KR20010105503A - Rapid Multiplex Genotyping Kit System for the detection of Y-chromosome STRs - Google Patents
Rapid Multiplex Genotyping Kit System for the detection of Y-chromosome STRs Download PDFInfo
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Abstract
Description
본 발명은 사람의 Y-염색체에 위치하는 STRs (Short Tandem Repeats) 유전자의 변이를 경제적이고도 신속하고 정확하게 분석할 수 있는 방법 및 키트 (allelic ladder 포함)에 관한 것으로, 더욱 상세하게는 여러 STRs 좌위를 동시에 타이핑(typing)하기 위하여 멀티플렉스(multiplex) 중합효소연쇄반응(PCR; polymerase chain reaction)을 이용하여 증폭 후, 얻어진 allelic ladder와 비교분석하는 것으로 구성되는, 개인식별 및 법과학 분야에 활용되는 분석 방법 및 키트에 관한 것이다.The present invention relates to methods and kits (including allelic ladders) that can economically, quickly and accurately analyze mutations in STRs (Short Tandem Repeats) genes located on human Y-chromosomes. Analytical methods used in the field of personal identification and forensic science, consisting of amplification using a multiplex polymerase chain reaction (PCR) to compare at the same time, followed by comparison with the obtained allelic ladder And kits.
최근 분자 생물학적 기법이 발전됨에 따라, 한국을 비롯한 아시아, 미국 및 유럽 대부분의 국가에서는 DNA 프로필(profile) 분석결과를 개인식별 및 법의학 분야에 적용시키고 있다. 현재 국제적으로 대부분의 경우, 상염색체 유전자 마커(marker) (STRs)를 통한 DNA 프로필 분석 방법이 주로 활용되고 있으며, 또한 이러한 상염색체 STR 분석 키트 시스템이 상업적으로 개발되어 있다. 한편, Y-염색체의 NRPY (non-recombining portion of the Y-chromosome) 부위는 X-염색체와 교차되지 않기 때문에, 이 부위에 위치한 여러 유전자들의 변이를 단상형(haplotype) 상태로 비교 분석함으로써 남자와 관련된 개인식별 및 법과학 분야에 매우 유용한 수단으로써 활용될 수 있다. 이와 같이 NRPY 상에 위치한 Y-STR 마커는 개인에 따라 매우 다양한 유전적 변이를 나타내기 때문에, 개인식별 및 법과학 분야에서는 Y-STRs에 관한 유전적 변이를 표준화된 기법에 의해 분석 및 판정할 필요가 있다. 아직까지 상염색체와는 달리 Y-염색체의 STR 분석방법 및 분석 키트 시스템 (allelic ladders)은 개발되어 있지 않다.With the recent development of molecular biological techniques, DNA profiles analysis results have been applied to the field of personal identification and forensics in many countries including Korea, Asia, USA and Europe. Currently in most cases internationally, methods for analyzing DNA profiles via autosomal gene markers (STRs) are mainly utilized, and such autosomal STR assay kit systems have been developed commercially. On the other hand, since the non-recombining portion of the Y-chromosome (NRPY) region of the Y-chromosome does not intersect with the X-chromosome, mutations of various genes located in this region are compared with the male by a haplotype. It can be used as a very useful tool in the field of personal identification and forensic science involved. Since the Y-STR markers located on NRPY show a wide variety of genetic variations according to the individual, it is necessary in the personal identification and forensic science fields to analyze and determine the genetic variation of Y-STRs by standardized techniques. have. Unlike the autosomal, no Y-chromosome STR assay and assay kit system (allelic ladders) have been developed.
따라서, 본 발명의 목적은 남자와 관련된 개인식별 및 법과학 분야에서 필요한 Y-STRs 분석 방법을 제공하는 것이다.Accordingly, it is an object of the present invention to provide a method for analyzing Y-STRs necessary in the field of personal identification and forensic science related to men.
본 발명의 다른 목적은 남자와 관련된 개인식별을 위한 Y-STRs 분석 키트를 제공하는 것이다.Another object of the present invention is to provide a Y-STRs analysis kit for personal identification associated with men.
