運用自動移液器進行聚合酶反應完成1000株水稻突變體篩選

30 PCR Reactions for Detecting 1 Mutant Out Of 1000 Plants by Automatic Pipetting Robot

10.30061/CEB.202312_19.0001

賴冠傑(Kuan-Chieh Lai)；林寬諺(Kuan-Yan Lin)；陳亮君(Liang-Chun Chen)；常玉強(Yuh-Chyang Charng)

三維陣列PCR ； 突變體篩選 ； 自動移液器 ； 3-dimensional pooling PCR ； Mutation screening ； Automatic pipetting robot

作物、環境與生物資訊

19卷（2023 / 12 / 31）

1 - 10

繁體中文;英文

自動移液器可代替人力執行大量、重複性的液體分裝、製備工作，作者先前曾將125株水稻樣品以移液器完成三維陣列混合分裝入15個PCR反應試管，成功篩選出唯一之突變體，本研究進一步挑戰分析1000個樣品，以10×10×10三維排列，XY平面、XZ平面、YZ平面分別混合成各群樣品，每群100個樣品共30群，只須執行30個PCR反應即可找出轉殖株，唯依據先前移液模式耗時約19小時，降低移液器之優勢；因此，本研究優化移液流程，以Python語言編寫指令，先前每一樣品取一份、移一份放入PCR試管，改變成為同一樣品取三份、分別移入三個標的試管，移液耗時縮減為7小時20分；更進一步改變操作流程，先取足每一PCR標的試管所需之100個樣品，專一移入該試管，移液耗時縮減為3小時。以上設計均可完成30個PCR反應篩選千分之一中之唯一水稻突變株，達到分析大量樣品時，精簡所需之耗材、人力及時間。

Pooled PCR technique has been widely used to test the population with limited resources by pooling multiple individual samples. Previously, we have developed a 3D pool sampling by an automatic pipetting robot to single out 1 mutant out of 125 rice plant with 15 PCR reactions. Here, we extend this concept to analyze 1000 rice plants with 30 PCR reactions. Yet, the pipetting process is time consuming by the original liquid handler program, for more than 19 hours. To this, we develop new pipetting programs by Python programming language. First, a population of 1000 individuals was organized by a 10×10×10 3-dimentional pattern to yield 30 PCR pooled samples. A time-saving program, for about 7 hours, was developed by picking 3 copies of a sample then dispense equally to 3 PCR target tubes. Furthermore, a 3-hours program was developed by the idea to pick once and for all samples of each PCR target tube. Both programs were determined to successfully single out 1 mutant out of 1000 rice plant with 30 PCR reactions. These results indicate that it is possible to perform sampling, PCR reactions and electrophoresis for analyzing 1000 rice samples within one day.