect the single nucleotide polymorphism of the ApoE gene associated with Alzheimer's disease and cardiovascular disease, the single nucleic acid is related to the ApoE genotype. It is applicable to kits or compositions for diagnosing various diseases caused by Alzheimer's disease and cardiovascular diseases, and is useful for providing information on whether related diseases such as Alzheimer's and the like occur by performing diagnosis of related diseases by ApoE genotype in real time can be

이하, 실시예를 통하여 본 발명의 구성 및 효과를 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것일 뿐, 본 발명의 범위가 이들 실시예에 의해 한정되는 것은 아니다.Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

실시예 1: 1형 단일핵산을 이용한 ApoE 분석Example 1: ApoE analysis using type 1 mononucleic acid

1형 단일핵산을 아포리포단백질 E(Apolipoprotein E, ApoE) 유전자의 6종의 표현형 E2/E2, E3/E3, E4/E4, E2/E3, E2/E4, E3/E4를 분석하는데 이용하였다.Type 1 mononucleic acid was used to analyze the six phenotypes E2/E2, E3/E3, E4/E4, E2/E3, E2/E4, and E3/E4 of the apolipoprotein E (ApoE) gene.

인간 염색체 19번에 위치한 ApoE 유전자는 심혈관계 및 알츠하이머 질병과 관련된 유전자이다. ApoE 유전자는 코돈(codon) 112(cys/arg), 코돈 158(cys/arg)의 DNA의 단일염기다형성(SNP)[게놈 DNA 위치 586(T/C), 724(T/C)번째]에 의해 세 가지의 대립 유전자 이형체(isoform)인 ApoEε2, ApoEε3, ApoEε4를 가지게 되며, 이 대립 유전자의 조합에 의해 6종의 표현형(E2/E2, E3/E3, E4/E4, E2/E3, E2/E4, E3/E4)을 갖게 된다.The ApoE gene, located on human chromosome 19, is a gene associated with cardiovascular and Alzheimer's disease. The ApoE gene is a single nucleotide polymorphism (SNP) of DNA of codon 112 (cys/arg) and codon 158 (cys/arg) [genomic DNA positions 586 (T/C), 724 (T/C)] It has three allele isoforms, ApoE ε2, ApoE ε3, and ApoE ε4, and the combination of these alleles results in six phenotypes (E2/E2, E3/E3, E4/E4, E2/ E3, E2/E4, E3/E4).

ApoE 다형성ApoE polymorphism 코돈 112codon 112 코돈 158codon 158 ApoE2/2ApoE2/2 Cys(TGC)Cys(TGC) Cys(TGC)Cys(TGC) ApoE3/3ApoE3/3 Cys(TGC)Cys(TGC) Arg(CGC)Arg(CGC) ApoE4/4ApoE4/4 Arg(CGC)Arg(CGC) Arg(CGC)Arg(CGC) ApoE2/3ApoE2/3 Cys(TGC)Cys(TGC) Cys(TGC)/Arg(CGC)Cys(TGC)/Arg(CGC) ApoE2/4ApoE2/4 Cys(TGC)/Arg(CGC)Cys(TGC)/Arg(CGC) Cys(TGC)/Arg(CGC)Cys(TGC)/Arg(CGC) ApoE3/4ApoE3/4 Cys(TGC)/Arg(CGC)Cys(TGC)/Arg(CGC) Arg(CGC)Arg(CGC)

상기 ApoE 유전자의 각 6종의 표현형을 구분하기 위하여, 5'-말단을 각각의 형광 염료(dye)가 부착된 4종의 개선된 형태의 1형 단일핵산을 이용하여 4-plex로 분석이 가능하도록 하였다. 기존의 분석법에서는 통상적으로 4-plex에 의한 분석법의 민감도 및 특이도를 충족시키기 쉽지 않았으나, 본 발명에서는 이에 대하여 충족할 만한 결과를 보여주었다.In order to distinguish each of the 6 types of phenotypes of the ApoE gene, it is possible to analyze the 5'-end in 4-plex using 4 types of improved type 1 mononucleic acids to which each fluorescent dye (dye) is attached. made to do Conventionally, it was not easy to satisfy the sensitivity and specificity of the 4-plex assay, but the present invention showed satisfactory results.

상세하게는 ApoE 유전자의 코돈 112 및 코돈 158의 대립 유전자형에 대한 SNP 여부를 측정하기 위하여, 본 발명에 따른 1형 단일핵산과 프라이머(primer)를 하기 표 8과 같이 IDT(Integrated DNA Technologies, USA)에 의뢰하여 제조하였다. 여기서 1형 단일핵산의 경우 X-Y-Z의 구조를 가지는 프로브로서 5'-말단에는 각각 6-FAM, HEX, TexasRed, Cy5를, 그리고 각 3'-말단에는 IABkFQ를 부착하였다. 또한, 리보뉴클레오티드(RNA)는 데옥시리보뉴클레오티드(DNA)와의 구별을 위하여 서열 앞에 첨자 "r"로 표시하였다. 112번 코돈의 SNP를 검출하기 위한 1형 단일핵산은 서열번호 3 및 4이며, 158번 코돈의 SNP를 검출하기 위한 1형 단일핵산은 서열번호 5 및 6이다.Specifically, in order to measure whether SNP for the allele of codon 112 and codon 158 of the ApoE gene, type 1 mononucleic acid and primers according to the present invention were used as shown in Table 8 below IDT (Integrated DNA Technologies, USA) Manufactured by request. Here, in the case of type 1 mononucleic acid, as a probe having an X-Y-Z structure, 6-FAM, HEX, TexasRed, and Cy5 were attached to the 5'-end, respectively, and IABkFQ was attached to each 3'-end. In addition, ribonucleotides (RNA) are indicated by a subscript "r" in front of the sequence to distinguish them from deoxyribonucleotides (DNA). Type 1 mononucleic acids for detecting the SNP of codon 112 are SEQ ID NOs: 3 and 4, and type 1 mononucleic acids for detecting the SNP of codon 158 are SEQ ID NOs: 5 and 6.