도 1은 본 발명에 따른 Multiplex system 중, Triplex systemⅠ (DYS19/DYS389Ⅰ/DYS389Ⅱ)에 의해 나타난 전기영동상1 is an electrophoretic image shown by Triplex system I ( DYS19 / DYS389I / DYS389II ) among the multiplex systems according to the present invention.
도 2는 본 발명에 따른 Multiplex system 중, Triplex systemⅡ (DYS390/DYS391/DYS393)에 의해 나타난 전기영동상Figure 2 is an electrophoretic image represented by Triplex system II ( DYS390 / DYS391 / DYS393 ) of the Multiplex system according to the present invention
도 3은 본 발명에 따른 Multiplex system 중, Triplex systemⅢ (DYS19/DYS388/DYS392)에 의해 나타난 전기영동상3 is an electrophoretic image shown by Triplex system III ( DYS19 / DYS388 / DYS392 ) of the Multiplex system according to the present invention.
이러한 목적을 위해, 상이한 인류집단을 대상 (약 2,000여명)으로 8 STR 유전자좌들에서 PCR 및 염기서열 분석방법으로 확인된 모든 대립유전자(DYS19: 7 alleles,DYS388: 7 alleles,DYS389Ⅰ: 5 alleles,DYS389Ⅱ: 7 alleles,DYS390: 8 alleles,DYS391: 5 alleles,DYS392: 10 alleles,DYS393: 7 alleles) 들을 선별하여 플라스미드 벡터에 클로닝한 후, 각각의 클론(clone)으로부터 PCR 방법에 의해 이들 대립유전자를 증폭시켰다. 이후 이들 PCR 산물을 각기 분리 및 정제과정을 거침으로써 Y-STRs의 대립유전자 판정에 표준화될 수 있는 allelic ladders를 개발한 것이다.For this purpose, alleles ( DYS19 : 7 alleles, DYS388 : 7 alleles, DYS389Ⅰ : 5 alleles, DYS389Ⅱ) identified by PCR and sequencing at 8 STR loci in different human populations (approximately 2,000). : 7 alleles, DYS390 : 8 alleles, DYS391 : 5 alleles, DYS392 : 10 alleles, DYS393 : 7 alleles) were selected and cloned into plasmid vectors, and then amplified these alleles from each clone by PCR method. I was. The PCR products were then separated and purified to develop allelic ladders that can be standardized for allelic determination of Y-STRs.
본 발명의 멀티플렉스 PCR 시스템을 이용한 Y-STRs 분석방법은DYS19,DYS388,DYS389Ⅰ,DYS389Ⅱ,DYS390,DYS391,DYS392, 및DYS393유전자좌들로 구성된 군으로부터 동시에 증폭될 수 있는 적어도 3개 이상의Y-STR 유전자좌들을 갖고 있는 하나 이상의 분석 DNA 샘플을 얻는 단계; 상기 DNA 샘플에서 특정 프라이머들을 사용하여 Y-STR 서열을 PCR 증폭하는 단계; 및 증폭된 산물을 전기영동을 통해 검출하여 allelic ladders와 비교함으로써 증폭된 대립유전자들을 평가하고, 이를 통해 DNA 샘플내의 분석된 유전자좌들 조합의 각각의 유전자형들을 결정하는 단계를 포함하는 것을 특징으로 한다.Y-STRs analysis method using the multiplex PCR system of the present invention is at least three or more Y-STR loci that can be amplified simultaneously from the group consisting of DYS19 , DYS388 , DYS389I , DYS389II , DYS390 , DYS391 , DYS392 , and DYS393 locus Obtaining one or more analytical DNA samples having these; PCR amplifying a Y-STR sequence using specific primers in the DNA sample; And evaluating the amplified alleles by detecting the amplified products by electrophoresis and comparing them with allelic ladders, thereby determining the respective genotypes of the analyzed locus combinations in the DNA sample.
