KR102543976B1 - HLA-B genotype analysis method using Korean-specific SNPs and optimized pipeline - Google Patents
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Abstract
본 발명은 한국인 특이적 SNP 정보 및 최적화된 파이프라인을 이용한 HLA-B 유전자형 분석 방법에 관한 것으로, 본 발명의 963개의 SNP 정보를 기반으로 HLA-B type을 분석하는 방법은 한국인 특이적인 SNP 정보 및 최적화된 분석 파이프라인을 제공함으로써 장기이식 대상자 및 공여자에 대해 사전 검사 등에 유용하게 활용 할 수 있으며, 낮은 비용과 적은 소요 시간으로 HLA type을 제공함으로써 약물유전학, 면역 질환, 관련 만성질환 등 다양한 분야에서 연구에 활용 할 수 있다.The present invention relates to a method for analyzing HLA-B genotyping using Korean-specific SNP information and an optimized pipeline. By providing an optimized analysis pipeline, it can be usefully used for pre-tests for organ transplant recipients and donors, and by providing HLA types at low cost and time, it can be used in various fields such as pharmacogenomics, immune diseases, and related chronic diseases. can be used for research.
Description
본 발명은 한국인 특이적 SNP 정보 및 최적화된 파이프라인을 이용한 HLA-B 유전자형 분석 방법에 관한 것이다.The present invention relates to an HLA-B genotyping method using Korean-specific SNP information and an optimized pipeline.
인간 백혈구 항원 (HLA) 유전자는 면역계의 주요 기능, 외래 또는 비정상 항원의 검출을 담당하는 세포 표면 항원 단백질을 암호화합니다. 이 유전자는 MHC (major histocompatibility complex)로 알려진 영역에 있는 6번 염색체 short arm에 위치해있다.The human leukocyte antigen (HLA) gene encodes a cell surface antigen protein responsible for a major function of the immune system, the detection of foreign or abnormal antigens. This gene is located on the short arm of chromosome 6 in a region known as the major histocompatibility complex (MHC).
HLA type은 장기이식 수여자와 공여자의 장기 및 조직 이식 적합성 확인에 중요한 정보이며 면역 질환의 위험요인 분석 및 다양한 연구에 활용되고 있다. 기존의 HLA typing 방법은 sequence-specific oligonucleotide (SSO), sequence-specific primer (SSP), sanger sequencing, high throughput next generation sequencing (NGS) 등의 기술을 통해 제공되었으나 높은 정확도에 비해 가격이 비싸고, 고해상도 HLA type의 경우 실험에 소요되는 시간이 길다는 단점이 있다. 또한, 기존 SNP(Single Nucleotide Polymorphism) 정보로부터 HLA type을 imputation을 통해 예측하는 방법(특허문헌 01)이 제공된 바 있으나, 분석에 사용되는 SNP 및 분석 절차에 필수적인 참조패널 정보에 따라 정확도가 상이하다는 단점이 있다. 특히 기존 SNP 정보들은 주로 서양인 연구 결과에서 확인된 것을 사용하기 때문에 한국인에서 높은 정확도로 HLA type을 제공할 수 없다는 문제점이 있다.HLA type is important information for confirming organ and tissue transplant suitability of organ transplant recipients and donors, and is used in various studies and risk factor analysis of immune diseases. Existing HLA typing methods have been provided through technologies such as sequence-specific oligonucleotide (SSO), sequence-specific primer (SSP), Sanger sequencing, and high throughput next generation sequencing (NGS), but they are expensive compared to high accuracy and high-resolution HLA In the case of the type, there is a disadvantage that the time required for the experiment is long. In addition, a method for predicting HLA type through imputation from existing SNP (Single Nucleotide Polymorphism) information has been provided (Patent Document 01), but the disadvantage is that the accuracy differs depending on the SNP used for analysis and the reference panel information essential for the analysis procedure. there is In particular, there is a problem in that the HLA type cannot be provided with high accuracy in Koreans because existing SNP information mainly uses those confirmed in Western research results.
따라서, 본 발명에서는 한국인 특이적인 SNP 정보 및 최적화된 분석 파이프라인을 제공함으로써 한국인 HLA 지역 맞춤형 유전정보 약 963개의 SNP를 사용하고 최적화된 HLA imputation 파이프라인을 이용하여 HLA type을 예측하여 4일 이내에 기존 방법 대비 20배 낮은 가격(약 5만원)에 99.1% 수준 정확도(2-digit 기준)의 HLA type을 제공 할 수 있다는 것을 확인하고 본 발명을 완성하였다.Therefore, in the present invention, by providing Korean-specific SNP information and an optimized analysis pipeline, about 963 SNPs of genetic information tailored to the Korean HLA region are used and the HLA type is predicted using an optimized HLA imputation pipeline, and the existing existing information within 4 days. The present invention was completed by confirming that it was possible to provide an HLA type with 99.1% level accuracy (2-digit standard) at a price 20 times lower than the method (about 50,000 won).
본 발명의 목적은 HLA-B type 관련 단일염기다형성(SNP)을 포함하는 HLA-B 유전자형 판별용 마커 조성물을 제공하는 데 있다.An object of the present invention is to provide a marker composition for discriminating HLA-B genotype, including an HLA-B type related single nucleotide polymorphism (SNP).
또한, 본 발명의 다른 목적은 상기 마커 조성물을 검출할 수 있는 제제를 포함하는 HLA-B 유전자형 분석용 조성물을 제공하는 데 있다.In addition, another object of the present invention is to provide a composition for HLA-B genotyping analysis comprising an agent capable of detecting the marker composition.
또한, 본 발명의 다른 목적은 상기 HLA-B 유전자형 분석용 조성물을 포함하는 HLA-B 유전자형 분석용 키트를 제공하는 데 있다.In addition, another object of the present invention is to provide a kit for analysis of HLA-B genotyping comprising the composition for analysis of HLA-B genotyping.
또한, 본 발명의 다른 목적은 HLA-B 유전자형 분석 방법을 제공하는 데 있다.In addition, another object of the present invention is to provide an HLA-B genotyping method.
상기와 같은 목적을 달성하기 위해, 본 발명은 HLA-B type 관련 단일염기다형성(SNP)을 포함하는 HLA-B 유전자형 판별용 마커 조성물을 제공한다.In order to achieve the above object, the present invention provides a marker composition for determining HLA-B genotype including a single nucleotide polymorphism (SNP) related to HLA-B type.
이어서, 본 발명은 상기 마커 조성물을 검출할 수 있는 제제를 포함하는 HLA-B 유전자형 분석용 조성물을 제공한다.Next, the present invention provides a composition for analysis of HLA-B genotyping comprising an agent capable of detecting the marker composition.
나아가, 본 발명은 상기 HLA-B 유전자형 분석용 조성물을 포함하는 HLA-B 유전자형 분석용 키트를 제공한다.Furthermore, the present invention provides a kit for analysis of HLA-B genotyping comprising the composition for analysis of HLA-B genotyping.
마지막으로, 본 발명은 분석대상으로부터 생물학적 시료를 수득하는 1단계; 상기 생물학적 시료로부터 유전체(genome DNA)를 추출하는 2단계; 상기 유전체(genome DNA)의 서열정보를 수득하는 3단계; 상기 서열정보로부터 HLA-B type 관련 단일염기다형성(SNP)에 대한 유전자형을 분석(genotyping)하여 분석대상의 SNP 데이터를 수집하는 4단계; 및 상기 분석대상의 SNP 데이터를 분석하여 HLA 대립유전자를 결정하는 5단계;를 포함하는 HLA-B 유전자형 분석 방법을 제공한다.Finally, the present invention is a first step of obtaining a biological sample from the analyte; Step 2 of extracting genome DNA from the biological sample; Step 3 of obtaining sequence information of the genome DNA; Step 4 of analyzing genotypes for single nucleotide polymorphisms (SNPs) related to HLA-B type from the sequence information and collecting SNP data of the analysis target; It provides an HLA-B genotyping method comprising a; and step 5 of determining the HLA allele by analyzing the SNP data of the analyte.
본 발명의 963개의 SNP 정보를 기반으로 HLA-B type을 분석하는 방법은 한국인 특이적인 SNP 정보 및 최적화된 분석 파이프라인을 제공함으로써 장기이식 대상자 및 공여자에 대해 사전 검사 등에 유용하게 활용 할 수 있으며, 낮은 비용과 적은 소요 시간으로 HLA type을 제공함으로써 약물유전학, 면역 질환, 관련 만성질환 등 다양한 분야에서 연구에 활용 할 수 있다.The method of analyzing HLA-B type based on 963 SNP information of the present invention can be usefully used for prior examination of organ transplant recipients and donors by providing Korean-specific SNP information and an optimized analysis pipeline, By providing HLA type at low cost and short lead time, it can be used for research in various fields such as pharmacogenetics, immune diseases, and related chronic diseases.
도 1은 한국인칩 유전체정보 정도관리 흐름도 및 HLA typing 정확도 측정 파이프라인을 도식화하여 나타낸 것이다.