ApoE 특이적 프라이머 세트 및 4종의 ApoE 단일핵산(1형)ApoE-specific primer set and four ApoE mononucleic acids (type 1) 프라이머명/프로브명Primer name/probe name 서열order 서열번호SEQ ID NO: ApoE_F 프라이머ApoE_F primer 5'-GAAGGCCTACAAATCGGAACT-3' 5'-GAAGGCCTACAAATCGGAACT-3' 1One ApoE_R 프라이머ApoE_R primer 5'-GCCACCTGCTCCTTCAC-3' 5'-GCCACCTGCTCCTTCAC-3' 22 ApoE 단일핵산 1ApoE mononucleic acid 1 56-FAM/-GAGGACGTGrUGCGGCC-/3IABkFQ 56-FAM/-GAGGACGTGrUGCGGCC-/3IABkFQ 33 ApoE 단일핵산 2ApoE mononucleic acid 2 5-HEX/-GAGGACGTGrCGCGGCC-/3IABkFQ 5-HEX/-GAGGACGTGrCGCGGCC-/3IABkFQ 44 ApoE 단일핵산 3ApoE mononucleic acid 3 5-TexRd/-CTGCAGAAGrUGCCTGGCA-/3IABkFQ 5-TexRd/-CTGCAGAAGrUGCCTGGCA-/3IABkFQ 55 ApoE 단일핵산 4ApoE mononucleic acid 4 5-Cy5/-GCAGAAGrCGCCTGGCA-/3IABkFQ 5-Cy5/-GCAGAAGrCGCCTGGCA-/3IABkFQ 66

분석을 위하여 한국세포주은행으로부터 인간 세포주 PC3(E2/E2), A549(E3/E3), U937(E4/E4)을 분양받았고, 이로부터 게놈 DNA(genomic DNA)를 수득하였다. 6종의 표현형에 대한 분석을 위하여, 동형 표현형은 PC3(E2/E2), A549(E3/E3), U937(E4/E4)을 사용하였고, 이형 표현형은 각 게놈 DNA의 혼합형인 PC3+A549(E2/E3), PC3+U937(E2/E4), A549+U937(E3/E4)을 고농도로 반응당 32ng (약 104 카피(copy))으로 포함되도록 하여 분석에 사용하였다.For analysis, human cell lines PC3 (E2/E2), A549 (E3/E3), and U937 (E4/E4) were purchased from the Korea Cell Line Bank, and genomic DNA was obtained therefrom. For the analysis of the six phenotypes, the isotype phenotypes PC3 (E2/E2), A549 (E3/E3), and U937 (E4/E4) were used, and the heterozygous phenotype was PC3+A549 (a mixture of each genomic DNA). E2/E3), PC3+U937 (E2/E4), and A549+U937 (E3/E4) were used in the analysis at a high concentration of 32 ng (about 10 4 copies) per reaction.

상기 표 8의 ApoE 단일핵산 1, 2, 3, 4 각각의 0.2μM, 0.15μM, 0.15μM, 및 0.075μM과, ApoE 정방향 및 역방향 프라이머 각각의 0.35μM의 최종농도 존재하에서, 상기 정량한 게놈 DNA, 0.5U RNase-H, AptaTaq DNA Master(Roche) 4㎕, GC rich solution(Roche) 3㎕, nuclease-free water로 총 부피(total volume)가 20㎕로 되도록 조정한 후 중합효소 연쇄반응(Polymerase Chain Reaction; PCR)을 수행하였다. 이때, PCR 반응 조건은 95℃에서 5분, 95℃에서 15초, 65℃에서 70초로 45 사이클(cycle)을 수행하였다. 이에 대한 결과를 도 1에 나타내었다.The quantified genomic DNA in the presence of 0.2 μM, 0.15 μM, 0.15 μM, and 0.075 μM of each of ApoE mononucleic acids 1, 2, 3, and 4 of Table 8 and 0.35 μM of each of the ApoE forward and reverse primers. , 0.5U RNase-H, AptaTaq DNA Master (Roche) 4 μl, GC rich solution (Roche) 3 μl, nuclease-free water to adjust the total volume to 20 μl, and then polymerase chain reaction (Polymerase) Chain Reaction (PCR) was performed. At this time, the PCR reaction conditions were 5 minutes at 95°C, 15 seconds at 95°C, and 45 cycles at 65°C for 70 seconds. The results for this are shown in FIG. 1 .

그 결과, 한 개의 반응 웰(well)에서 ApoE 각 대립 유전자의 조합 6종에 대한 분석이 가능한 것을 확인하였다. 이 결과에서 단일염기다형성의 호모 또는 헤테로인 경우 개선된 프로브(probe) 형태를 사용할 경우 구분능이 우수함을 확인할 수 있었다.As a result, it was confirmed that analysis of 6 combinations of ApoE alleles was possible in one reaction well. From this result, it was confirmed that in the case of homo or hetero of the single nucleotide polymorphism, the ability to discriminate was excellent when the improved probe type was used.

실시예 2: 2형 단일핵산을 이용한 ApoE 분석Example 2: ApoE analysis using type 2 mononucleic acid

ApoE 유전자의 코돈(codon) 112 및 코돈 158의 대립 유전자형에 대한 SNP 여부를 측정하기 위하여, 본 발명에 따른 2형 단일핵산을 사용하였다. 하기 표 9와 같이, IDT(Integrated DNA Technologies, USA)에 의뢰하여 2형 단일핵산을 제조하였다.In order to determine whether or not the SNP for the alleles of codon 112 and codon 158 of the ApoE gene, type 2 mononucleic acid according to the present invention was used. As shown in Table 9 below, type 2 mononucleic acids were prepared by requesting IDT (Integrated DNA Technologies, USA).

여기서, 2형 단일핵산의 경우 X-Y-Z의 구조를 가지는 프라이머 및 프로브로서, 5'-말단에는 각각 6-FAM, HEX, TexasRed를, 그리고 각 3'-말단에는 IABkFQ를 부착하였다. 또한, 리보뉴클레오티드(RNA)는 데옥시리보뉴클레오티드(DNA)와의 구별을 위하여 서열 앞에 첨자 "r"로 표시하였다. 112번 코돈의 SNP를 검출하기 위한 2형 단일핵산은 서열번호 7 및 8이며, 158번 코돈의 SNP를 검출하기 위한 2형 단일핵산은 서열번호 9 및 10이다.Here, in the case of type 2 mononucleic acid, as primers and probes having the structure of X-Y-Z, 6-FAM, HEX, and TexasRed were attached to the 5'-end, respectively, and IABkFQ was attached to each 3'-end. In addition, ribonucleotides (RNA) are indicated by a subscript "r" in front of the sequence to distinguish them from deoxyribonucleotides (DNA). The type 2 mononucleic acids for detecting the SNP of codon 112 are SEQ ID NOs: 7 and 8, and the type 2 mononucleic acids for detecting the SNP of codon 158 are SEQ ID NOs: 9 and 10.