상기 적어도 3개 이상의 유전자좌들로 구성된 멀티플렉스 STR 시스템은 바람직하게는 트리플렉스 STR 시스템이며, 본 발명의 트리플렉스 STR 시스템은 바람직하게는 다음과 같은 3종의 유전자좌들의 조합으로 구성된 군으로부터 선택된 1종 이상의 유전자좌들의 조합을 포함하는 것을 특징으로 한다:The multiplex STR system consisting of the at least three or more loci is preferably a triplex STR system, and the triplex STR system of the present invention is preferably one selected from the group consisting of the following three locus combinations: It is characterized by including a combination of the above loci:
DYS19/DYS389Ⅰ/DYS389ⅡDYS19 / DYS389Ⅰ / DYS389Ⅱ
DYS390/DYS391/DYS393DYS390 / DYS391 / DYS393
DYS19/DYS388/DYS392DYS19 / DYS388 / DYS392
본 발명은 또한DYS19,DYS388,DYS389Ⅰ,DYS389Ⅱ,DYS390,DYS391,DYS392, 및DYS393유전자좌들로 구성된 군으로부터 선택된 적어도 3개 이상의 유전자좌들의 조합 및 선택된 유전자좌 각각에 해당되는 프라이머들을 가지는 용기를 포함하는 Y-STRs 분석 키트에 관한 것이다.The present invention also provides a container comprising a container having a combination of at least three or more loci selected from the group consisting of DYS19 , DYS388 , DYS389I , DYS389II , DYS390 , DYS391 , DYS392 , and DYS393 loci and primers corresponding to each of the selected loci. STRs analysis kit.
상기 분석 키트의 상기 유전자좌에 해당하는 프라이머는 하기의 서열 쌍을 갖는다 :Primers corresponding to the locus of the assay kit have the following sequence pairs:
유전자좌가DYS19인 경우에는 상류 5'-CTACTgAgTTTCTgTTATAgT-3',If the locus is DYS19 upstream 5'-CTACTgAgTTTCTgTTATAgT-3 ',
하류 5'-ATggCATgTAgTgAggACA-3' ;Downstream 5'-ATggCATgTAgTgAggACA-3 ';
유전자좌가DYS388인 경우에는 상류 5'-gTgAgTTAgCCgTTTAgC gA-3', 하류 5'-CAgATCgCAACCACTgCg-3';Upstream 5′-gTgAgTTAgCCgTTTAgC gA-3 ′, downstream 5′-CAgATCgCAACCACTgCg-3 ′ when the locus is DYS388 ;
유전자좌가DYS389Ⅰ및DYS389Ⅱ인 경우에는 상류 5'-CCAACTCTC ATCTgTATTATCTAT-3', 하류 5'-TCTTATCTCCACCCACCAgA-3';Upstream 5′-CCAACTCTC ATCTgTATTATCTAT-3 ′, downstream 5′-TCTTATCTCCACCCACCAgA-3 ′ when the loci are DYS389I and DYS389II ;
유전자좌가DYS390인 경우에는 상류 5'-TATATTTTACACATTTTTggg CC-3', 하류 5'-TgACAgTAAAATgAACACATTgC-3';Upstream 5′-TATATTTTACACATTTTTggg CC-3 ′, downstream 5′-TgACAgTAAAATgAACACATTgC-3 ′ when the locus is DYS390 ;
유전자좌가DYS391인 경우에는 상류 5'-CTATTCATTCAATCATACAC CCA-3', 하류 5'-gATTCTTTgTggTgggTCTg-3';Upstream 5′-CTATTCATTCAATCATACAC CCA-3 ′, downstream 5′-gATTCTTTgTggTgggTCTg-3 ′ when the locus is DYS391 ;
유전자좌가DYS392인 경우에는 상류 5'-TCATTAATCTAgCTTTTAAAAA CAA-3', 하류 5'-A gACCCAgTTgATgCAATgT-3';Upstream 5′-TCATTAATCTAgCTTTTAAAAA CAA-3 ′, downstream 5′-A gACCCAgTTgATgCAATgT-3 ′ when the locus is DYS392 ;
유전자좌가DYS393인 경우에는 상류 5'-gTggTCTTCTACTTgTgTCAA TAC-3', 하류 5'-AACTCAAgTCCAAAAAATgAgg-3'.Upstream 5′-gTggTCTTCTACTTgTgTCAA TAC-3 ′ and downstream 5′-AACTCAAgTCCAAAAAATgAgg-3 ′ when the locus is DYS393 .
