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作物学报 ›› 2013, Vol. 39 ›› Issue (07): 1200-1205.doi: 10.3724/SP.J.1006.2013.01200

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

高通量的PCR模板植物基因组DNA制备方法

王慧娜,初志战,马兴亮,李日清,刘耀光*   

  1. 亚热带农业生物资源保护与利用国家重点实验室 / 华南农业大学生命科学学院,广东广州510642
  • 收稿日期:2012-10-09 修回日期:2013-01-15 出版日期:2013-07-12 网络出版日期:2013-03-22
  • 基金资助:

    本研究由国家重点基础研究发展计划(973计划)项目(2011CB100203)资助。

A High Through-Put Protocol of Plant Genomic DNA Preparation for PCR

WANG Hui-Na,CHU Zhi-Zhan,MA Xing-Liang,LI Ri-Qing,LIU Yao-Guang*   

  1. State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources; College of Life Sciences, South China Agricultural University, Guangzhou 510642, China
  • Received:2012-10-09 Revised:2013-01-15 Published:2013-07-12 Published online:2013-03-22

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 mm40 mm,与96方孔板的孔深大致相同) 或一小块(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0.5~1.0 µL此粗制gDNA溶液(含有2~3 ng gDNA µL-1)96PCR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)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(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

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. 0.5–1.0 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.

Key words: Genomic DNA preparation, PCR, Genotyping

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