4종의 ApoE 단일핵산(2형)4 types of ApoE mononucleic acids (type 2) 단일핵산명single nucleic acid name 서열order 서열번호SEQ ID NO: ApoE 단일핵산1ApoE mononucleic acid 1 56-FAM/-CGGTCATGGAGGACGTGrUGC-/3IABkFQ 56-FAM/-CGGTCATGGAGGACGTGrUGC-/3IABkFQ 77 ApoE 단일핵산2ApoE mononucleic acid 2 5TexRd-XN/-GCGGATATGGAGGACGTrGCG-/3IABkFQ 5TexRd-XN/-GCGGATATGGAGGACGTrGCG-/3IABkFQ 88 ApoE 단일핵산3ApoE mononucleic acid 3 56-FAM/-CTGGTACACTGCCAGGCArCTT-/3IABkFQ 56-FAM/-CTGGTACACTGCCAGGCArCTT-/3IABkFQ 99 ApoE 단일핵산4ApoE mononucleic acid 4 5HEX/-CTGGTACACTGCCAGGCrGATTC-/3IABkFQ 5HEX/-CTGGTACACTGCCAGGCrGATTC-/3IABkFQ 1010

분석을 위하여 한국세포주은행으로부터 인간 세포주 PC3(E2/E2), A549(E3/E3), U937(E4/E4)을 분양받았고, 이로부터 게놈 DNA를 수득하였다. 6종의 표현형에 대한 분석을 위하여, 동형 표현형은 PC3(E2/E2), A549(E3/E3), U937(E4/E4)을 사용하였고, 이형 표현형은 각 게놈 DNA의 혼합형인 PC3+A549(E2/E3), PC3+U937(E2/E4), A549+U937(E3/E4)을 고농도로 반응당 32ng(약 104 카피)으로 포함되도록 하여 분석에 사용하였다.For analysis, human cell lines PC3 (E2/E2), A549 (E3/E3), and U937 (E4/E4) were purchased from the Korea Cell Line Bank, and genomic DNA was obtained therefrom. For the analysis of the six phenotypes, the isotype phenotypes PC3 (E2/E2), A549 (E3/E3), and U937 (E4/E4) were used, and the heterozygous phenotype was PC3+A549 (a mixture of each genomic DNA). E2/E3), PC3+U937 (E2/E4), and A549+U937 (E3/E4) were used in the analysis at a high concentration of 32 ng (about 10 4 copies) per reaction.

상기 표 9의 ApoE 단일핵산 1, 2, 3, 4 각각의 0.375μM, 0.1μM, 0.25μM, 및 0.25μM의 최종농도 존재하에서, 상기 게놈 DNA와 0.1ng의 내열성 RNase H, 그리고 AptaTaq DNA Master w/o MgCl2(Roche) 4㎕, GC rich solution(Roche) 4㎕, 2.75mM MgCl2, 62.5nM Low ROX에 nuclease-free water로 총 부피가 20㎕로 되도록 조정한 후 중합효소 연쇄반응(PCR)을 수행하였다. 이때, PCR 반응 조건은 95℃에서 10분, 95℃에서 15초, 64℃에서 55초로 40 사이클을 수행하였다. 이에 대한 결과를 도 2에 나타내었다.In the presence of final concentrations of 0.375 μM, 0.1 μM, 0.25 μM, and 0.25 μM of ApoE mononucleic acids 1, 2, 3, and 4 of Table 9, respectively, the genomic DNA, 0.1 ng of heat-resistant RNase H, and AptaTaq DNA Master w /o MgCl 2 (Roche) 4 μl, GC rich solution (Roche) 4 μl, 2.75 mM MgCl 2 , 62.5 nM Low ROX After adjusting the total volume to 20 μl with nuclease-free water, polymerase chain reaction (PCR) ) was performed. At this time, 40 cycles of PCR reaction conditions were performed at 95° C. for 10 minutes, 95° C. for 15 seconds, and at 64° C. for 55 seconds. The results for this are shown in FIG. 2 .

그 결과, 본 발명에 따른 2형 단일핵산을 이용한 ApoE 각 대립 유전자의 조합 6종에 대한 분석에서, 코돈 112의 586T 변이와 코돈 158의 724T 변이의 경우 형광 염료 차이에 의한 분석이 아닌 동일 형광 염료에 의한 종료점(end point) 형광 값의 차이로 구분이 가능한 제한점이 있었지만, 1형 단일핵산과 더불어 2형 단일핵산도 단일염기다형성의 호모 또는 헤테로의 구분능이 우수함을 확인할 수 있었다.As a result, in the analysis of 6 combinations of each ApoE allele using the type 2 mononucleic acid according to the present invention, in the case of the 586T mutation of codon 112 and the 724T mutation of codon 158, the same fluorescent dye was not analyzed by the difference in the fluorescent dye. Although there were limitations that could be distinguished by the difference in end point fluorescence values by , it was confirmed that the homo or hetero of single nucleotide polymorphisms were excellent in type 2 mononucleic acids as well as type 1 mononucleic acids.

