Anatomy & Biological Anthropology (해부∙생물인류학)

Korean Association of Physical Anthropologists (대한체질인류학회)
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Determination of Repeat Numbers of (CA)n in Mitochondrial D-loop using Polymerase Chain Reaction-single Strand Conformational Polymorphism (PCR-SSCP)

중합효소연쇄반응-단일가닥입체형태다형태 방법을 이용한 미토콘드리아 D-고리에서의 CA 디뉴클레오티드 반복수의 결정

  • Kim, Joo-Young (Department of Anatomy, College of Medicine, Yeungnam University) ;
  • Kim, Dae-Kwang (Department of Medical Genetics, School of Medicine, Keimyung University)
  • 김주영 (영남대학교 의과대학 해부학교실) ;
  • 김대광 (계명대학교 의과대학 의학유전학교실)
  • Received : 2018.07.11
  • Accepted : 2018.09.11
  • Published : 2018.09.30
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Abstract

Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis is a kind of sensitive mutation detection method that has been usually used in field of medical genetics. A single DNA strand with a mutation or nucleotide polymorphism has a different conformation from its wild-type counterpart, and these conformational differences result in different electrophoretic mobility. In previous study of mitochondrial microsatellite instability in 50 uterine leiomyomas, PCR-SSCP showed 4 types of band mobility at (CA)n of the mitochondrial D-loop. In type 1 and 4, positions of the lower single stand of both were same but those of upper strand were different. In sequencing analysis, repeat number of (CA)n in type 1 was 4, 5 in type 2, 6 in type 3, and 4 in type 4, respectively. Without using expensive sequencing analysis, PCR-SSCP method can be used to detect the repeat number of(CA)n in mitochondrial D-loop.

중합효소연쇄반응-단일가닥입체형태다형태 (Polymerase Chain Reaction-Single Strand Conformation Polymorphism, PCR-SSCP) 방법은 돌연변이를 민감하게 검출하는 방법으로 의학유전학 영역에서 많이 사용되었다. 돌연변이나 단일뉴클레오티드다형태(single nucleotide polymorphism, SNP)를 가지는 DNA 단일가닥조각은 전기영동 상에서 정상 야생 DNA 조각과는 다른 이동 양상을 보인다. 미토콘드리아의 복제와 전사는 D-고리에서 일어나며, 이 부위에 시토신-아데노신(cytosine-adenosien, CA) 디뉴클레오티드의 반복수 다형태 ((CA)n)가 나타난다. 이 연구에서는 자궁근종의 미토콘드리아 현미부수체의 불안정 논문에서 나타난 SSCP 양상을 재분석하고, 각 SSCP 양상에 대한 PCR 산물을 DNA 염기서열분석을 실시하여 SSCP 양상과 (CA)n 다형태를 비교 조사하고자 한다. 총 50례의 자궁근종의 (CA)n 다형태의 SSCP 양상은 단일가닥의 이동을 기준으로 네 가지 유형으로 분류할 수 있었는데, 아래쪽 단일가닥의 이동속도가 빠른 순으로 3가지 형으로 분류하였다. 제1 형의 아래쪽 단일가닥의 높이는 서로 같았으나, 위쪽 단일가닥의 높이는 일치하지 않은 경우를 제 4형으로 분류하였다. DNA 염기서열 분석에서 제 1형의 (CA)의 반복수는 4회, 제 2형은 5회, 제 3형은 6회 그리고 제 4형은 제 1형과 같이 (CA)n 반복수는 4회였다. 이와 같은 결과는 값비싼 염기서열을 이용하지 않고 단순히 PCR-SSCP 방법으로 미토콘드리아 D-고리의 (CA)n 반복수를 구할 수 있다는 것을 보여준다.

Keywords

References

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