運用自動移液器改善水稻突變體篩選效率

聚合酶連鎖反應（Polymerase Chain Reaction, PCR）可以擴增基因體中指定的DNA序列，因此可用於篩選大量野生型植株中之基改植株或突變體，當突變效率低時（例如千分之一或更低），在大量樣品中選拔突變體耗時費工。本研究改良篩選步驟，以大量水稻樣品中篩選唯一基改植株為例，將樣品以三維陣列排列分群，例如125株水稻樣品中僅1株為基改水稻，一般以PCR配合專一引子分析各樣品共125個PCR反應，在此則以5×5×5三維排列，採XY平面、XZ平面、YZ平面分別混合成各群樣品，每群25個樣品共15群，只需執行15個PCR反應即可找出轉殖株。然若以人工操作上述樣品混合分群，耗費人力且容易操作失誤，因此本研究利用自動移液分注器（OT-2 Robot）完成混合分群，撰寫程式碼將96孔排放之樣品轉換為上述三維陣列之排列執行樣品混合，以已知野生／轉基因水稻DNA為材料，分別完成64（4^3）或125（5^3）個DNA樣品混合至12或15個試管，PCR反應後成功篩選群體中唯一轉基因植株，明確精簡在大量樣品中篩選少數個體所需之耗材、人力及時間，大大提升篩選效能。

關鍵字

三維陣列PCR ；突變體篩選；自動移液器

並列摘要

Polymerase Chain Reaction (PCR) has been widely adopted for plant mutant screening. With large number of plants to be screened, sampling of PCR reactions of each individual and subsequent electrophoresis analysis are time and labor consuming. To this, in this study, the process of was refined that the screens are not done on individuals but on pools of plants to reduce the number of PCR reactions, termed as a 3- dimensional pooling PCR (3D PCR). The process of PCR sampling was performed by an automatic pipetting robot (OT-2). With designed software, a population of 64 samples is organized in a 3D (4× 4×4) consisting of 4 blocks, rows and columns consisting of 4 plants. Thus, 12 (4+4+4) PCR tubes, instead of 64, were needed for running PCR and gel electrophoresis analyses. To extend this concept, a population of 125 (5×5×5) samples can be arranged into 15 (5+5+5) PCR tubes. To demonstrate this, the experiments were performed by two steps. Firstly, we designed a single positive sample, dye (or plasmid) vs H_2O, in a population of 64 to perform the 3D pooling and obtained the predicted results. Finally, we created a population of 125 rice DNA samples containing only one transgenic plant. With performing 3D pool sampling by OT-2, the single transgenic rice plant was single out efficiently and correctly without performing 125 PCR reactions but 15.

並列關鍵字

3-Dimensional pooling PCR ； Mutation screening ； Automatic pipetting robot

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