<110> NuriBio Co., Ltd. <120> Promer for real-time detection for SNP analysis of ApoE gene and detection method using the same <130> PA-20-0124 <160> 104 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ApoE_F primer <400> 1 gaaggcctac aaatcggaac t 21 <210> 2 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> ApoE_R primer <400> 2 gccacctgct ccttcac 17 <210> 3 <211> 16 <212> DNA_RNA <213> Artificial Sequence <220> <223> Single nucleic acid type 1 for detecting SNP in 112 codon of ApoE gene <220> <223> RNA is located at 10th of sequence <400> 3 gaggacgtgu gcggcc 16 <210> 4 <211> 16 <212> DNA_RNA <213> Artificial Sequence <220> <223> Single nucleic acid type 1 for detecting SNP in 112 codon of ApoE gene <220> <223> RNA is located at 10th of sequence <400> 4 gaggacgtgc gcggcc 16 <210> 5 <211> 18 <212> DNA_RNA <213> Artificial Sequence <220> <223> Single nucleic acid type 1 for detecting SNP in 158 codon of ApoE gene <220> <223> RNA is located at 10th of sequence <400> 5 ctgcagaagu gcctggca 18 <210> 6 <211> 16 <212> DNA_RNA <213> Artificial Sequence <220> <223> Single nucleic acid type 1 for detecting SNP in 158 codon of ApoE gene <220> <223> RNA is located at 8th of sequence <400> 6 gcagaagcgc ctggca 16 <210> 7 <211> 20 <212> DNA_RNA <213> Artificial Sequence <220> <223> Single nucleic acid type 2 for detecting SNP in 112 codon of ApoE gene <220> <223> RNA is located at 18th of sequence <400> 7 cggtcatgga ggacgtgugc 20 <210> 8 <211> 20 <212> DNA_RNA <213> Artificial Sequence <220> <223> Single nucleic acid type 2 for detecting SNP in 112 codon of ApoE gene <220> <223> RNA is located at 18th of sequence <400> 8 gcggatatgg aggacgtgcg 20 <210> 9 <211> 21 <212> DNA_RNA <213> Artificial Sequence <220> <223> Single nucleic acid type 2 for detecting SNP in 158 codon of ApoE gene <220> <223> RNA is located at 19th of sequence <400> 9 ctggtacact gccaggcact t 21 <210> 10 <211> 22 <212> DNA_RNA <213> Artificial Sequence <220> <223> Single nucleic acid type 2 for detecting SNP in 158 codon of ApoE gene <220> <223> RNA is located at 18th of sequence <400> 10 ctggtacact gccaggcgat tc 22 <210> 11 <211> 4 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 11 cgtg 4 <210> 12 <211> 5 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 12 acgtg 5 <210> 13 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 13 gacgtg 6 <210> 14 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 14 ggacgtg 7 <210> 15 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 15 aggacgtg 8 <210> 16 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 16 gaggacgtg 9 <210> 17 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 17 ggaggacgtg 10 <210> 18 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 18 tggaggacgt g 11 <210> 19 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 19 atggaggacg tg 12 <210> 20 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 20 catggaggac gtg 13 <210> 21 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 21 acatggagga cgtg 14 <210> 22 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 22 gacatggagg acgtg 15 <210> 23 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 23 ggacatggag gacgtg 16 <210> 24 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 24 cggacatgga ggacgtg 17 <210> 25 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 25 gcggacatgg aggacgtg 18 <210> 26 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 26 cgcggacatg gaggacgtg 19 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 27 gcgcggacat ggaggacgtg 20 <210> 28 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 28 ggcgcggaca tggaggacgt g 21 <210> 29 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 29 gggcgcggac atggaggacg tg 22 <210> 30 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 30 tgggcgcgga catggaggac gtg 23 <210> 31 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 31 ctgggcgcgg acatggagga cgtg 24 <210> 32 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 32 gctgggcgcg gacatggagg acgtg 25 <210> 33 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 33 ggctgggcgc ggacatggag gacgtg 26 <210> 34 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 34 cggctgggcg cggacatgga ggacgtg 27 <210> 35 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 35 ccggctgggc gcggacatgg aggacgtg 28 <210> 36 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 36 cccggctggg cgcggacatg gaggacgtg 29 <210> 37 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 37 gcccggctgg gcgcggacat ggaggacgtg 30 <210> 38 <211> 4 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 38 gaag 4 <210> 39 <211> 5 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 39 agaag 5 <210> 40 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 40 cagaag 6 <210> 41 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 41 gcagaag 7 <210> 42 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 42 tgcagaag 8 <210> 43 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 43 ctgcagaag 9 <210> 44 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 44 cctgcagaag 10 <210> 45 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 45 acctgcagaa g 11 <210> 46 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 46 gacctgcaga ag 12 <210> 47 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 47 tgacctgcag aag 13 <210> 48 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 48 atgacctgca gaag 14 <210> 49 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 49 gatgacctgc agaag 15 <210> 50 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 50 cgatgacctg cagaag 16 <210> 51 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 51 ccgatgacct gcagaag 17 <210> 52 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 52 gccgatgacc tgcagaag 18 <210> 53 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 53 tgccgatgac ctgcagaag 19 <210> 54 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 54 atgccgatga cctgcagaag 20 <210> 55 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 55 gatgccgatg acctgcagaa g 21 <210> 56 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 56 cgatgccgat gacctgcaga ag 22 <210> 57 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 57 gcgatgccga tgacctgcag aag 23 <210> 58 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 58 cgcgatgccg atgacctgca gaag 24 <210> 59 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 59 ccgcgatgcc gatgacctgc agaag 25 <210> 60 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 60 tccgcgatgc cgatgacctg cagaag 26 <210> 61 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 61 ctccgcgatg ccgatgacct gcagaag 27 <210> 62 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 62 cctccgcgat gccgatgacc tgcagaag 28 <210> 63 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 63 tcctccgcga tgccgatgac ctgcagaag 29 <210> 64 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 64 ctcctccgcg atgccgatga cctgcagaag 30 <210> 65 <211> 1 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 65 g 1 <210> 66 <211> 2 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 66 gc 2 <210> 67 <211> 3 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 67 gcg 3 <210> 68 <211> 4 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 68 gcgg 4 <210> 69 <211> 5 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 69 gcggc 5 <210> 70 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 70 gcggcc 6 <210> 71 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 71 gcggccg 7 <210> 72 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 72 gcggccgc 8 <210> 73 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 73 gcggccgcc 9 <210> 74 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 74 gcggccgcct 10 <210> 75 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 75 gcggccgcct g 11 <210> 76 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 76 gcggccgcct gg 12 <210> 77 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 77 gcggccgcct ggt 13 <210> 78 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 78 gcggccgcct ggtg 14 <210> 79 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 79 gcggccgcct ggtgc 15 <210> 80 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 80 gcggccgcct ggtgca 16 <210> 81 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 81 gcggccgcct ggtgcag 17 <210> 82 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 82 gcggccgcct ggtgcagt 18 <210> 83 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 83 gcggccgcct ggtgcagta 19 <210> 84 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 84 gcggccgcct ggtgcagtac 20 <210> 85 <211> 1 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 85 g 1 <210> 86 <211> 2 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 86 gc 2 <210> 87 <211> 3 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 87 gcc 3 <210> 88 <211> 4 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 88 gcct 4 <210> 89 <211> 5 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 89 gcctg 5 <210> 90 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 90 gcctgg 6 <210> 91 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 91 gcctggc 7 <210> 92 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 92 gcctggca 8 <210> 93 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 93 gcctggcag 9 <210> 94 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 94 gcctggcagt 10 <210> 95 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 95 gcctggcagt g 11 <210> 96 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 96 gcctggcagt gt 12 <210> 97 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 97 gcctggcagt gta 13 <210> 98 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 98 gcctggcagt gtac 14 <210> 99 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 99 gcctggcagt gtacc 15 <210> 100 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 100 gcctggcagt gtacca 16 <210> 101 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 101 gcctggcagt gtaccag 17 <210> 102 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 102 gcctggcagt gtaccagg 18 <210> 103 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 103 gcctggcagt gtaccaggc 19 <210> 104 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 104 gcctggcagt gtaccaggcc 20 <110> NuriBio Co., Ltd. <120> Promer for real-time detection for SNP analysis of ApoE