高通量的PCR模板植物基因组DNA制备方法（一）

摘  要：制备大量生物样品的模板DNA用于PCR检测是费时费人工的工作。本文介绍一种高通量的植物基因组DNA(gDNA)快速制备及其用于PCR基因型检测的操作方法。将一小段单子叶植物苗叶片(长度约30 mm或40 mm，与96方孔板的孔深大致相同) 或一小块(约2~4 mg)双子叶植物叶片放入96方孔板的各孔中、放入一粒钨合金珠和150 µL制备缓冲液，盖好硅橡胶盖，在涡旋器振动3~5 min破碎组织。用96针复制器(或多通道移液器)转移每样品约0.5~1 µL此粗制gDNA溶液(含有2~3 ng gDNA µL^-1)到96孔PCR板的反应液中，适合用各种类型的PCR标记(如简单序列重复SSR，插入缺失InDel等)进行基因型检测，或较大的DNA片段(>1 kb)的扩增，可以得到良好的效果。本方法的关键是控制合适的破碎叶片量与制备溶液量比例(约2~5 mg, 但不超过10 mg 150 µL^-1溶液)，以及不要加入过多量的gDNA(不超过PCR反应液量的1/10)，以免带入过量的杂质抑制PCR。因此，这种从种植材料，制备gDNA, 转移样品gDNA，到PCR都是96格式化操作的高通量、低成本方法适合于大量植物样品的规模化的基因型检测。

关键词：基因组DNA制备；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检测技术已在如基因定位、亲缘关系的分析、分子标记辅助育种、转基因植株检测、以及种子纯度鉴定等基础和应用研究领域广泛利用。在大规模基于PCR的基因型检测操作中，模板DNA制备是一个影响工作效率的最重要因素之一。多年来,人们一直致力于对DNA抽提方法的探讨，并先后报道了一些相关的方法或简易制备法^[1-5]。但需多步骤操作，且使用微量离心管，达不到高通量的要求，样品间DNA浓度差异大，重现性也不理想。本文介绍一种从种苗制备基因组DNA (gDNA) 模板、到转移模板DNA溶液至PCR反应液全程都采用96格式化高通量模板DNA制备的操作方法。该法不需纯化和乙醇沉淀DNA，因而操作简单快速，同时还适合于从干燥植物叶片制备模板DNA。所制备的模板DNA不仅可用于大规模分子标记(简单序列重复/微卫星SSR, 插入缺失InDel, 酶切扩增多态序列CAPS等)的基因型检测，大大提高基因型分析的效率，也可用于较大DNA片段(>1 kb)的扩增。

1  材料与方法

1.1  器具和试剂

    (1) 96方孔板(2.2 mL或1.6 mL容积)及其配套硅橡胶盖(建议使用国产品以降低成本)。

    (2) 96针复制器(方统科技生产或自制，见附录I)。

    (3) 钨合金珠(直径3 mm，Qiagen, Cat. 69997)或不锈钢珠（5直径mm）。

    (4) 普通涡旋器(其林贝尔QL-861)，或MTM-96研磨器(方统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(国产品)。

    (7) 其他PCR扩增仪器和试剂及其电泳检测设备。

    (8) 10´制备缓冲母液：100 mmol L^-1 Tris(pH9.5), 5 mmol L^-1 EDTA, 1 mol L^-1 KCl；1´制备缓冲溶液：使用前将母液稀释10倍。

    (9) 普通Taq酶(如上海申能博彩公司产品)及其他PCR试剂。

    (10) 其他常用试剂。