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중형저서동물에서 효율적인 DNA 추출 방법 비교 연구

Comparative Study of DNA Extraction Method in Meiofauna

이원철|백진욱|이승한
한국환경생물학회 (Korean Society Of Environmental Biology)|2011.09|환경생물 (Korean Journal of Environmental Biology)|제29권 제3호|pp. 138-143

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참고문헌

키워드

DNA extractionmeiofaunamtCOILudoxRose Bengal

초록

이번 연구에서는 중형저서동물의 생태학적 연구에 사용하는 Ludox와 Rose Bengal을 처리하였을 때, 고정액의 종류에 따른 DNA (mtCOI)의 추출 효율을 비교하고자 하였다. 실험을 위해 저서성 요각류 Tigriopus japonicus s.l.를 실험동물로 사용하였으며, 99% 에탄올과 4%포르말린의 두 종류의 고정액을 사용한 뒤 이들을 각각대조구로 삼았다. 그리고 (1) Ludox HS40, (2) Rose Bengal,(3) Ludox HS40+Rose Bengal을 각각 시료와 반응시킨뒤, mtCOI 유전자를 추출하였다. 이후 PCR을 진행하고산물을 전기영동하여 유전자의 증폭여부를 확인하였다. 또한 모든 실험은 30회 반복하여 실험간의 결과를 비교하였다. 그 결과, 에탄올의 경우에는 대조구를 포함한(1), (2), (3)의 실험 모두에서 96% 이상의 효율을 보였지만, 포르말린의 경우에는 대조구에서 27% 증폭되었으며,(1)과 (3)에서 약 3%, (2)에서 약 7%만 증폭되어 두 고정액에 따른 차이가 뚜렷하게 나타났다. 결과적으로 현재의 연구를 통해서 99% 에탄올이 중형저서동물에서DNA를 추출하는 데 적합한 고정액임을 확인하였다.

Abstract

The efficiency of mtCOI amplication after DNA extraction of benthic harpacticoid Tigriopus japonicus s.l. was tested under different conditions depending on fixative (99% Ethanol,or 4% Formalin) and additional chemicals (Ludox or Rose Bengal). Each experimental group by the fixative was subdivided into four groups, respectively: 1) Control (fixative only), 2) processed with Ludox HS40, 3) processed with Rose Bengal, and 4) processed with both Ludox HS40 and Rose Bengal. For the 99% ethanol-fixed sample, overall success rate of amplification by PCR was 96% or above, while for the 4% formalin-fixed one, success rate was much lower than those of ethanol-fixed: 1) Control: 27%, 2) Ludox HS40: 3%, 3) Rose Bengal: 7%, and 4) Ludox HS40 and Rose Bengal: 3%. As a result present study verify that 99% ethanol is a proper fixative for DNA extraction in meiofauna organisms.

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