gene and detection method using the same <130> PA-20-0124 <160> 104 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ApoE_F primer <400> 1 gaaggcctac aaatcggaac t 21 <210> 2 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> ApoE_R primer <400> 2 gccacctgct ccttcac 17 <210> 3 <211> 16 <212> DNA_RNA <213> Artificial Sequence <220> <223> Single nucleic acid type 1 for detecting SNP in 112 codon of ApoE gene <220> <223> RNA is located at 10th of sequence <400> 3 gaggacgtgu gcggcc 16 <210> 4 <211> 16 <212> DNA_RNA <213> Artificial Sequence <220> <223> Single nucleic acid type 1 for detecting SNP in 112 codon of ApoE gene <220> <223> RNA is located at 10th of sequence <400> 4 gaggacgtgc gcggcc 16 <210> 5 <211> 18 <212> DNA_RNA <213> Artificial Sequence <220> <223> Single nucleic acid type 1 for detecting SNP in 158 codon of ApoE gene <220> <223> RNA is located at 10th of sequence <400> 5 ctgcagaagu gcctggca 18 <210> 6 <211> 16 <212> DNA_RNA <213> Artificial Sequence <220> <223> Single nucleic acid type 1 for detecting SNP in 158 codon of ApoE gene <220> <223> RNA is located at 8th of sequence <400> 6 gcagaagcgc ctggca 16 <210> 7 <211> 20 <212> DNA_RNA <213> Artificial Sequence <220> <223> Single nucleic acid type 2 for detecting SNP in 112 codon of ApoE gene <220> <223> RNA is located at 18th of sequence <400> 7 cggtcatgga ggacgtgugc 20 <210> 8 <211> 20 <212> DNA_RNA <213> Artificial Sequence <220> <223> Single nucleic acid type 2 for detecting SNP in 112 codon of ApoE gene <220> <223> RNA is located at 18th of sequence <400> 8 gcggatatgg aggacgtgcg 20 <210> 9 <211> 21 <212> DNA_RNA <213> Artificial Sequence <220> <223> Single nucleic acid type 2 for detecting SNP in 158 codon of ApoE gene <220> <223> RNA is located at 19th of sequence <400> 9 ctggtacact gccaggcact t 21 <210> 10 <211> 22 <212> DNA_RNA <213> Artificial Sequence <220> <223> Single nucleic acid type 2 for detecting SNP in 158 codon of ApoE gene <220> <223> RNA is located at 18th of sequence <400> 10 ctggtacact gccaggcgat tc 22 <210> 11 <211> 4 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 11 cgtg 4 <210> 12 <211> 5 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 12 acgtg 5 <210> 13 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 13 gacgtg 6 <210> 14 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 14 ggacgtg 7 <210> 15 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 15 aggacgtg 8 <210> 16 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 16 gaggacgtg 9 <210> 17 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 17 ggaggacgtg 10 <210> 18 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 18 tggaggacgt g 11 <210> 19 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 19 atggaggacg tg 12 <210> 20 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 20 catggaggac gtg 13 <210> 21 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 21 acatggagga cgtg 14 <210> 22 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 22 gacatggagg acgtg 15 <210> 23 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 23 ggacatggag gacgtg 16 <210> 24 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 24 cggacatgga ggacgtg 17 <210> 25 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 25 gcggacatgg aggacgtg 18 <210> 26 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 26 cgcggacatg gaggacgtg 19 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 27 gcgcggacat ggaggacgtg 20 <210> 28 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 28 ggcgcggaca tggaggacgt g 21 <210> 29 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 29 gggcgcggac atggaggacg tg 22 <210> 30 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 30 tgggcgcgga catggaggac gtg 23 <210> 31 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 31 ctgggcgcgg acatggagga cgtg 24 <210> 32 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 32 gctgggcgcg gacatggagg acgtg 25 <210> 33 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 33 ggctgggcgc ggacatggag gacgtg 26 <210> 34 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 34 cggctgggcg cggacatgga ggacgtg 27 <210> 35 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 35 ccggctgggc gcggacatgg aggacgtg 28 <210> 36 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 36 cccggctggg cgcggacatg gaggacgtg 29 <210> 37 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 37 gcccggctgg gcgcggacat ggaggacgtg 30 <210> 38 <211> 4 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 38 gaag 4 <210> 39 <211> 5 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 39 agaag 5 <210> 40 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 40 cagaag 6 <210> 41 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 41 gcagaag 7 <210> 42 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 42 tgcagaag 8 <210> 43 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 43 ctgcagaag 9 <210> 44 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 44 cctgcagaag 10 <210> 45 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 45 acctgcagaa g 11 <210> 46 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 46 gacctgcaga ag 12 <210> 47 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 47 tgacctgcag aag 13 <210> 48 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 48 atgacctgca gaag 14 <210> 49 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 49 gatgacctgc agaag 15 <210> 50 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 50 cgatgacctg cagaag 16 <210> 51 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 51 ccgatgacct gcagaag 17 <210> 52 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 52 gccgatgacc tgcagaag 18 <210> 53 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 53 tgccgatgac ctgcagaag 19 <210> 54 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 54 atgccgatga cctgcagaag 20 <210> 55 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 55 gatgccgatg acctgcagaa g 21 <210> 56 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 56 cgatgccgat gacctgcaga ag 22 <210> 57 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 57 gcgatgccga tgacctgcag aag 23 <210> 58 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 58 cgcgatgccg atgacctgca gaag 24 <210> 59 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 59 ccgcgatgcc gatgacctgc agaag 25 <210> 60 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 60 tccgcgatgc cgatgacctg cagaag 26 <210> 61 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 61 ctccgcgatg ccgatgacct gcagaag 27 <210> 62 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 62 cctccgcgat gccgatgacc tgcagaag 28 <210> 63 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 63 tcctccgcga tgccgatgac ctgcagaag 29 <210> 64 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> X part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 64 ctcctccgcg atgccgatga cctgcagaag 30 <210> 65 <211> 1 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 65 g 1 <210> 66 <211> 2 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 66 gc 2 <210> 67 <211> 3 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 67 gcg 3 <210> 68 <211> 4 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 68 gcgg 4 <210> 69 <211> 5 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 69 gcggc 5 <210> 70 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 70 gcggcc 6 <210> 71 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 71 gcggccg 7 <210> 72 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 72 gcggccgc 8 <210> 73 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 73 gcggccgcc 9 <210> 74 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 74 gcggccgcct 10 <210> 75 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 75 gcggccgcct g 11 <210> 76 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 76 gcggccgcct gg 12 <210> 77 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 77 gcggccgcct ggt 13 <210> 78 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 78 gcggccgcct ggtg 14 <210> 79 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 79 gcggccgcct ggtgc 15 <210> 80 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 80 gcggccgcct ggtgca 16 <210> 81 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 81 gcggccgcct ggtgcag 17 <210> 82 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 82 gcggccgcct ggtgcagt 18 <210> 83 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 83 gcggccgcct ggtgcagta 19 <210> 84 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 112 codon of ApoE gene <400> 84 gcggccgcct ggtgcagtac 20 <210> 85 <211> 1 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 85 g 1 <210> 86 <211> 2 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 86 gc 2 <210> 87 <211> 3 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 87 gcc 3 <210> 88 <211> 4 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 88 gcct 4 <210> 89 <211> 5 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 89 gcctg 5 <210> 90 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 90 gcctgg 6 <210> 91 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 91 gcctggc 7 <210> 92 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 92 gcctggca 8 <210> 93 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 93 gcctggcag 9 <210> 94 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 94 gcctggcagt 10 <210> 95 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 95 gcctggcagt g 11 <210> 96 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 96 gcctggcagt gt 12 <210> 97 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 97 gcctggcagt gta 13 <210> 98 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 98 gcctggcagt gtac 14 <210> 99 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 99 gcctggcagt gtacc 15 <210> 100 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 100 gcctggcagt gtacca 16 <210> 101 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 101 gcctggcagt gtaccag 17 <210> 102 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 102 gcctggcagt gtaccagg 18 <210> 103 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 103 gcctggcagt gtaccaggc 19 <210> 104 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Z part of Single nucleic acid for detecting SNP in 158 codon of ApoE gene <400> 104 gcctggcagt gtaccaggcc 20

