Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Development of In situ PCR Method Using Primer Polymers

프라이머 중합체를 이용한 원위치 중합효소 연쇄반응 In situ PCR 방법의 개발

  • Published : 2004.06.01
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Abstract

Reduction in the leakage of the amplified PCR product out of cell is required for effective in situ PCR. For this purpose, primers with complementary tail sequences at their 5' sides were utilized to synthesize high molecular weight PCR products, but it is time-consuming and causes deterioration of cellular appearance with many PCR cycles. Therefore, it is required to optimize the PCR condition with minimal PCR cycles. To achieve the pur-pose, primer polymers were made without the target DNA in tube from nonspecific amplification with tailed primers and treated onto the fixed Molt/LAV cells on the glass slide for the 20 cycle-in situ PCR, in which the appropriate target signals were observed for the possible use of primer polymers in in situ PCR.

효과적인 원위치 중합효소 연쇄반응 (In situ PCR)을 위해서는 증폭된 PCR 산물의 세포외 유출을 감소시켜야 한다. 이를 위한 한 방법으로 거대분자 PCR 산물을 합성시키기 위한 5'쪽에 서로 상보적인 꼬리서열을 가진 프라이머(꼬리 프라이머; tailed primer)가 사용되었으나 많은 PCR 횟수로 인해 시간의 낭비와 세포조직의 형태보존성이 저하되는 문제가 발생하였다. 따라서 PCR 조건을 가능한 최적화시키고, 최소의 PCR 횟수로써 세포외 유출을 막을 수 없는 방법이 필요하게 되었다. 이러한 방법의 일환으로 꼬리 프라이머를 이용하여 PCR 튜브 속에서 목표 핵산없이 프라이머 중합체(primer polymers)의 형성을 유도하였고, 이를 유리 슬라이드위에 고정시킨 Molt/LAV 세포들에 처리하여 20 회의 짧은 시간에서도 적절한 탐침을 할 수 있게 되었다. 이로 인해 프라이머 중합체의 원위치 중합효소 연쇄반응에서의 사용가능성을 타진하였다.

Keywords

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