摘 要：制備大量生物樣品的模板DNA用于PCR檢測是費時費人工的工作。本文介紹一種高通量的植物基因組DNA(gDNA)快速制備及其用于PCR基因型檢測的操作方法。將一小段單子葉植物苗葉片(長度約30 mm或40 mm，與96方孔板的孔深大致相同) 或一小塊(約2~4 mg)雙子葉植物葉片放入96方孔板的各孔中、放入一粒鎢合金珠和150 μL制備緩沖液，蓋好硅橡膠蓋，在渦旋器振動3~5 min破碎組織。用96針復制器(或多通道移液器)轉移每樣品約0.5~1 μL此粗制gDNA溶液(含有2~3 ng gDNA μL-1)到96孔PCR板的反應液中，適合用各種類型的PCR標記(如簡單序列重復SSR，插入缺失InDel等)進行基因型檢測，或較大的DNA片段(>1 kb)的擴增，可以得到良好的效果。本方法的關鍵是控制合適的破碎葉片量與制備溶液量比例(約2~5 mg, 但不超過10 mg 150 μL-1溶液)，以及不要加入過多量的gDNA(不超過PCR反應液量的1/10)，以免帶入過量的雜質抑制PCR。因此，這種從種植材料，制備gDNA, 轉移樣品gDNA，到PCR都是96格式化操作的高通量、低成本方法適合于大量植物樣品的規模化的基因型檢測。
關鍵詞：基因組DNA制備；PCR；基因分型
A High Through-Put Protocol of Plant Genomic DNA Preparation for PCR
WANG Hui-Na, CHU Zhi-Zhan, MA Xing-Liang, LI Ri-Qing, and LIU Yao-Guang*
State Key Laboratory
for Conservation and Utilization of Subtropical Agro-bioresources;
College of Life Sciences, South China Agricultural University, Guangzhou
510642, China
Abstract: Preparation of large numbers of plant genomic DNA (gDNA) samples for PCR in basic researches and molecular breeding in crops is a time-consuming and laborious work. In this study we developed a protocol for rapid and high through-put preparation of plant gDNA for PCR. A piece (about 30 or 40 mm in length, the same as the depth of the used 96-deep well plates) of rice (or other monocot plants) seedling leaf, or about 2-4 mg of dicot plant leaf tissue, was put into each well of 96-deep well plates. After adding a tungsten bead and 150 μL buffer (10 mmol L-1 Tris (pH 9.5), 0.5 mmol L-1 EDTA, 100 mmol L-1 KCl) in each well, the plates were sealed with silicon rubber caps, and vigorously shaken with a vortex shaker for 3–5 min, followed by a brief centrifugation for a few seconds. For the pieces of monocot seedling leaves (30 or 40 mm in length), only the bottom parts (about 8 mm, ca. 2–4 mg) could be broken by the tungsten beads. Small amounts (ca. 0.5–1 μL each) of the crude gDNA solutions containing about 2–3 ng gDNA μL-1 were directly transferred with a 96-pin replicator (or a multiple-channel pipetter) to 96-well PCR plates containing PCR solution (15–20 μL each well) for various types of PCR markers, such as Simple Sequence Repeat (SSR) and Insertion Deletion (InDel). Our tests showed that too large amounts (2 μL or more) or too high concentration (>10 mg broken tissue in 150 μL solution) of the gDNA
in PCR could suppress the amplification reaction, due to the carrying-in of higher levels of inhibitory materials from the crude gDNA solution. Therefore, it is important to control a suitable ratio of the amount of broken plant to the volume of tissue/preparation solution (ca. 2–5 mg, but no more than 10 mg in 150 μL solution). The PCR amplifications with the template gDNAs prepared by this protocol are reliable for amplification of PCR markers and relatively large (>1 kb) DNA. This 96-formated high through-put/low-cost method is especially suitable for genotyping large numbers of plant samples.
Keywords: Genomic DNA preparation; PCR; Genotyping
基于PCR的DNA檢測技術已在如基因定位、親緣關系的分析、分子標記輔助育種、轉基因植株檢測、以及種子純度鑒定等基礎和應用研究領域廣泛利用。在大規模基于PCR的基因型檢測操作中，模板DNA制備是一個影響工作效率的最重要因素之一。多年來,人們一直致力于對DNA抽提方法的探討，并先后報道了一些相關的方法或簡易制備法[1-5]。但需多步驟操作，且使用微量離心管，達不到高通量的要求，樣品間DNA濃度差異大，重現性也不理想。本文介紹一種從種苗制備基因組DNA
(gDNA)
模板、到轉移模板DNA溶液至PCR反應液全程都采用96格式化高通量模板DNA制備的操作方法。該法不需純化和乙醇沉淀DNA，因而操作簡單快速，同時還適合于從干燥植物葉片制備模板DNA。所制備的模板DNA不僅可用于大規模分子標記(簡單序列重復/微衛星SSR,
插入缺失InDel, 酶切擴增多態序列CAPS等)的基因型檢測，大大提高基因型分析的效率，也可用于較大DNA片段(>1 kb)的擴增。
1 材料與方法
1.1 器具和試劑
(1) 96方孔板(2.2 mL或1.6 mL容積)及其配套硅橡膠蓋(建議使用國產品以降低成本)。
(2) 96針復制器(方統科技生產或自制，見附錄I)。
(3) 鎢合金珠(直徑3 mm，Qiagen, Cat. 69997)或不銹鋼珠（5直徑mm）。
(4) 普通渦旋器(其林貝爾QL-861)，或MTM-96研磨器(方統生物科技有限公司制造)。
(5) 8通道或12通道移液器。
(6) 96孔PCR板(國產品)。
(7) 其他PCR擴增儀器和試劑及其電泳檢測設備。
(8) 10′制備緩沖母液：100 mmol L-1 Tris(pH9.5), 5 mmol L-1 EDTA, 1 mol L-1 KCl；1′制備緩沖溶液：使用前將母液稀釋10倍。
(9) 普通Taq酶(如上海申能博彩公司產品)及其他PCR試劑。
(10) 其他常用試劑。