Claims (24)

ApoE(apolipoprotein E) 유전자의 단일염기다형성 검출용 단일핵산으로서,
상기 단일핵산은 ⅰ) X-Y-Z의 구조를 가지며,
ⅱ) 단일염기다형성 부위를 포함한 ApoE 유전자의 염기서열 일부 또는 전체에 상보적 결합을 하고,
ⅲ) 양 말단 또는 내부에 동일하거나 적어도 2개의 상이한 탐지 가능한 마커가 부착되어 있으며,
iv) 상기 Y는 ApoE 유전자에 위치하는 1개 또는 2개의 염기서열로 구성된 RNA로서 ApoE 유전자와 혼성화시 절단 효소에 의해 절단되며,
상기 ApoE 유전자의 단일염기다형성을 확인하기 위한 1형 단일핵산의 경우에, 상기 X는 4 내지 20개의 염기서열로 구성되는 DNA이며, 상기 Z는 1 내지 20개의 염기서열로 구성되는 DNA이며, 상기 ApoE 유전자와 단일핵산이 혼성화된 후 절단 시약에 의해 Y가 절단될 때 X 및 Z도 ApoE 유전자로부터 분리되는 것을 특징으로 하며,
상기 ApoE 유전자의 단일염기다형성을 확인하기 위한 2형 단일핵산의 경우에, 상기 X는 10 내지 30개의 염기서열로 구성되는 DNA이며, 상기 Z는 1 내지 5개의 염기서열로 구성되는 DNA이며, 상기 ApoE 유전자와 단일핵산이 혼성화된 후 절단 시약에 의해 Y가 절단될 때 Z는 ApoE 유전자로부터 분리되지만 X는 분리되지 않고 프라이머 및 프로브로 작동하는 것을 특징으로 하는, 단일핵산.
A single nucleic acid for detecting single nucleotide polymorphisms of ApoE (apolipoprotein E) gene,
The mononucleic acid has the structure of i) XYZ,
ii) complementary binding to some or all of the nucleotide sequence of the ApoE gene including the single nucleotide polymorphism site;
iii) the same or at least two different detectable markers are attached to both ends or inside,
iv) Y is RNA composed of one or two base sequences located in the ApoE gene, and is cleaved by a cleavage enzyme when hybridizing with the ApoE gene,
In the case of type 1 mononucleic acid for confirming the single nucleotide polymorphism of the ApoE gene, X is DNA consisting of 4 to 20 nucleotide sequences, Z is DNA consisting of 1 to 20 nucleotide sequences, and It is characterized in that X and Z are also separated from the ApoE gene when Y is cleaved by a cleavage reagent after the ApoE gene and the mononucleic acid are hybridized,
In the case of type 2 mononucleic acid for confirming the single nucleotide polymorphism of the ApoE gene, X is DNA consisting of 10 to 30 nucleotide sequences, Z is DNA consisting of 1 to 5 nucleotide sequences, and A mononucleic acid, characterized in that when Y is cleaved by a cleavage reagent after hybridization of the ApoE gene and the mononucleic acid, Z is separated from the ApoE gene but X is not separated, and acts as a primer and a probe.
 제1항에 있어서,
상기 ApoE 유전자의 단일염기다형성(SNP)을 탐지하기 위해 Y부위가 단일염기다형성 부위에 직접 혼성화 되는 것을 특징으로 하는, 단일핵산.
According to claim 1,
A single nucleic acid, characterized in that the Y site is directly hybridized to the single nucleotide polymorphism site in order to detect the single nucleotide polymorphism (SNP) of the ApoE gene.
 제1항에 있어서, 단일핵산은 서열번호 3, 4, 5 및 6; 또는 서열번호 7, 8, 9 및 10으로 구성된 것인, 단일핵산.
According to claim 1, wherein the mononucleic acid is SEQ ID NOs: 3, 4, 5 and 6; Or consisting of SEQ ID NOs: 7, 8, 9 and 10, a mononucleic acid.
 제1항에 있어서, ApoE 유전자의 112번 코돈의 단일염기다형성을 확인하기 위한 1형 단일핵산의 X는 서열번호 27 또는 상기 서열번호 27의 염기서열 3‘ 말단을 기준으로 4개 내지 19개의 염기를 포함하는 염기서열로 이루어진 군으로부터 선택된 어느 하나의 염기서열인 것을 특징으로 하는, 단일핵산.
According to claim 1, wherein X of the type 1 mononucleic acid for confirming the single nucleotide polymorphism of codon 112 of the ApoE gene is 4 to 19 bases based on SEQ ID NO: 27 or the 3' end of the nucleotide sequence of SEQ ID NO: 27 A single nucleic acid, characterized in that it is any one base sequence selected from the group consisting of a base sequence comprising a.
 제4항에 있어서, ApoE 유전자의 112번 코돈의 단일염기다형성을 확인하기 위한 1형 단일핵산의 X는 서열번호 11 내지 27로 이루어진 군으로부터 선택된 어느 하나의 염기서열인 것을 특징으로 하는, 단일핵산.
The single nucleic acid according to claim 4, wherein X of the type 1 single nucleic acid for confirming the single nucleotide polymorphism of codon 112 of the ApoE gene is any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 11 to 27. .
 제1항에 있어서, ApoE 유전자의 112번 코돈의 단일염기다형성을 확인하기 위한 2형 단일핵산의 X는 서열번호 37 또는 상기 서열번호 37의 염기서열 3‘ 말단을 기준으로 10개 내지 29개의 염기를 포함하는 염기서열로 이루어진 군으로부터 선택된 어느 하나의 염기서열인 것을 특징으로 하는, 단일핵산.
According to claim 1, wherein X of the type 2 mononucleic acid for confirming the single nucleotide polymorphism of codon 112 of the ApoE gene is SEQ ID NO: 37 or 10 to 29 bases based on the 3' end of the nucleotide sequence of SEQ ID NO: 37 A single nucleic acid, characterized in that it is any one base sequence selected from the group consisting of a base sequence comprising a.