도 2는 한국인 특이적 SNP 정보를 이용한 HLA-B 유전자형 분석 방법의 예시를 나타낸 것이다.Figure 1 schematically shows a Korean chip genome information quality control flow chart and an HLA typing accuracy measurement pipeline.
Figure 2 shows an example of an HLA-B genotyping method using Korean-specific SNP information.
이하, 본 발명에서 사용되는 용어를 상세히 설명한다.Hereinafter, terms used in the present invention will be described in detail.
또한, 본 명세서에서 사용되는 용어 (terminology)들은 본 발명의 바람직한 실시 예를 적절히 표현하기 위해 사용된 용어들로서, 이는 사용자, 운용자의 의도 또는 본 발명이 속하는 분야의 관례 등에 따라 달라질 수 있다. 따라서 본 용어들에 대한 정의는 본 명세서 전반에 걸친 내용을 토대로 내려져야 할 것이다. 명세서 전체에서, 어떤 부분이 어떤 구성요소를 “포함”한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다.In addition, the terms (terminology) used in this specification are terms used to appropriately express preferred embodiments of the present invention, which may vary according to the intention of a user or operator or customs in the field to which the present invention belongs. Therefore, definitions of these terms should be made based on the contents throughout this specification. Throughout the specification, when a certain component is said to "include", it means that it may further include other components without excluding other components unless otherwise stated.
본 발명에서 사용되는 용어 “다형성 (polymorphism)”이란 하나의 유전자 좌위 (locus)에 두 가지 이상의 대립 유전자 (allele)가 존재하는 경우를 말하며 다형성 부위 중에서, 사람에 따라 단일 염기만이 다른 것을 단일 염기 다형성 (single nucleotide polymorphism, SNP)이라 한다. 바람직한 다형성 마커는 선택된 집단에서 1% 이상, 더욱 바람직하게는 5% 또는 10% 이상의 발생 빈도를 나타내는 두 가지 이상의 대립 유전자를 가진다. 따라서, 다형성 마커에 대한 유전적 연관 (genetic association)은 특정한 다형성 마커의 하나 이상의 특정한 대립형질에 대해 연관이 있다는 것을 의미한다. 마커는 단일 염기 다형성 (SNP), 마이크로새틀라이트 (microsatellite), 삽입, 결실, 중복 및 전위 (translocation)를 포함한, 게놈에서 발견되는 임의의 변이 형태의 대립형질을 포함할 수 있다.The term "polymorphism" used in the present invention refers to the case where two or more alleles exist in one locus, and among the polymorphic sites, only a single base differs from person to person. This is called a single nucleotide polymorphism (SNP). Preferred polymorphic markers have two or more alleles that exhibit an incidence of greater than 1%, more preferably greater than 5% or 10% in a selected population. Thus, genetic association to a polymorphic marker means that there is an association to one or more specific alleles of a specific polymorphic marker. Markers can include alleles of any variant form found in the genome, including single nucleotide polymorphisms (SNPs), microsatellites, insertions, deletions, duplications and translocations.
본 발명에서 사용되는 용어 “대립 유전자 (allele)” 또는 “대립형질”이란 상동 염색체의 동일한 유전자 좌위에 존재하는 한 유전자의 여러 타입을 말한다. 대립 유전자는 다형성을 나타내는데 사용되기도 하며, 예컨대, SNP는 두 종류의 대립 인자 (biallele)를 갖는다.As used herein, the term "allele" or "allele" refers to several types of a gene present in the same locus of a homologous chromosome. Alleles are also used to indicate polymorphism, for example, SNPs have two types of alleles.
본 발명에서 사용되는 용어 “rs_id”란 1998년부터 SNP 정보를 축적하기 시작한 NCBI가 초기에 등록되는 모든 SNP에 대하여 부여한 독립된 표지자인 rs-ID를 의미한다. 본 발명에서는 rs831571와 같은 형태로 기재하였다. 이와 같은 표에 기재된 rs_id는 본 발명의 다형성 마커인 SNP 마커를 의미한다. 당업자라면 상기 rs_id를 이용하여 SNP의 위치 및 서열을 용이하게 확인할 수 있을 것이다. NCBI의 dbSNP (The Single Nucleotide Polymorphism Database) 번호인 rs_id에 해당하는 구체적인 서열은 시간이 지남에 따라 약간 변경될 수 있다. 본 발명의 범위가 상기 변경된 서열에도 미치는 것은 당업자에게 자명할 것이다.The term “rs_id” used in the present invention means rs-ID, an independent indicator assigned to all SNPs initially registered by NCBI, which began accumulating SNP information in 1998. In the present invention, it is described in the form of rs831571. rs_id described in this table means a SNP marker, which is a polymorphic marker of the present invention. A person skilled in the art can easily identify the location and sequence of the SNP using the rs_id. The specific sequence corresponding to NCBI's dbSNP (The Single Nucleotide Polymorphism Database) number, rs_id, may change slightly over time. It will be apparent to those skilled in the art that the scope of the present invention also extends to the altered sequence.
이하, 첨부된 도면을 참조하여 본 발명의 구현예로 본 발명을 상세히 설명하기로 한다. 다만, 하기 구현 예는 본 발명에 대한 예시로 제시되는 것으로, 당업자에게 주지 저명한 기술 또는 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우에는 그 상세한 설명을 생략할 수 있고, 이에 의해 본 발명이 제한되지는 않는다. 본 발명은 후술하는 특허 청구범위의 기재 및 그로부터 해석되는 균등 범주 내에서 다양한 변형 및 응용이 가능하다.Hereinafter, the present invention will be described in detail as an embodiment of the present invention with reference to the accompanying drawings. However, the following implementation examples are presented as examples of the present invention, and if it is determined that a detailed description of a well-known technology or configuration may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the description of the claims described later and equivalent scopes interpreted therefrom.
본 발명은 HLA-B type 관련 단일염기다형성(SNP)을 포함하는 HLA-B 유전자형 판별용 마커 조성물을 제공한다.The present invention provides a marker composition for discriminating HLA-B genotype including a single nucleotide polymorphism (SNP) related to HLA-B type.
또한, 상기 HLA-B type 관련 단일염기다형성(SNP)은 인간 6번 염색체상에 존재하는 단일염기다형성(SNP)일 수 있으며, 바람직하게는 인간 6번 염색체의 30321652 ~ 32324956 지역(Genome Reference Consortium : GRCh37.p13, hg19 version, Genbank sequence: CM000668.1, RefSeq sequence: NC_000006.11)에 존재하는 단일염기다형성(SNP)일수 있으며, 더욱 바람직하게는 rs3130375, rs3094626, rs117973624, rs2158285, rs1012411, rs4711214, rs2022082, rs3129808, rs2844748, rs17839997, rs2844746, rs79168747, rs2844745, rs16897402, rs970270, rs7744914, rs2844743, rs28780099, rs3130136, rs3130140, rs9404965, rs9391683, rs2524202, rs2516688, rs1264570, rs11961853, rs3130118, rs1264568, rs1034325, rs1034324, rs147409284, rs10947072, rs9261752, rs1264562, rs57700953, rs1592415, rs1264547, rs1150769, rs4713325, rs117323106, rs1264537, rs116866167, rs9295863, rs17410877, rs1264518, rs17476793, rs9380178, rs17195173, rs4959049, rs9295878, rs2844730, rs1078792, rs1076832, rs138184092, rs2844729, rs2516677, rs9501336, rs2516676, rs2516675, rs59865400, rs2524172, rs7761210, rs9295895, rs12174433, rs3757335, rs762324, rs1264459, rs117058374, rs3131115, rs10484552, rs142126210, rs115484417, rs2524222, rs3888777, rs9262113, rs3130041, rs1264451, rs3130244, rs188748372, rs17195467, rs4148249, rs9262119, rs6934466, rs7759666, rs1264420, rs1264419, rs2267637, rs146574978, rs6919835, rs3895681, rs2075016, rs3814959, rs13201129, rs2394390, rs3132595, rs117993568, rs117013517, rs1076829, rs3094097, rs60557285, rs9262141, rs2239888, rs2269706, rs61572689, rs2269705, rs1075496, rs2269703, rs9405048, rs78556678, rs9262152, rs187119487, rs3094127, rs9500867, rs148850465, rs9380182, rs41315800, rs147510149, rs12111310, rs8512, rs9468821, rs2301277, rs2535319, rs4713358, rs9262164, rs2535324, rs2535323, rs113033684, rs2394401, rs3095341, rs3095340, rs3131036, rs11966040, rs3095338, rs13204228, rs12210947, rs3094117, rs3094114, rs58954828, rs12526481, rs73425733, rs13212094, rs17189441, rs140096325, rs3094112, rs3131061, rs3131063, rs3131064, rs1264377, rs1264375, rs12197154, rs9380191, rs3094123, rs1264362, rs9391696, rs2394