논문 상세보기
pp.36-44
Environmental DNA (eDNA) can exist in both intracellular and extracellular forms in natural ecosystems. When targeting harmful cyanobacteria, extracellular eDNA indicates the presence of traces of cyanobacteria, while intracellular eDNA indicates the potential for cyanobacteria to occur. However, identifying the “actual” potential for harmful cyanobacteria to occur is difficult using the existing sediment eDNA analysis method, which uses silica beads and cannot distinguish between these two forms of eDNA. This study analyzes the applicability of a density gradient centrifugation method (Ludox method) that can selectively analyze intracellular eDNA in sediment to overcome the limitations of conventional sediment eDNA analysis. PCR was used to amplify the extracted eDNA based on the two different methods, and the relative amount of gene amplification was compared using electrophoresis and Image J application. While the conventional bead beating method uses sediment as it is to extract eDNA, it is unknown whether the mic gene amplified from eDNA exists in the cyanobacterial cell or only outside of the cell. However, since the Ludox method concentrates the intracellular eDNA of the sediment through filtration and density gradient, only the mic gene present in the cyanobacteria cells could be amplified. Furthermore, the bead beating method can analyze up to 1 g of sediment at a time, whereas the Ludox method can analyze 5 g to 30 g at a time. This gram of sediments makes it possible to search for even a small amount of mic gene that cannot be searched by conventional bead beating method. In this study, the Ludox method secured sufficient intracellular gene concentration and clearly distinguished intracellular and extracellular eDNA, enabling more accurate and detailed potential analysis. By using the Ludox method for environmental RNA expression and next-generation sequencing (NGS) of harmful cyanobacteria in the sediment, it will be possible to analyze the potential more realistically.
서 론
재료 및 방 법
1. 조사지점
2. 퇴적물 채집방법
3. 물리적 충격 분리법 (Silica Bead Beating method:SBB method)
4. 밀도 구배 원심분리법 (Ludox method)
5. PCR 증폭
6. 2-MIB 생합성 유전자 (mic) 염기서열 계통분석
결과 및 고 찰
1. 퇴적물 전처리 방법에 따른 DNA 증폭효율
2. 밀도 구배 원심분리법의 현장탐색 효율
3. PCR product 염기서열의 계통학적 분석
결 론
적 요
REFERENCES