 제6항에 있어서, ApoE 유전자의 112번 코돈의 단일염기다형성을 확인하기 위한 2형 단일핵산의 X는 서열번호 17 내지 37로 이루어진 군으로부터 선택된 어느 하나의 염기서열인 것을 특징으로 하는, 단일핵산.
The single nucleic acid according to claim 6, wherein X of the type 2 mononucleic acid for confirming the single nucleotide polymorphism of codon 112 of the ApoE gene is any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 17 to 37. .
 제1항에 있어서, ApoE 유전자의 158번 코돈의 단일염기다형성을 확인하기 위한 1형 단일핵산의 X는 서열번호 54 또는 상기 서열번호 54의 염기서열 3‘ 말단을 기준으로 4개 내지 19개의 염기를 포함하는 염기서열로 이루어진 군으로부터 선택된 어느 하나의 염기서열인 것을 특징으로 하는, 단일핵산.
According to claim 1, wherein X of the type 1 mononucleic acid for confirming the single nucleotide polymorphism of codon 158 of the ApoE gene is 4 to 19 bases based on SEQ ID NO: 54 or the 3' end of the nucleotide sequence of SEQ ID NO: 54 A single nucleic acid, characterized in that it is any one base sequence selected from the group consisting of a base sequence comprising a.
 제8항에 있어서, ApoE 유전자의 158번 코돈의 단일염기다형성을 확인하기 위한 1형 단일핵산의 X는 서열번호 38 내지 54로 이루어진 군으로부터 선택된 어느 하나의 염기서열인 것을 특징으로 하는, 단일핵산.
The mononucleic acid according to claim 8, wherein X of the type 1 mononucleic acid for confirming the single nucleotide polymorphism of codon 158 of the ApoE gene is any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 38 to 54. .
 제1항에 있어서, ApoE 유전자의 158번 코돈의 단일염기다형성을 확인하기 위한 2형 단일핵산의 X는 서열번호 64 또는 상기 서열번호 64의 염기서열의 3‘ 말단을 기준으로 10개 내지 29개의 염기를 포함하는 염기서열로 이루어진 군으로부터 선택된 어느 하나의 염기서열인 것을 특징으로 하는, 단일핵산.
According to claim 1, wherein X of the type 2 mononucleic acid for confirming the single nucleotide polymorphism of codon 158 of the ApoE gene is 10 to 29 based on SEQ ID NO: 64 or the 3' end of the nucleotide sequence of SEQ ID NO: 64 A single nucleic acid, characterized in that it is any one base sequence selected from the group consisting of base sequences including bases.
 제10항에 있어서, ApoE 유전자의 158번 코돈의 단일염기다형성을 확인하기 위한 2형 단일핵산의 X는 서열번호 44 내지 64로 이루어진 군으로부터 선택된 어느 하나의 염기서열인 것을 특징으로 하는, 단일핵산.
The mononucleic acid according to claim 10, wherein X of the type 2 mononucleic acid for confirming the single nucleotide polymorphism of codon 158 of the ApoE gene is any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 44 to 64. .
 제1항에 있어서, ApoE 유전자의 112번 코돈의 단일염기다형성을 확인하기 위한 1형 단일핵산의 Z는 서열번호 84 또는 상기 서열번호 84의 염기서열 5‘ 말단을 기준으로 1개 내지 19개의 염기를 포함하는 염기서열로 이루어진 군으로부터 선택된 어느 하나의 염기서열인 것을 특징으로 하는, 단일핵산.
According to claim 1, wherein Z of the type 1 mononucleic acid for confirming the single nucleotide polymorphism of codon 112 of the ApoE gene is SEQ ID NO: 84 or 1 to 19 bases based on the 5' end of the nucleotide sequence of SEQ ID NO: 84 A single nucleic acid, characterized in that it is any one base sequence selected from the group consisting of a base sequence comprising a.
 제12항에 있어서, ApoE 유전자의 112번 코돈의 단일염기다형성을 확인하기 위한 1형 단일핵산의 Z는 서열번호 65 내지 84로 이루어진 군으로부터 선택된 어느 하나의 염기서열인 것을 특징으로 하는, 단일핵산.
The mononucleic acid according to claim 12, wherein Z of the type 1 mononucleic acid for confirming the single nucleotide polymorphism of codon 112 of the ApoE gene is any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 65 to 84. .
 제1항에 있어서, ApoE 유전자의 112번 코돈의 단일염기다형성을 확인하기 위한 2형 단일핵산의 Z는 서열번호 65 내지 69로 이루어진 군으로부터 선택된 어느 하나의 염기서열인 것을 특징으로 하는, 단일핵산.
The single nucleic acid according to claim 1, wherein Z of the type 2 mononucleic acid for confirming the single nucleotide polymorphism of codon 112 of the ApoE gene is any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 65 to 69. .
 제1항에 있어서, ApoE 유전자의 158번 코돈의 단일염기다형성을 확인하기 위한 1형 단일핵산의 Z는 서열번호 104 또는 상기 서열번호 104의 염기서열 5‘ 말단을 기준으로 1개 내지 19개의 염기를 포함하는 염기서열로 이루어진 군으로부터 선택된 어느 하나의 염기서열인 것을 특징으로 하는, 단일핵산.
The method of claim 1, wherein Z of the type 1 mononucleic acid for confirming the single nucleotide polymorphism of codon 158 of the ApoE gene is 1 to 19 bases based on SEQ ID NO: 104 or the 5' end of the nucleotide sequence of SEQ ID NO: 104 A single nucleic acid, characterized in that it is any one base sequence selected from the group consisting of a base sequence comprising a.