412, rs3094111, rs16897919, rs1264352, rs4713391, rs1264350, rs12198842, rs9368644, rs7749924, rs1264347, rs3130648, rs9348844, rs3130787, rs11752162, rs55745908, rs117589205, rs2517576, rs2844663, rs3095350, rs3130654, rs11759095, rs9468841, rs9262256, rs3095354, rs28362611, rs1264327, rs1264323, rs1264319, rs2301755, rs9468843, rs9378150, rs73727123, rs3130780, rs3218822, rs138449990, rs3218826, rs1264298, rs7766094, rs885905, rs148172787, rs2532934, rs4711247, rs2517459, rs2532929, rs2253588, rs3095152, rs12525616, rs2844651, rs3131784, rs3132571, rs11753943, rs3131921, rs4711249, rs2844648, rs2844701, rs3130781, rs2517451, rs3130782, rs2517449, rs11970154, rs2240804, rs2240803, rs45627239, rs12190030, rs12212418, rs11753326, rs11752177, rs117677608, rs4711250, rs6928738, rs2844697, rs3095150, rs2532924, rs17196137, rs2530715, rs2254847, rs3131933, rs2532919, rs3131932, rs2530710, rs118150420, rs117480691, rs2844680, rs2844679, rs9295938, rs112483846, rs76095072, rs12196237, rs6927057, rs1634719, rs1624944, rs41291746, rs1632854, rs117712789, rs9394026, rs1619376, rs9262493, rs9262498, rs9295942, rs4713411, rs9262539, rs12198448, rs3094672, rs115194949, rs2517554, rs2523898, rs12179536, rs78457009, rs9501039, rs62399431, rs2523897, rs11753789, rs146685560, rs58508214, rs12110785, rs4713421, rs34247365, rs3909113, rs10947126, rs59356242, rs11754586, rs6933349, rs7768644, rs11969717, rs4713423, rs28732081, rs3869098, rs4248153, rs4248154, rs28894977, rs9357105, rs2844670, rs12197587, rs4711253, rs9262559, rs2517552, rs2517549, rs2508015, rs12528584, rs17190106, rs10947130, rs2844645, rs2523868, rs2523866, rs4713429, rs2523857, rs16898614, rs2523848, rs9262636, rs16898629, rs78725346, rs3906263, rs117633310, rs62399065, rs2246330, rs9378152, rs117598544, rs3132564, rs9380215, rs117631086, rs12202207, rs3130955, rs2535298, rs67565791, rs2535294, rs118014968, rs9263509, rs9263600, rs6457327, rs2517485, rs35944195, rs2517529, rs1265063, rs1265062, rs2233984, rs2233978, rs2233974, rs1265052, rs2270190, rs2233967, rs2233956, rs2233955, rs1265048, rs79499809, rs3130975, rs3130977, rs3130553, rs3130554, rs3130980, rs3132555, rs3094217, rs117951780, rs3094216, rs33941312, rs4713436, rs707913, rs144847588, rs3130989, rs3094211, rs6913137, rs3095313, rs3131000, rs3778630, rs3094204, rs3778638, rs9263702, rs9263703, rs6931921, rs3131005, rs9263717, rs9263719, rs3131009, rs4959053, rs3823418, rs3132569, rs28732100, rs1265100, rs1265099, rs2074478, rs2233954, rs2233950, rs1265098, rs79592231, rs3130573, rs1265097, rs1265095, rs1265093, rs2233945, rs2233941, rs7757012, rs1265086, rs116969494, rs9263740, rs142986308, rs147733073, rs886401, rs3132538, rs2240064, rs2240062, rs12523890, rs2240061, rs1265071, rs130071, rs1265112, rs142126856, rs1265109, rs56205037, rs17190769, rs9263794, rs1419881, rs17190776, rs9263796, rs2106074, rs9501063, rs76364340, rs1265163, rs3757349, rs3130931, rs17840029, rs3130503, rs12215963, rs879882, rs885950, rs885948, rs1265158, rs2269713, rs887468, rs3094188, rs1265156, rs3131018, rs9501066, rs4713438, rs3130457, rs9263846, rs9461653, rs4122190, rs4122189, rs11961983, rs10807081, rs9263871, rs9263875, rs17191195, rs3132506, rs3887152, rs35718543, rs3132503, rs9468898, rs9295967, rs74504771, rs28360994, rs9263966, rs36001438, rs9263969, rs9461662, rs9263980, rs3130517, rs12662501, rs6904669, rs6927444, rs4373355, rs3130521, rs3130942, rs28397289, rs3130941, rs3130527, rs3868078, rs3130473, rs3130474, rs11967600, rs11967684, rs2394888, rs3869115, rs3130685, rs117767616, rs7756999, rs3130531, rs2894186, rs3095250, rs3130712, rs114343342, rs114751866, rs3134762, rs3130695, rs3095245, rs3130423, rs72502552, rs9264416, rs2894196, rs2524118, rs2844623, rs67524139, rs76235944, rs12663884, rs9394047, rs9264643, rs28626310, rs6457358, rs2074489, rs2074488, rs9357121, rs7454526, rs6923313, rs2524084, rs2524082, rs3134745, rs3132486, rs2524075, rs13203895, rs2524070, rs6906846, rs2524067, rs9378228, rs6912893, rs9348859, rs2853939, rs117477942, rs9468919, rs9468922, rs9468924, rs2524042, rs2853928, rs16899168, rs117484768, rs9468925, rs2524162, rs115464798, rs115748458, rs78489254, rs9348863, rs9357123, rs2853926, rs28732109, rs28894990, rs2246954, rs2524123, rs9461685, rs4406273, rs2524095, rs16899202, rs9380238, rs28367639, rs2524145, rs3915971, rs4523128, rs9368677, rs9264904, rs79409503, rs6905036, rs9264927, rs7761965, rs16899241, rs3873386, rs1634761, rs6930944, rs9264942, rs9468942, rs3094691, rs2524168, rs3094683, rs9265569, rs9265579, rs1625792, rs112587708, rs2507997, rs28732138, rs4540292, rs2156875, rs2844586, rs2523619, rs4394275, rs2523618, rs112985609, rs9391846, rs2442719, rs9266093, rs2596501, rs3819294, rs3819285, rs1051488, rs2523605, rs2523590, rs114783056, rs2523589, rs6901719, rs2596548, rs2523557, rs2523554, rs2596551, rs9266359, rs9266380, rs2844575, rs114042808, rs2523535, rs2844569, rs2922997, rs13215664, rs2523644, rs4610605, rs13198903, rs2523638, rs7382258, rs2844545, rs13193697, rs2507982, rs2442736, rs9501587, rs9266658, rs4713462, rs9266722, rs1521, rs17193507, rs3016013, rs2442752, rs2507976, rs2442749, rs9266772, rs9266773, rs7741091, rs118061248, rs9266774, rs2428486, rs6932730, rs4711269, rs2428484, rs2523477, rs56403814, rs2263323, rs77997850, rs2596480, rs9267123, rs2395030, rs2516518, rs117545169, rs9404989, rs147770380, rs17206980, rs116096501, rs3130922, rs3095234, rs3828903, rs2534674, rs2534673, rs3828913, rs2516497, rs3828917, rs2534657, rs3132468, rs2844498, rs115707901, rs3131633, rs2246986, rs117257265, rs2516491, rs3093998, rs3132454, rs3093992, rs2516482, rs3219190, rs3853601, rs57256697, rs7753431, rs2075580, rs2239709, rs2523508, rs2523505, rs2844509, rs12662306, rs74290517, rs2251824, rs6916921, rs2239707, rs2230365, rs115625921, rs4947325, rs17200733, rs928815, rs2857709, rs2857708, rs909253, rs2229092, rs1800629, rs3093664, rs3093672, rs3093561, rs769177, rs3093553, rs2256965, rs2256974, rs1052248, rs986475, rs11575839, rs77107611, rs2736191, rs2844480, rs2857595, rs2736186, rs9348876, rs2857609, rs2280801, rs2736171, rs75044749, rs2242657, rs1046089, rs2261033, rs11229, rs2736155, rs1046756, rs2178899, rs115247800, rs755714, rs1077394, rs7770976, rs760293, rs3130048, rs2242656, rs2844463, rs3130617, rs707974, rs28732158, rs141943810, rs9469032, rs9267536, rs79515906, rs11552113, rs150576099, rs805281, rs805273, rs145012921, rs805295, rs9267547, rs2242653, rs805287, rs118062293, rs1065356, rs376510, rs805293, rs805292, rs453098, rs2272592, rs409558, rs3749953, rs116847602, rs17201144, rs707937, rs707928, rs2736426, rs707927, rs34585796, rs181542871, rs75155700, rs11751198, rs534892, rs2075800, rs2227955, rs2075799, rs9469057, rs34791928, rs16867582, rs117317180, rs35294112, rs2763979, rs6457452, rs6921489, rs117792332, rs9501157, rs574914, rs494620, rs3130482, rs614549, rs12661281, rs2736428, rs6907185, rs115646381, rs555007, rs652888, rs2844458, rs486416, rs113973079, rs13194087, rs9267661, rs7887, rs116606