Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Cloning and DNA Sequencing for Unstable Minisatellites DNA Regions in E. coli.

대장균 내에서 불안정한 Minisatellite DNA 영역의 클론닝 및 DNA 염기서열 결정

  • 임선희 (동아대학교 자연과학대학 생물학과) ;
  • 김재우 (동아대학교병원 임상병리) ;
  • 김광섭 (동아대학교 자연과학대학 생물학) ;
  • 정윤희 (동아대학교 자연과학대학 생물학) ;
  • 윤세련 (동아대학교 자연과학대학 생물학) ;
  • 배호정 (동아대학교 자연과학대학 생물학) ;
  • 안태진 (동아대학교 자연과학대학 생물학) ;
  • 선우양일 (동아대학교 자연과학대학 생물학과)
  • Published : 2004.06.01
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Abstract

Instability of some eukaryotic sequence propagated in prokaryotic hosts is a frequently observed phenomenon. It is well documented that long inverted repeats, AT-rich sequences with structures like Z-DNA are extremely unstable in E. coli. These sequences may either be under-represented or even lost when cloned in E. coli. When we analyzed the polymorphic pattern for several tandom repeat (TR) in human SCKI gene, we found some TR regions were frequently deleted from plasmids and had difficult problem for their sequencing. These regions may result in non-clonability of the DNA sequence. Here we have cloned two difficult TR regions under low temperature and made two library for DNA sequencing using a nebulizer or sonicator. This study will help to determine the unstable genomic elements in complex mammalian genome.

진핵생물의 특정 염기배열을 원핵생물 내에서 증폭시킬 때 불안정성이 비교적 빈번히 관찰되어진다. 특히 long inverted repeats나 AT-rich sequences그리고 Z-DNA와 같은 구조를 지닌 염기배열은 대장균 내에서 매우 불안정하다. 이러한 염기서열은 대장균 내에서 부분적으로 결실되거나 완전히 손실된다. 본 연구실에서 human SCKI 유전자에 존재하는 몇 개의 tandem repeat (TR)에 대하여 다형성을 조사하였을 때, 어떤 TR 부분은 플라스미드로부터 빈번히 결실되어 그에 대한 염기서열 결정이 어려웠다. 그 결과 이러한 부분은 클론닝 될 수 없는 염기서열로 남게 되었다. 본 연구에서는 클론닝이 어려운 두 개의 TR 영역을 저온에서 클론닝하고 nebulizer나 sonicator를 이용하여 두 개의 library를 만들어 DNA 염기서열을 결정하였다. 이러한 연구는 복잡한 고등생물의 게놈연구에서 불안정한 게놈부분의 염기서열을 결정하는데 도움을 줄 것으로 사료된다.

Keywords

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