 제15항에 있어서, ApoE 유전자의 158번 코돈의 단일염기다형성을 확인하기 위한 1형 단일핵산의 Z는 서열번호 85 내지 104로 이루어진 군으로부터 선택된 어느 하나의 염기서열인 것을 특징으로 하는, 단일핵산.
The mononucleic acid according to claim 15, wherein Z of the type 1 mononucleic acid for confirming the single nucleotide polymorphism of codon 158 of the ApoE gene is any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 85 to 104. .
 제1항에 있어서, ApoE 유전자의 158번 코돈의 단일염기다형성을 확인하기 위한 2형 단일핵산의 Z는 서열번호 85 내지 89로 이루어진 군으로부터 선택된 어느 하나의 염기서열인 것을 특징으로 하는, 단일핵산.
The single nucleic acid according to claim 1, wherein Z of the type 2 mononucleic acid for confirming the single nucleotide polymorphism of codon 158 of the ApoE gene is any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 85 to 89. .
 제1항에 있어서,
상기 탐지 가능한 마커는 단일핵산에 공유 결합 또는 비공유 결합에 의해 결합하는 형광물질, 또는 형광물질과 소광물질로 이루어진 형광쌍인 것인, 단일핵산.
According to claim 1,
The detectable marker is a fluorescent material that binds to the mononucleic acid by covalent or non-covalent bonding, or a fluorescent pair consisting of a fluorescent material and a quenching material.
 제1항 내지 제18항 중 어느 한 항의 단일핵산을 포함하는, ApoE 유전자의 단일염기다형성 실시간 검출용 키트.
A kit for real-time detection of a single nucleotide polymorphism of the ApoE gene, comprising the single nucleic acid of any one of claims 1 to 18.
 제19항에 있어서,
상기 키트는 효소를 더 포함하는 키트로,
상기 효소는 RNaseH, RNase Ⅱ, RNase Ⅲ, RNase Ⅳ 또는 RNase T2의 RNA 가수분해효소(ribonuclease, RNase)인, 키트.
20. The method of claim 19,
The kit is a kit further comprising an enzyme,
wherein the enzyme is an RNA hydrolase (ribonuclease, RNase) of RNaseH, RNase II, RNase III, RNase IV or RNase T2.
 a) 생물학적 시료로부터 검출하고자 하는 ApoE 유전자의 단일염기다형성 부위를 포함하는 표적 핵산을 수득하는 단계;
b) 제1항 내지 제19항 중 어느 한 항의 단일핵산을 제조하는 단계;
c) 상기 단계 a)에서 수득한 표적 핵산, 상기 단계 b)에서 제조한 1형 단일핵산, ApoE 유전자 특이적 프라이머 세트, 및 절단시약과 혼합하거나 상기 단계 a)에서 수득한 표적 핵산, 상기 단계 b)에서 제조한 2형 단일핵산, 및 절단시약과 혼합한 후 신장반응을 통해 ApoE 유전자의 단일염기다형성 부위를 포함하는 표적 핵산-단일핵산 복합체를 증폭시키는 단계; 및
d) 상기 단계 c)에서 증폭된 ApoE 유전자의 단일염기다형성 부위를 포함하는 표적 핵산-단일핵산 복합체로부터 분리된 단일핵산 단편의 양을 측정하는 단계;를 포함하는, ApoE 유전자의 단일염기다형성 검출 방법.
a) obtaining a target nucleic acid comprising a single nucleotide polymorphism site of the ApoE gene to be detected from a biological sample;
b) preparing the mononucleic acid of any one of claims 1 to 19;
c) the target nucleic acid obtained in step a), the type 1 mononucleic acid prepared in step b), the ApoE gene-specific primer set, and the target nucleic acid obtained in step a) or mixed with the cleavage reagent, step b ) amplifying the target nucleic acid-mononucleic acid complex comprising the mononucleotide polymorphism site of the ApoE gene through elongation after mixing with the type 2 mononucleic acid prepared in ) and a cleavage reagent; and
d) measuring the amount of a single nucleic acid fragment isolated from a target nucleic acid-mononucleic acid complex including the mononucleotide polymorphism site of the ApoE gene amplified in step c); .
 제21항에 있어서,
상기 단계 a)의 단일염기다형성 부위를 포함하는 표적 핵산은 시료로부터 검출하고자 하는 RNA 또는 DNA이거나, 상기 RNA를 역전사 중합효소로 증폭하여 얻은 cDNA인 것인, ApoE 유전자의 단일염기다형성 검출 방법.
22. The method of claim 21,
The target nucleic acid including the single nucleotide polymorphism site of step a) is RNA or DNA to be detected from the sample, or cDNA obtained by amplifying the RNA with a reverse transcription polymerase.
 제21항에 있어서,
상기 단계 c)의 절단시약은 RNaseH, RNase Ⅱ, RNase Ⅲ, RNase Ⅳ 또는 RNase T2의 RNA 가수분해효소(ribonuclease, RNase)인, ApoE 유전자의 단일염기다형성 검출 방법.
22. The method of claim 21,
The cleavage reagent of step c) is a ribonuclease (RNase) of RNaseH, RNase II, RNase III, RNase IV or RNase T2, a method for detecting single nucleotide polymorphism in the ApoE gene.
 제21항에 있어서,
상기 단계 c)의 ApoE 유전자 특이적 프라이머 세트는 서열번호 1 및 2로 구성된 것인, ApoE 유전자의 단일염기다형성 검출 방법.22. The method of claim 21,
The ApoE gene-specific primer set of step c) is composed of SEQ ID NOs: 1 and 2, ApoE gene single nucleotide polymorphism detection method.
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