947, rs116078299, rs2844452, rs9267673, rs644045, rs7746553, rs12614, rs117314762, rs13194698, rs1048709, rs4151669, rs17201431, rs537160, rs2072634, rs4151657, rs149101394, rs116928087, rs2072633, rs4151672, rs760070, rs4151664, rs9501161, rs16867755, rs440454, rs2734331, rs429608, rs9296003, rs474534, rs6941112, rs389883, rs116851612, rs9267796, rs2066982, rs9469079, rs150876029, rs7766862, rs181025235, rs3130286, rs9267798, rs185819, rs1150754, rs2239688, rs204900, rs115461385, rs41270461, rs117767511, rs41268899, rs3134954, rs429150, rs2269426, rs3807039, rs17207986, rs8283, rs3830076, rs117160266, rs204892, rs4713505, rs204999, rs4713506, rs9296009, rs117604062, rs2269425, rs10947233, rs3134603, rs3134950, rs205000, rs3130283, rs34280838, rs3130284, rs408359, rs17493811, rs9469089, rs3134943, rs2071288, rs2269422, rs1800625, rs204995, rs204993, rs2022059, rs204992, rs176095, rs204989, rs45592033, rs8192579, rs2071278, rs9267820, rs9267822, rs2071287, rs3132935, rs2071277, rs206017, rs17604492, rs206015, rs3134798, rs431541, rs379464, rs394657, rs1109771, rs8192590, rs430916, rs423023, rs422951, rs520692, rs8192588, rs2071284, rs915894, rs3134931, rs3830041, rs375244, rs12055568, rs3134929, rs3096702, rs397081, rs2267645, rs9267845, rs499691, rs510321, rs9296012, rs365053, rs9378123, rs374205, rs495089, rs1426713, rs440261, rs377763, rs9267873, rs3134926, rs9501398, rs9267898, rs508445, rs693797, rs9267911, rs6901158, rs511027, rs4947337, rs424232, rs439303, rs507778, rs13194642, rs9267948, rs427037, rs454875, rs9267954, rs9267956, rs405875, rs2894239, rs6936204, rs9296015, rs4713514, rs3130320, rs28895018, rs3115562, rs3132933, rs144538125, rs9268145, rs926070, rs17422727, rs55987614, rs28366175, rs9268167, rs9268199, rs3864302, rs9268205, rs6910071, rs9268220, rs498422, rs7756177, rs72849457, rs4279480, rs521828, rs117123773, rs12524063, rs3132959, rs1003878, rs117322947, rs28361060, rs144849145, rs9368716, rs2273019, rs1033499, rs117067518, rs2273018, rs3132954, rs3117126, rs3117119, rs12662503, rs1265758 및 rs3129907으로 이루어진 군에서 선택된 어느 하나 이상의 단일염기다형성(SNP)일 수 있다.In addition, the HLA-B type related single nucleotide polymorphism (SNP) may be a single nucleotide polymorphism (SNP) present on human chromosome 6, and preferably, the region 30321652 to 32324956 (Genome Reference Consortium: GRCh37.p13, hg19 version, Genbank sequence: CM000668.1, RefSeq sequence: NC_000006.11), and more preferably rs3130375, rs3094626, rs117973624, rs2158285, rs1012411, rs47 11214, rs2022082 , rs3129808, rs2844748, rs17839997, rs2844746, rs79168747, rs2844745, rs16897402, rs970270, rs7744914, rs2844743, rs28780099, rs31301 36, rs3130140, rs9404965, rs9391683, rs2524202, rs2516688, rs1264570, rs11961853, rs3130118, rs1264568, rs1034325, rs1034324, rs1474092 84, rs10947072 , rs9261752, rs1264562, rs57700953, rs1592415, rs1264547, rs1150769, rs4713325, rs117323106, rs1264537, rs116866167, rs9295863, rs174 10877, rs1264518, rs17476793, rs9380178, rs17195173, rs4959049, rs9295878, rs2844730, rs1078792, rs1076832, rs138184092, rs2844729, rs2 516677, rs9501336 , rs2516676, rs2516675, rs59865400, rs2524172, rs7761210, rs9295895, rs12174433, rs3757335, rs762324, rs1264459, rs117058374, rs31311 15, rs10484552, rs142126210, rs115484417, rs2524222, rs3888777, rs9262113, rs3130041, rs1264451, rs3130244, rs188748372, rs17195467, rs 4148249, rs9262119 , rs6934466, rs7759666, rs1264420, rs1264419, rs2267637, rs146574978, rs6919835, rs3895681, rs2075016, rs3814959, rs13201129, rs23943 90, rs3132595, rs117993568, rs117013517, rs1076829, rs3094097, rs60557285, rs9262141, rs2239888, rs2269706, rs61572689, rs2269705, rs10 75496, rs2269703 , rs9405048, rs78556678, rs9262152, rs187119487, rs3094127, rs9500867, rs148850465, rs9380182, rs41315800, rs147510149, rs12111310, rs8512, rs9468821, rs2301277, rs2535319, rs4713358, rs9262164, rs2535324, rs2535323, rs113033684, rs2394401, rs3095341, rs3095340, rs313 1036, rs11966040 , rs3095338, rs13204228, rs12210947, rs3094117, rs3094114, rs58954828, rs12526481, rs73425733, rs13212094, rs17189441, rs140096325, rs3094112, rs3131061, rs3131063, rs3131064, rs1264377, rs1264375, rs12197154, rs9380191, rs3094123, rs1264362, rs9391696, rs2394412, rs3 094111, rs16897919 , rs1264352, rs4713391, rs1264350, rs12198842, rs9368644, rs7749924, rs1264347, rs3130648, rs9348844, rs3130787, rs11752162, rs557459 08, rs117589205, rs2517576, rs2844663, rs3095350, rs3130654, rs11759095, rs9468841, rs9262256, rs3095354, rs28362611, rs1264327, rs1264 323, rs1264319 , rs2301755, rs9468843, rs9378150, rs73727123, rs3130780, rs3218822, rs138449990, rs3218826, rs1264298, rs7766094, rs885905, rs148172 787, rs2532934, rs4711247, rs2517459, rs2532929, rs2253588, rs3095152, rs12525616, rs2844651, rs3131784, rs3132571, rs11753943, rs31319 21, rs4711249 , rs2844648, rs2844701, rs3130781, rs2517451, rs3130782, rs2517449, rs11970154, rs2240804, rs2240803, rs45627239, rs12190030, rs12212 418, rs11753326, rs11752177, rs117677608, rs4711250, rs6928738, rs2844697, rs3095150, rs2532924, rs17196137, rs2530715, rs2254847, rs31 31933, rs2532919 , rs3131932, rs2530710, rs118150420, rs117480691, rs2844680, rs2844679, rs9295938, rs112483846, rs76095072, rs12196237, rs6927057, rs 1634719, rs1624944, rs41291746, rs1632854, rs117712789, rs9394026, rs1619376, rs9262493, rs9262498, rs9295942, rs4713411, rs9262539, rs 12198448, rs3094672 , rs115194949, rs2517554, rs2523898, rs12179536, rs78457009, rs9501039, rs62399431, rs2523897, rs11753789, rs146685560, rs58508214, rs12110785, rs4713421, rs34247365, rs3909113, rs10947126, rs59356242, rs11754586, rs6933349, rs7768644, rs11969717, rs4713423, rs287320 81, rs3869098, rs4248153 , rs4248154, rs28894977, rs9357105, rs2844670, rs12197587, rs4711253, rs9262559, rs2517552, rs2517549, rs2508015, rs12528584, rs17190 106, rs10947130, rs2844645, rs2523868, rs2523866, rs4713429, rs2523857, rs16898614, rs2523848, rs9262636, rs16898629, rs78725346, rs390 6263, rs117633310 , rs62399065, rs2246330, rs9378152, rs117598544, rs3132564, rs9380215, rs117631086, rs12202207, rs3130955, rs2535298, rs67565791, rs2 535294, rs118014968, rs9263509, rs9263600, rs6457327, rs2517485, rs35944195, rs2517529, rs1265063, rs1265062, rs2233984, rs2233978, rs2 233974, rs1265052 , rs2270190, rs2233967, rs2233956, rs2233955, rs1265048, rs79499809, rs3130975, rs3130977, rs3130553, rs3130554, rs3130980, rs3132555 , rs3094217, rs117951780, rs3094216, rs33941312, rs4713436, rs707913, rs144847588, rs3130989, rs3094211, rs6913137, rs3095313, rs313100 0, rs3778630 , rs3094204, rs3778638, rs9263702, rs9263703, rs6931921, rs3131005, rs9263717, rs9263719, rs3131009, rs4959053, rs3823418, rs3132569, rs28732100, rs1265100, rs1265099, rs2074478, rs2233954, rs2233950, rs1265098, rs79592231, rs3130573, rs1265097, rs1265095, rs1265093, rs2233945 , rs2233941, rs7757012, rs1265086, rs116969494, rs9263740, rs142986308, rs147733073, rs886401, rs3132538, rs2240064, rs2240062, rs125 