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Molecular Techniques to Identify Freshwater Eels: RFLP Analyses of PCR-amplified DNA Fragments and Allele-specific PCR from Mitochondrial DNA

淡水鰻魚的分生鑑定技術:以粒線體去氧核醣核酸進行限制酵素片段長度多型性分析以及專一引子的聚合酵素連鎖反應分析

Abstracts


由於淡水鰻魚的鰻苗在外型上十分相似而難以區別,傳統的分類工作面臨了許多困難和不確定性。本篇研究參考已經發表的兩個粒線體基因(cytochrome b及12S rRNA)建立了鰻魚鑑定的分子標準。根據推測得到的限制酵素片段長度多型性,結合三個限制酵素就能在cytochrome b基因有很好的鑑別力。另外我們也在這兩個基因上共找到39個特別的核苷酸位址,可用來設計專一的引子進行聚合酵素連鎖反應分析。為了檢驗這些預測的正確性,我們選取四種共58個樣本進行DpnII和HaeIII的限制酵素片段長度多型性分析以及專一引子的聚合酵素連鎖反應分析。結果顯示,他們分別都有超過95%和99%的正確率,和根據突變率預測的結果相符合。因此,本篇研究所提出來的分生鑑定技術,對鰻魚養殖業以及生態研究工作應有一定的參考價值。

Parallel abstracts


Because of morphological ambiguity, traditional classification of freshwater eel elvers (of the genus Anguilla) has always been difficult and unreliable. This study analyzes 2 mitochondrial genes viz., cytochrome b and 12S ribosomal RNA (rRNA) genes from previous studies, to establish molecular standards for eel identification. Prediction of restriction fragment length polymorphism (RFLP) indicated that a combination of 3 enzyme results for cytochrome b revealed good resolution power. We also selected 39 specific nucleotide sites of the 2 genes at which the site is unique for individual species: thus these can potentially be used for identification by polymerase chain reaction (PCR). To verify the accuracy of our predictions, we examined 58 specimens from 4 species by DpnII and HaeIII digestions and by PCR amplification using species-specific primers. Consistent with the putative mutation rates, more than 95% and 99% of the specimens could be successfully identified by the RFLP and PCR methods, respectively. The molecular techniques developed here can be helpful in eel aquaculture and ecological studies.

Parallel keywords

PCR RFLP Identification Anguilla Elver

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