본 발명의 분석 키트는 바람직하게는 은 염색을 통하여 유전자형들을 가시화하는 것을 특징으로 한다.The assay kit of the present invention is preferably characterized by visualizing genotypes via silver staining.
본 발명, 즉 "Y-STRs 분석 키트"의 구성은 다음과 같다.The configuration of the present invention, that is, the "Y-STRs analysis kit" is as follows.
[8 종류 Y-STR 마커의 트리플렉스 은 감지 시스템][Triplex Silver Detection System for 8 Types of Y-STR Markers]
본 발명품에는TaqDNA 중합효소와 분석대상 DNA를 제외한, 각 시스템 별로 50회 PCR이 가능한 10× 프라이머 쌍, Y-STR 10× 완충액, 2× loading solution, positive control DNA, allelic ladder가 한 키트로 구성된다.Except for Taq DNA polymerase and DNA to be analyzed, the present invention consists of a kit of 10 × primer pairs, Y-STR 10 × buffer, 2 × loading solution, positive control DNA, and allelic ladder that can be PCR 50 times for each system. do.
1) Y-Triplex system kitⅠ:DYS19/DYS389Ⅰ/DYS389Ⅱ 1) Y-Triplex system kitⅠ: DYS19 / DYS389Ⅰ / DYS389Ⅱ
2) Y-Triplex system kitⅡ:DYS390/DYS391/DYS393 2) Y-Triplex system kitⅡ: DYS390 / DYS391 / DYS393
3) Y-Triplex system kitⅢ:DYS19/DYS388/DYS392 3) Y-Triplex system kit III: DYS19 / DYS388 / DYS392
* DYS393-16, 17 대립유전자: 지금까지 세계적으로 보고된 바 없는 본 발명품의 한국인의 특정 대립유전자. * DYS393--16 , 17 allele: Korean specific allele of the present invention that has not been reported worldwide.
실시예 1-3Example 1-3
본 발명의 실시예에서 사용된 인간 게놈 DNA는 한국인의 경우는 단국대 병원의 기증자, 단국대 학생, 동아시인의 경우는 일본, 중국, 몽고, 베트남, 필리핀, 인도네시아, 태국 등지의 공동 연구자들로부터 혈액을 공급받아 추출한 것을 사용하였다. 상기 DNA 샘플은 하나의 반응용기에서 증폭된다. PCR 증폭은 2.5 ??l의 10× PCR 완충용액, 0.2 mM dNTPs, 0.25 유니트의 Taq 중합효소, 1 내지 1O ng DNA 주형 및 0.015-0.5 ??M의 각 해당 프라이머로 구성된 전체 부피가 25 ??l인 반응용액에서 수행하였다. PCR 사이클은 다음과 같은 증폭 프로토콜을 채택하였다Human genomic DNA used in the embodiment of the present invention is a blood donor from Dankook University donors, Dankook University students, cooperative researchers in Japan, China, Mongolia, Vietnam, Philippines, Indonesia, Thailand, etc. The one that was supplied and extracted was used. The DNA sample is amplified in one reaction vessel. PCR amplification was performed using a total volume of 25 ?? of 10 × PCR buffer, 0.2 mM dNTPs, 0.25 units of Taq polymerase, 1 to 10 ng DNA template, and 0.015-0.5 ?? M of each corresponding primer. The reaction solution was carried out in l. The PCR cycle adopts the following amplification protocol.
94℃에서 3분간 예열;Preheat at 94 ° C. for 3 minutes;
94℃에서 30초, 58℃에서 30초, 72℃에서 45초로 30회 반복; 및30 repetitions of 30 seconds at 94 ° C, 30 seconds at 58 ° C, 45 seconds at 72 ° C; And
72℃에서 5분간 최종 연장.Final extension for 5 minutes at 72 ° C.
증폭된 산물들은 전기영동장치를 사용하여 50W로 자일렌 시아놀 염색약(Xylene Cyanol Dye)이 겔 (20 cm x 43 cm)의 밑바닥에서 약 4Omm 내지 60mm 될 때까지 전기영동을 수행하였다. 겔은 6T4, 7M 우레아 아크릴아미드 겔이며 런닝버퍼 및 겔버퍼는 TBE 버퍼를 사용하였다.The amplified products were subjected to electrophoresis using an electrophoresis apparatus at 50W until the xylene cyanol dye (Xylene Cyanol Dye) was about 40-60 mm at the bottom of the gel (20 cm x 43 cm). The gel was a 6T4, 7M urea acrylamide gel and the running buffer and the gel buffer used TBE buffer.