23890, rs2240061, rs1265071, rs130071, rs1265112, rs142126856, rs1265109, rs56205037, rs17190769, rs9263794, rs1419881, rs17190776, rs9 263796, rs2106074 , rs9501063, rs76364340, rs1265163, rs3757349, rs3130931, rs17840029, rs3130503, rs12215963, rs879882, rs885950, rs885948, rs1265158, rs2269713, rs887468, rs3094188, rs1265156, rs3131018, rs9501066, rs4713438, rs3130457, rs9263846, rs9461653, rs4122190, rs4122189, rs11 961983 , rs10807081, rs9263871, rs9263875, rs17191195, rs3132506, rs3887152, rs35718543, rs3132503, rs9468898, rs9295967, rs74504771, rs2836 0994, rs9263966, rs36001438, rs9263969, rs9461662, rs9263980, rs3130517, rs12662501, rs6904669, rs6927444, rs4373355, rs3130521, rs3130 942, rs28397289 , rs3130941, rs3130527, rs3868078, rs3130473, rs3130474, rs11967600, rs11967684, rs2394888, rs3869115, rs3130685, rs117767616, rs7756 999, rs3130531, rs2894186, rs3095250, rs3130712, rs114343342, rs114751866, rs3134762, rs3130695, rs3095245, rs3130423, rs72502552, rs92 64416, rs2894196 , rs2524118, rs2844623, rs67524139, rs76235944, rs12663884, rs9394047, rs9264643, rs28626310, rs6457358, rs2074489, rs2074488, rs9357 121, rs7454526, rs6923313, rs2524084, rs2524082, rs3134745, rs3132486, rs2524075, rs13203895, rs2524070, rs6906846, rs2524067, rs937822 8, rs6912893 , rs9348859, rs2853939, rs117477942, rs9468919, rs9468922, rs9468924, rs2524042, rs2853928, rs16899168, rs117484768, rs9468925, rs252 4162, rs115464798, rs115748458, rs78489254, rs9348863, rs9357123, rs2853926, rs28732109, rs28894990, rs2246954, rs2524123, rs9461685, rs4406273, rs2524095 , rs16899202, rs9380238, rs28367639, rs2524145, rs3915971, rs4523128, rs9368677, rs9264904, rs79409503, rs6905036, rs9264927, rs77619 65, rs16899241, rs3873386, rs1634761, rs6930944, rs9264942, rs9468942, rs3094691, rs2524168, rs3094683, rs9265569, rs9265579, rs1625792 , rs112587708 , rs2507997, rs28732138, rs4540292, rs2156875, rs2844586, rs2523619, rs4394275, rs2523618, rs112985609, rs9391846, rs2442719, rs92660 93, rs2596501, rs3819294, rs3819285, rs1051488, rs2523605, rs2523590, rs114783056, rs2523589, rs6901719, rs2596548, rs2523557, rs252355 4, rs2596551 , rs9266359, rs9266380, rs2844575, rs114042808, rs2523535, rs2844569, rs2922997, rs13215664, rs2523644, rs4610605, rs13198903, rs2523 638, rs7382258, rs2844545, rs13193697, rs2507982, rs2442736, rs9501587, rs9266658, rs4713462, rs9266722, rs1521, rs17193507, rs3016013, rs2442752 , rs2507976, rs2442749, rs9266772, rs9266773, rs7741091, rs118061248, rs9266774, rs2428486, rs6932730, rs4711269, rs2428484, rs252347 7, rs56403814, rs2263323, rs77997850, rs2596480, rs9267123, rs2395030, rs2516518, rs117545169, rs9404989, rs147770380, rs17206980, rs11 6096501, rs3130922 , rs3095234, rs3828903, rs2534674, rs2534673, rs3828913, rs2516497, rs3828917, rs2534657, rs3132468, rs2844498, rs115707901, rs313163 3, rs2246986, rs117257265, rs2516491, rs3093998, rs3132454, rs3093992, rs2516482, rs3219190, rs3853601, rs57256697, rs7753431, rs207558 0, rs2239709 , rs2523508, rs2523505, rs2844509, rs12662306, rs74290517, rs2251824, rs6916921, rs2239707, rs2230365, rs115625921, rs4947325, rs1720 0733, rs928815, rs2857709, rs2857708, rs909253, rs2229092, rs1800629, rs3093664, rs3093672, rs3093561, rs769177, rs3093553, rs2256965, rs2256974 , rs1052248, rs986475, rs11575839, rs77107611, rs2736191, rs2844480, rs2857595, rs2736186, rs9348876, rs2857609, rs2280801, rs2736171 , rs75044749, rs2242657, rs1046089, rs2261033, rs11229, rs2736155, rs1046756, rs2178899, rs115247800, rs755714, rs1077394, rs7770976, rs 760293 , rs3130048, rs2242656, rs2844463, rs3130617, rs707974, rs28732158, rs141943810, rs9469032, rs9267536, rs79515906, rs11552113, rs1505 76099, rs805281, rs805273, rs145012921, rs805295, rs9267547, rs2242653, rs805287, rs118062293, rs1065356, rs376510, rs805293, rs805292, rs453098 , rs2272592, rs409558, rs3749953, rs116847602, rs17201144, rs707937, rs707928, rs2736426, rs707927, rs34585796, rs181542871, rs751557 00, rs11751198, rs534892, rs2075800, rs2227955, rs2075799, rs9469057, rs34791928, rs16867582, rs117317180, rs35294112, rs2763979, rs645 7452, rs6921489 , rs117792332, rs9501157, rs574914, rs494620, rs3130482, rs614549, rs12661281, rs2736428, rs6907185, rs115646381, rs555007, rs652888, rs2844458, rs486416, rs113973079, rs13194087, rs9267661, rs7887, rs116606947, rs116078299, rs2844452, rs9267673, rs644045, rs7746553, rs12614 , rs117314762, rs13194698, rs1048709, rs4151669, rs17201431, rs537160, rs2072634, rs4151657, rs149101394, rs116928087, rs2072633, rs4 151672, rs760070, rs4151664, rs9501161, rs16867755, rs440454, rs2734331, rs429608, rs9296003, rs474534, rs6941112, rs389883, rs11685161 2, rs9267796 , rs2066982, rs9469079, rs150876029, rs7766862, rs181025235, rs3130286, rs9267798, rs185819, rs1150754, rs2239688, rs204900, rs115461 385, rs41270461, rs117767511, rs41268899, rs3134954, rs429150, rs2269426, rs3807039, rs17207986, rs8283, rs3830076, rs117160266, rs2048 92, rs4713505 , rs204999, rs4713506, rs9296009, rs117604062, rs2269425, rs10947233, rs3134603, rs3134950, rs205000, rs3130283, rs34280838, rs313028 4, rs408359, rs17493811, rs9469089, rs3134943, rs2071288, rs2269422, rs1800625, rs204995, rs204993, rs2022059, rs204992, rs176095, rs204 989 , rs45592033, rs8192579, rs2071278, rs9267820, rs9267822, rs2071287, rs3132935, rs2071277, rs206017, rs17604492, rs206015, rs3134798, rs431541, rs379464, rs394657, rs1109771, rs8192590, rs430916, rs423023, rs422951, rs520692, rs8192588, rs2071284, rs915894, rs3134931 , rs3830041, rs375244, rs12055568, rs3134929, rs3096702, rs397081, rs2267645, rs9267845, rs499691, rs510321, rs9296012, rs365053, rs93 78123, rs374205, rs495089, rs1426713, rs440261, rs377763, rs9267873, rs3134926, rs9501398, rs9267898, rs508445, rs693797, rs9267911 , rs6901158, rs511027, rs4947337, rs424232, rs439303, rs507778, rs13194642, rs9267948, rs427037, rs454875, rs9267954, rs9267956, rs405 875, rs2894239, rs6936204, rs9296015, rs4713514, rs3130320, rs28895018, rs3115562, rs3132933, rs144538125, rs9268145, rs926070, rs17422 727 , rs55987614, rs28366175, rs9268167, rs9268199, rs3864302, rs9268205, rs6910071, rs9268220, rs498422, rs7756177, rs72849457, rs427948 0, rs521828, rs117123773, rs12524063, rs3132959, rs1003878, rs117322947, rs28361060, rs144849145, rs9368716, rs2273019, rs1033499, rs11 7067518, rs2273018 , rs3132954, rs3117126, rs3117119, rs12662503, rs1265758 and rs3129907 may be any one or more single nucleotide polymorphisms (SNPs) selected from the group consisting of.
또한, 상기 HLA-B 유전자형 판별용 마커는 한국인 특이적인 것일 수 있으나, 이에 제한되는 것은 아니다.In addition, the marker for determining the HLA-B genotype may be Korean-specific, but is not limited thereto.
이어서, 본 발명은 상기 마커 조성물을 검출할 수 있는 제제를 포함하는 HLA-B 유전자형 분석용 조성물을 제공한다.Next, the present invention provides a composition for analysis of HLA-B genotyping comprising an agent capable of detecting the marker composition.
또한, 상기 HLA-B 유전자형 분석용 조성물은 한국인 대상인 것일 수 있으나, 이에 제한되는 것은 아니다.In addition, the HLA-B genotyping composition may be for Koreans, but is not limited thereto.
또한, 상기 마커 조성물을 검출할 수 있는 제제는 SNP 마커를 포함하는 10개 이상의 연속적인 염기서열로 구성된 폴리뉴클레오티드 또는 그의 상보적 폴리뉴클레오티드에 특이적으로 결합하는 프라이머 또는 프로브일 수 있으나, 이에 제한되는 것은 아니다.In addition, the agent capable of detecting the marker composition may be a primer or probe that specifically binds to a polynucleotide composed of 10 or more consecutive nucleotide sequences including a SNP marker or a polynucleotide complementary thereto, but is limited thereto. It is not.
또한, 상기 폴리뉴클레오티드 또는 그의 상보적 폴리뉴클레오티드와 특이적으로 결합하는 프라이머 또는 프로브는 대립형질 특이적 (allele-specific)이다.In addition, primers or probes that specifically bind to the polynucleotide or its complementary polynucleotide are allele-specific.
상기 "프라이머"란 짧은 자유 수산화기를 가지는 핵산서열로서 상보적인 템플리트와 염기쌍을 형성할 수 있고 템플리트 가닥 복사를 위한 시작 지점으로 기능하는 짧은 핵산서열을 말한다. 본 발명의 프라이머는 예를 들면, 포스포르아미다이트 고체 지지체 방법과 같은 당 분야에 공지된 방법을 이용하여 화학적으로 합성할 수 있다.The term "primer" refers to a short nucleic acid sequence having a short free hydroxyl group capable of forming base pairs with a complementary template and serving as a starting point for copying a template strand. The primers of the present invention can be chemically synthesized using methods known in the art, such as, for example, the phosphoramidite solid support method.
상기 "프로브"는 mRNA와 특이적으로 결합할 수 있는 수개 내지 수백 개의 염기로 이루어진 RNA 또는 DNA 등의 핵산 단편을 의미하며 라벨링되어 있어 특정 mRNA의 존재유무를 확인할 수 있다. 프로브는 올리고뉴클레오타이드 프로브, 단쇄 DNA 프로브, 이중쇄 DNA 프로브, RNA 프로브 등의 형태로 제작될 수 있고 비오틴, FITC, 로다민, DIG 등으로 표지되거나 방사선 동위 원소 등으로 표지될 수 있다.The "probe" refers to a nucleic acid fragment such as RNA or DNA composed of several to hundreds of bases that can specifically bind to mRNA, and is labeled so that the presence or absence of a specific mRNA can be confirmed. The probe may be prepared in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, and the like, and may be labeled with biotin, FITC, rhodamine, DIG, or the like, or labeled with a radioactive isotope.
또한, 상기 프로브는 검출 가능한 물질 예를 들면, 적합한 신호를 제공하고 충분한 반감기를 갖는 방사성 표지로 표지할 수 있다. 표지된 프로브는 문헌 (Sambook et al., Molecular Cloning, A LaboratoryMannual, 1989)에 공지된 바와 같은 고체 지지체 상의 핵산에 혼성화시킬 수 있다.Alternatively, the probe may be labeled with a detectable substance, for example, a radioactive label that provides an appropriate signal and has a sufficient half-life. The labeled probe can be hybridized to a nucleic acid on a solid support as known in the literature (Sambook et al., Molecular Cloning, A Laboratory Manual, 1989).
또한, 상기 프로브는 대립유전자 특이적 프로브로서, 핵산 단편 중에 다형성 부위가 존재하여, 하나의 대립유전자를 포함한 핵산 단편에는 혼성화하지만, 다른 대립유전자를 포함한 핵산 단편에는 혼성화하지 않을 수 있다. 이 경우 혼성화 조건은 대립형질 간의 혼성화 강도에 있어서 유의한 차이를 보여 대립형질 중 하나에만 혼성화되도록 충분히 엄격해야 한다. 바람직하게는 프로브는 혼성화에서의 최대 효율을 위하여 단일 가닥일 수 있으나, 이에 제한되지 않는다.In addition, the probe is an allele-specific probe, and since a polymorphic site exists in a nucleic acid fragment, it hybridizes to a nucleic acid fragment containing one allele, but may not hybridize to a nucleic acid fragment containing another allele. In this case, hybridization conditions should be sufficiently stringent to hybridize to only one of the alleles by showing a significant difference in hybridization strength between alleles. Preferably, the probe may be single-stranded for maximum efficiency in hybridization, but is not limited thereto.
또한, 프라이머를 이용한 특정 핵산의 검출은 PCR과 같은 증폭 방법을 사용하여 목적 유전자의 서열을 증폭한 다음 당 분야에 공지된 방법으로 유전자의 증폭여부를 확인함으로써 수행될 수 있다. 또한, 프로브를 이용한 특정 핵산의 검출은 적합한 조건하에서 시료 핵산을 프로브와 접촉시킨 후 혼성화되는 핵산의 존재여부를 확인함으로써 수행될 수 있다.In addition, detection of a specific nucleic acid using a primer can be performed by amplifying the sequence of a target gene using an amplification method such as PCR, and then confirming whether the gene is amplified by a method known in the art. In addition, detection of a specific nucleic acid using a probe may be performed by contacting a sample nucleic acid with the probe under suitable conditions and confirming the existence of hybridized nucleic acids.
또한, 상기 프로브나 프라이머를 이용하여 특정 핵산을 검출할 수 있는 방법으로는 예를 들면, 이에 한정되지는 않으나 중합효소 연쇄반응 (PCR), DNA 시퀀싱, RT-PCR, 프라이머 연장법 (Nikiforeov et al., Nucl Acids Res 22, 4167-4175, 1994), 올리고뉴클레오타이드 연장 분석 (Nickerson et al., Pro Nat Acad Sci USA, 87, 8923-8927, 1990), 대립형질 특이적 PCR법 (Rust et al., Nucl Acids Res, 6, 3623-3629, 1993), RNase 불일치절단 (RNase mismatch cleavage, Myers et al., Science, 230, 1242-1246, 1985), 단일가닥 입체 다형화 (single strand conformation lymorphism, Orita et al., Pro Nat Acad Sci USA, 86, 2766-2770, 1989), SSCP 및 헤테로두플렉스 동시 분석법 (Lee et al., Mol Cells, 5:668-672, 1995), 변성 구배 젤 전기영동 (DGGE, Cariello et al., Am J Hum Genet, 42, 726-734, 1988), 변성 고압 액체 크로마토그래피 (Underhill et al., Genome Res, 7, 996-1005, 1997), 혼성화 반응, DNA 칩 등이 있다. 상기 혼성화 반응의 예로는 노던 하이브리다이제이션 (Maniatis T. et al., Molecular Cloning, Cold Spring Habor Laboratory, NY, 1982), 인시츄 하이브리다이제이션 (Jacquemier et al., Bull Cancer, 90:31-8, 2003) 및 마이크로어레이 (Macgregor, Expert, Rev Mol Diagn 3:185-200, 2003) 방법 등이 있다.In addition, as a method for detecting a specific nucleic acid using the probe or primer, for example, but not limited to, polymerase chain reaction (PCR), DNA sequencing, RT-PCR, primer extension method (Nikiforeov et al. ., Nucl Acids Res 22, 4167-4175, 1994), oligonucleotide extension assay (Nickerson et al., Pro Nat Acad Sci USA, 87, 8923-8927, 1990), allele-specific PCR method (Rust et al. , Nucl Acids Res, 6, 3623-3629, 1993), RNase mismatch cleavage (Myers et al., Science, 230, 1242-1246, 1985), single strand conformation lymorphism (Orita et al.) al., Pro Nat Acad Sci USA, 86, 2766-2770, 1989), SSCP and heteroduplex simultaneous analysis (Lee et al., Mol Cells, 5:668-672, 1995), denaturing gradient gel electrophoresis (DGGE , Cariello et al., Am J Hum Genet, 42, 726-734, 1988), denaturing high pressure liquid chromatography (Underhill et al., Genome Res, 7, 996-1005, 1997), hybridization reaction, DNA chip, etc. there is. Examples of such hybridization reactions include Northern hybridization (Maniatis T. et al., Molecular Cloning, Cold Spring Habor Laboratory, NY, 1982), in situ hybridization (Jacquemier et al., Bull Cancer, 90:31-8 , 2003) and microarray (Macgregor, Expert, Rev Mol Diagn 3:185-200, 2003) method.
또한, HLA-B 유전자형 분석용 조성물은 상술한 핵산을 검출하는 방법에 일반적으로 사용되는 시약을 추가로 포함할 수 있다. 예를 들면, PCR 반응에 요구되는 dNTP (deoxynulceotide triphosphate), 내열성 중합효소 (polymerase), 염화마그네슘 등의 금속이온염이 포함할 수 있으며, 시퀀싱에 요구되는 dNTP, 시쿼나제 (sequenase) 등을 포함할 수 있다.In addition, the HLA-B genotyping composition may further include a reagent generally used in the above-described method for detecting nucleic acids. For example, metal ion salts such as dNTP (deoxynulceotide triphosphate), heat-resistant polymerase, and magnesium chloride required for PCR reaction may be included, and dNTP required for sequencing, sequencenase, and the like may be included. can
나아가, 본 발명은 상기 HLA-B 유전자형 분석용 조성물을 포함하는 HLA-B 유전자형 분석용 키트를 제공한다.Furthermore, the present invention provides a kit for analysis of HLA-B genotyping comprising the composition for analysis of HLA-B genotyping.