전기영동이 끝난 겔은 은 염색과정을 거쳐 밴드를 확인하는데 우선 10의 에탄올로 10분 동안 수세시켰다. 그런 다음 증류수로 3회 수세한 후,0.2질산은(silver nitrate)에서 20분 동안 반응시키고 증류수로 다시 한번 세척하였다. 수세된 겔은 0.28M 무수 탄산나트륨와 0.019포르말린 용액에서 환원시키면서 나타나는 밴드의 양상에 따라 10아세트산 용액으로 염색반응을 중지시켰다. 이렇게 염색된 겔은 3M 종이에 흡착시켜 겔 건조기에서 말린 후 보관하였다. 시험 결과를 도 1 내지 도 3에 도시하였다.After electrophoresis, the gel was subjected to silver staining to identify a band. The gel was first washed with 10 ethanol for 10 minutes. Then, washed three times with distilled water, and reacted for 0.2 minutes in 0.2 silver nitrate and washed once again with distilled water. The washed gel was stopped with 10 acetic acid solution according to the appearance of the band while reducing in 0.28 M anhydrous sodium carbonate and 0.019 formalin solution. This dyed gel was adsorbed onto 3M paper, dried in a gel drier and stored. Test results are shown in FIGS.
Y-STR 마커는 개인에 따라 매우 다양한 유전적 변이를 나타내기 때문에, 특히 남자와 관련된 개인식별 및 법과학 분야에서는 Y-STRs에 관한 유전적 변이를 표준화된 기법에 의해 발명 개발될 필요가 있다. 일반적으로 강력 사건이나 성범죄가 남자에 의해 일어나는 경우가 많다고 볼 때, Y-염색체의 DNA 정보는 이러한 문제 해결에 매우 적절한 마커가 될 수 있으며, 특히 본 발명품은 남자와 여자의 세포조직이 혼합된 시료에서 남자의 DNA만을 특이적으로 분석할 수 있는 장점이 있다. 현재 국내외적으로 상염색체 유전자 마커(STRs)를 분석하는 키트 시스템은 개발 및 제품화되어 있으나, 상염색체와는 달리 아직까지 Y-염색체의 STR 분석 키트 시스템(allelic ladders)은 개발되어 있지 않다. 따라서 개발된 본 발명품의 제품화에 따라 국내 생명공학 산업의 선진화 및 경쟁력 확보에도 기여할 것이며, 이에 따른 고용증대 및 경제적인 부가가치는 물론 분석기법의 효율성과 신뢰도를 높일 수 있을 것으로 기대된다. 또한 향후 지속적인 기술개발을 유도함으로써 Bio-chip 개발 등과 같은 보다 신속하고도 경제적인 유전자 타이핑 기법에 의해, 남북 통일을 대비한 이산가족 찾기 및 지문을 대체할 수 있는 전국민의 DNA profiling 사업의 수행을 가능하게 할 수 있다.Since Y-STR markers represent a wide variety of genetic variations, the genetic variation of Y-STRs needs to be invented and developed by standardized techniques, particularly in the field of personal identification and forensic science involving men. In general, strong events and sex crimes are often caused by men, so the DNA information of Y-chromosome can be a very suitable marker for solving this problem. In particular, there is an advantage that can specifically analyze the DNA of a man. Currently, kit systems for analyzing autosomal gene markers (STRs) have been developed and commercialized at home and abroad. Unlike autosomals, STR assay kit systems (allelic ladders) of Y-chromosomes have not been developed yet. Therefore, the development of the present invention will contribute to the advancement and competitiveness of the domestic biotechnology industry, and it is expected to increase the efficiency and reliability of the analytical method as well as increase employment and economic added value. In addition, by inducing continuous technology development in the future, faster and more economical genetic typing techniques such as bio-chip development, DNA profiling business of nationwide who can search for separated families and replace fingerprints in preparation for unification of South and North Korea. You can do that.
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