또한, 상기 키트는 다형성 마커 중 1종 이상의 마커를 확인하기 위한 폴리뉴클레오티드, 프라이머 또는 프로브뿐만 아니라 분석 방법에 적합한 한 종류 또는 그 이상의 다른 구성 성분 조성물, 용액 또는 장치가 포함될 수 있다.In addition, the kit may include polynucleotides, primers, or probes for identifying one or more of the polymorphic markers, as well as one or more other component compositions, solutions, or devices suitable for the analysis method.
예를 들어, 본 발명의 키트는 PCR을 수행하기 위해 필요한 필수 요소를 포함하는 키트일 수 있다. PCR 키트는, 상기 다형성 마커에 대한 특이적인 폴리뉴클레오티드, 프라이머 또는 프로브 외에도 테스트 튜브 또는 다른 적절한 컨테이너, 반응 완충액 (pH 및 마그네슘 농도는 다양), 데옥시뉴클레오타이드 (dNTPs), Taq-폴리머라아제 및 역전사효소와 같은 효소, DNase, RNAse 억제제, DEPC-수 (DEPC-water) 및 멸균수 등을 포함할 수 있다.For example, the kit of the present invention may be a kit containing essential elements required to perform PCR. The PCR kit contains, in addition to polynucleotides, primers or probes specific for the above polymorphic markers, a test tube or other suitable container, reaction buffer (with varying pH and magnesium concentration), deoxynucleotides (dNTPs), Taq-polymerase and reverse transcription enzymes such as enzymes, DNase, RNAse inhibitors, DEPC-water and sterile water, and the like.
마지막으로, 본 발명은 분석대상으로부터 생물학적 시료를 수득하는 1단계; 상기 생물학적 시료로부터 유전체(genome DNA)를 추출하는 2단계; 상기 유전체(genome DNA)의 서열정보를 수득하는 3단계; 상기 서열정보로부터 HLA-B type 관련 단일염기다형성(SNP)에 대한 유전자형을 분석(genotyping)하여 분석대상의 SNP 데이터를 수집하는 4단계; 및 상기 분석대상의 SNP 데이터를 분석하여 HLA 대립유전자를 결정하는 5단계;를 포함하는 HLA-B 유전자형 분석 방법을 제공한다.Finally, the present invention is a first step of obtaining a biological sample from the analyte; Step 2 of extracting genome DNA from the biological sample; Step 3 of obtaining sequence information of the genome DNA; Step 4 of analyzing genotypes for single nucleotide polymorphisms (SNPs) related to HLA-B type from the sequence information and collecting SNP data of the analysis target; It provides an HLA-B genotyping method comprising a; and step 5 of determining the HLA allele by analyzing the SNP data of the analyte.
또한, 상기 생물학적 시료는 혈액, 조직, 세포, 혈청, 혈장, 타액, 소변 등을 사용할 수 있으며, 이에 한정되는 것은 아니다.In addition, the biological sample may be blood, tissue, cell, serum, plasma, saliva, urine, etc., but is not limited thereto.
또한, 상기 방법은 2자리(digit) 또는 4자리(digit) 대립유전자 해상도로 HLA-B 유전자형을 결정하는 것일 수 있고, 바람직하게는 2자리(digit) 대립유전자 해상도로 HLA-B 유전자형을 결정하는 것일 수 있으나, 이에 제한되는 것은 아니다.In addition, the method may be to determine the HLA-B genotype with 2-digit or 4-digit allele resolution, preferably determining the HLA-B genotype with 2-digit allele resolution. It may be, but is not limited thereto.
또한, 상기 방법은 한국인을 대상으로 하는 것일 수 있으나, 이에 제한되는 것은 아니다.In addition, the method may be for Koreans, but is not limited thereto.
또한, 상기 5단계는 분석대상의 SNP 데이터와 참조 패널(reference panel)과 비교분석하여 HLA 대립유전자를 결정하는 것일 수 있다.In addition, the fifth step may be to determine the HLA allele by comparing and analyzing the SNP data of the subject to be analyzed with a reference panel.
상기 참조 패널(reference panel)은 유전자형의 연관성에 대한 사전 정보(predetermined information)를 제공하는 레퍼런스(reference) 데이터로서, 당업계에 알려진 Han(Chinese) MHC reference panel, Pan-Asian MHC reference panel, Korean MHC reference panel 등을 사용할 수 있다.The reference panel is reference data that provides predetermined information on the association of genotypes, and includes Han (Chinese) MHC reference panel, Pan-Asian MHC reference panel, and Korean MHC known in the art. A reference panel can be used.
이하, 본 발명의 실시예를 첨부된 도면을 참고하여 보다 상세하게 설명하도록 한다. 그러나, 하기의 실시예는 본 발명의 내용을 구체화 하기 위한 것일 뿐, 이에 의해 본 발명이 한정되는 것은 아닐 것이다.Hereinafter, embodiments of the present invention will be described in more detail with reference to the accompanying drawings. However, the following examples are only intended to specify the content of the present invention, and the present invention will not be limited thereby.
<실시예 1> 유전체정보의 정도관리<Example 1> Quality control of genome information
도 1을 참조하여 134,721명의 유전체역학조사사업 참여자를 대상으로 한국인칩을 이용하여 유전체정보를 생산하였다. 상기 한국인 칩은 국립보건연구원에서 개발된 약 83만개의 유전변이 정보를 포함하고 있는 korea biobank array를 사용하였다 (Moon et al. Scientific Reports 2019).Referring to FIG. 1, genome information was produced using Korean chips for 134,721 participants in the genome epidemiological investigation project. The Korean chip used the korea biobank array, which contains about 830,000 genetic mutation information developed by the National Institute of Health (Moon et al. Scientific Reports 2019).
생산된 유전체정보는 도 1을 참조하여 정도관리 파이프라인에 따라서 genotype calling, low quality 유전변이 제거, low quality 샘플 제거, MDS (Multi-dimensional scaling)/PCA (Principal Component Analysis)를 통한 이상치 제거 등을 수행하여 정제하였다.Genotype calling, low quality genetic mutation removal, low quality sample removal, outlier removal through MDS (Multi-dimensional scaling) / PCA (Principal Component Analysis), etc. Purification was carried out.
정제 후 Minor allele frequency가 0.01 이상, HWE 1e-6 미만, genotype call rate가 0.05 미만인 SNP을 선정하여 HLA-B region QC 분석을 진행하였으며, 염색체 6번, 30321652 ~ 32324956 지역(Genome Reference Consortium : GRCh37.p13, hg19 version, Genbank sequence: CM000668.1, RefSeq sequence: NC_000006.11)의 SNP 추출하였다.After purification, HLA-B region QC analysis was performed by selecting SNPs with a minor allele frequency of 0.01 or more, HWE less than 1e-6, and a genotype call rate of less than 0.05. p13, hg19 version, Genbank sequence: CM000668.1, RefSeq sequence: NC_000006.11) SNPs were extracted.
<실시예 2> SNP 정보 기반 HLA-B typing 정보 생산<Example 2> Production of HLA-B typing information based on SNP information
실시예 1에서 정제된 유전체정보의 phasing 분석은 Eagle 소프트웨어를 사용하여 수행하였으며, HLA imputation(HLA type 분석)은 Impute v4 소프트웨어를 사용하여 수행하였다. reference panel은 Han(Chinese) MHC reference panel을 이용하였으며, Han(Chinese) MHC reference panel은 10,689명 시퀀싱 정보를 사용하여 구성되었다 (Zhou et al. Nature Genetics 2016). Phasing analysis of the genomic information refined in Example 1 was performed using Eagle software, and HLA imputation (HLA type analysis) was performed using Impute v4 software. The reference panel used the Han (Chinese) MHC reference panel, and the Han (Chinese) MHC reference panel was constructed using sequencing information from 10,689 people (Zhou et al. Nature Genetics 2016).
구체적으로, HLA-B type 분석을 위해 Han Chinese HLA reference panel과 QC(quality-control)된 한국인칩 유전체정보에서 공통된 HLA-B type 관련 963개의 SNP 마커 추출하였다 (Genome Reference Consortium : GRCh37.p13, hg19 version, Genbank sequence: CM000668.1, RefSeq sequence: NC_000006.11, Chromosome 6, 30321652 ~ 32324956 지역). 상기 HLA-B type 관련 SNP 마커 정보는 표 1에 나타내었다.Specifically, for HLA-B type analysis, 963 SNP markers related to common HLA-B type were extracted from the Han Chinese HLA reference panel and QC (quality-control) Korean chip genome information (Genome Reference Consortium: GRCh37.p13, hg19 version, Genbank sequence: CM000668.1, RefSeq sequence: NC_000006.11, Chromosome 6, region 30321652 to 32324956). SNP marker information related to the HLA-B type is shown in Table 1.
그 후, 실시예 1에서 정제된 유전체정보는 Eagle 소프트웨어를 사용하여 phasing 분석하였다. 또한, HLA imputation은 한국인 칩(Moon et al. Scientific Reports 2019)에서 장기이식코호트 참여자의 genome data를 이용하였으며, genome DNA로부터 표 1의 SNP 마커를 검출할 수 있는 프로브(probe)를 사용하여 SNP 데이터를 수집하고, 상기 SNP 데이터는 reference panel과 표 1의 SNP 마커에 대한 유전자형 비교분석을 통해 HLA 대립유전자를 결정하였다(도 2 참조).Then, the genomic information purified in Example 1 was analyzed by phasing using Eagle software. In addition, HLA imputation used genome data of organ transplant cohort participants from Korean chips (Moon et al. Scientific Reports 2019), and SNP data using probes capable of detecting the SNP markers in Table 1 from genomic DNA. was collected, and the HLA allele was determined through comparative genotype analysis of the SNP data for the reference panel and the SNP markers in Table 1 (see FIG. 2).
<실시예 3> HLA-B typing 정보 제공<Example 3> Providing HLA-B typing information
상기 실시예 2의 방법으로 HLA typing을 수행하고 HLA-B 유전자에 대한 HLA typing 정보를 저해상도인 2 digit 및 고해상도인 4 digit 정보로 정리하였다. 결과는 각 HLA type 및 digit 별 확률값 형태로 나타나며, 그 중 가장 높은 확률을 보이는 HLA allele을 HLA type 정보로 제공한다.HLA typing was performed by the method of Example 2, and HLA typing information for the HLA-B gene was organized into low-resolution 2-digit and high-resolution 4-digit information. The result is displayed in the form of probability values for each HLA type and digit, and among them, the HLA allele with the highest probability is provided as HLA type information.
<실시예 4> HLA-B typing 정확도 검증<Example 4> HLA-B typing accuracy verification
NGS 기반 HLA typing 방법과 SNP 정보 기반 HLA typing 방법의 정확도 비교를 위해, 장기이식코호트 참여자 중에서 상기 한국인칩 유전체정보 중 254명을 선별하여 실시예 3의 SNP 정보 기반 HLA typing 정보와 실제 HLA typing 정보와 비교하여 정확도 검증을 수행하였다. In order to compare the accuracy of the NGS-based HLA typing method and the SNP information-based HLA typing method, 254 of the Korean chip genome information were selected from among the organ transplant cohort participants, and the SNP information-based HLA typing information and actual HLA typing information and actual HLA typing information of Example 3 were selected. Accuracy verification was performed by comparison.
HLA-B 유전자형으로 판별된 샘플의 HLA allele 정확도를 비교해본 결과, SNP 정보 기반 HLA typing 방법을 통해 HLA-B 유전자형으로 결정하는 경우 2 digit 기준은 99.14%, 4 digit 기준 97.79%의 정확도를 보였다. 정확도는 하기 수학식 1의 계산식으로 도출하였다.As a result of comparing the HLA allele accuracy of the samples determined by the HLA-B genotype, when determining the HLA-B genotype through the SNP information-based HLA typing method, the accuracy of 99.14% for the 2-digit standard and 97.79% for the 4-digit standard was shown. Accuracy was derived by the calculation formula of Equation 1 below.
이러한 결과를 통해 본 발명의 963개의 SNP 정보를 기반으로 HLA-B type을 분석하는 방법은 한국인 특이적인 SNP 정보 및 최적화된 분석 파이프라인을 제공함으로써 4일 이내에 기존 방법 대비 20배 낮은 가격(약 5만원 미만)에 99.1% 수준 정확도(2-digit 기준)의 HLA type을 제공 할 수 있다는 것을 확인하였다. HLA-B type은 장기이식 시 공여자, 수여자간 장기 적합여부를 판단할 때 보는 주요 유전자이다. 따라서 본 발명의 방법을 이용하여 장기이식 대상자 및 공여자에 대해 사전 검사 등에 유용하게 활용 할 수 있으며, 낮은 비용과 적은 소요 시간으로 HLA type을 제공함으로써 약물유전학, 면역 질환, 관련 만성질환 등 다양한 분야에서 연구에 활용 할 수 있을 것으로 예상된다.Through these results, the method of analyzing HLA-B type based on 963 SNP information of the present invention provides Korean-specific SNP information and an optimized analysis pipeline, which is 20 times lower than the existing method within 4 days (about 5 It was confirmed that the HLA type with 99.1% level accuracy (2-digit standard) can be provided for less than 10,000 won). HLA-B type is a major gene to be looked at when determining organ compatibility between donor and recipient during organ transplantation. Therefore, by using the method of the present invention, it can be usefully used for pre-examination of organ transplant recipients and donors, and by providing HLA type at low cost and time, it can be used in various fields such as pharmacogenetics, immune diseases, and related chronic diseases. It is expected that it can be used in research.
이상에서 살펴본 바와 같이, 본 발명의 구체적인 실시예를 상세하게 설명되었으나, 본 발명의 사상을 이해하는 당업자는 동일한 사상의 범위 내에서 다른 구성요소를 추가, 변경, 삭제 등을 통하여, 퇴보적인 다른 발명이나 본 발명 사상의 범위 내에 포함되는 다른 실시예를 용이하게 제안할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. 본 발명의 범위는 상술한 상세한 설명보다는 후술하는 특허청구의 범위에 의하여 나타내어지며, 특허청구의 범위의 의미 및 범위 그리고 그 균등 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.As described above, the specific embodiments of the present invention have been described in detail, but those skilled in the art who understand the spirit of the present invention may add, change, delete, etc. other components within the scope of the same idea, and other degenerate inventions. However, other embodiments included within the scope of the present invention can be easily suggested. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting. The scope of the present invention is indicated by the claims to be described later rather than the above detailed description, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts thereof are included in the scope of the present invention. should be interpreted as
Claims (9)
Table 1 (Paragraph Identification Number [0061]) marker set composition for determining HLA-B genotype including single nucleotide polymorphism (SNP) related to HLA-B type.
상기 유전체(genome DNA)의 서열정보를 수득하는 2단계;
상기 서열정보로부터 표 1(문단식별번호 [0061])에 기재된 963개의 단일염기다형성(SNP)에 대한 유전자형을 분석(genotyping)하여 분석대상의 SNP 데이터를 수집하는 3단계; 및
상기 분석대상의 SNP 데이터를 분석하여 HLA 대립유전자를 결정하는 4단계;를 포함하는, HLA-B 유전자형 분석 방법.
Step 1 of extracting genome DNA from a biological sample obtained from an analyte;
Step 2 of obtaining sequence information of the genome DNA;
A third step of genotyping the 963 single nucleotide polymorphisms (SNPs) described in Table 1 (paragraph identification number [0061]) from the sequence information to collect SNP data of the analysis target; and
4 steps of determining the HLA allele by analyzing the SNP data of the analyte; containing, HLA-B genotyping method.
상기 생물학적 시료는 혈액, 조직, 세포, 혈청, 혈장, 타액 및 소변으로 이루어진 군으로부터 선택되는 어느 하나인 것을 특징으로 하는, HLA-B 유전자형 분석 방법.
According to claim 2,
The biological sample is characterized in that any one selected from the group consisting of blood, tissue, cells, serum, plasma, saliva and urine, HLA-B genotyping method.
상기 방법은 2자리(digit) 또는 4자리(digit) 대립유전자 해상도로 HLA-B 유전자형을 결정하는 것을 특징으로 하는, HLA-B 유전자형 분석 방법.
According to claim 2,
The method is characterized by determining the HLA-B genotype with 2-digit or 4-digit allele resolution, HLA-B genotyping method.
상기 분석대상은 한국인인 것을 특징으로 하는, HLA-B 유전자형 분석 방법.
According to claim 2,
Characterized in that the analysis target is a Korean, HLA-B genotyping method.
상기 4단계는 분석대상의 SNP 데이터와 참조 패널(reference panel)과 비교분석하여 HLA 대립유전자를 결정하는 것을 특징으로 하는, HLA-B 유전자형 분석 방법.
According to claim 2,
The step 4 is characterized in that the HLA allele is determined by comparing and analyzing the SNP data of the target and the reference panel (reference panel), HLA-B genotyping method.
상기 참조 패널(reference panel)은 Han(Chinese) MHC reference panel, Pan-Asian MHC reference panel 및 Korean MHC reference panel 이루어진 군에서 선택되는 것을 특징으로 하는, HLA-B 유전자형 분석 방법.
According to claim 6,
The reference panel is characterized in that selected from the group consisting of Han (Chinese) MHC reference panel, Pan-Asian MHC reference panel and Korean MHC reference panel, HLA-B genotyping method.
A composition for analysis of HLA-B genotyping, comprising an agent capable of detecting the marker set composition of claim 1.
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