高通量PCR模板植物基因组DNA制备方法

摘要

制备大量生物样品的模板DNA用于PCR检测是费时费工的工作.本文介绍一种快速高通量的植物基因组DNA (gDNA)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(与96方孔板的孔深大致相同)或一小块(约2～5 mg)双子叶植物叶片放入96方孔板的各孔中、放入一粒直径4 mm或3 mm的合金珠和150 μL制备缓冲液,盖上硅橡胶盖,在涡旋器或震动研磨器震动2～4 min破碎组织.此方法获得的粗制gDNA样品浓度约2～4 ngμL-1.用96针复制器或多通道移液器转移约0.5～1.0 μL的gDNA溶液到96孔PCR板的反应液中,利用各种类型的PCR标记(简单序列重复SSR,插入缺失InDel等)进行基因型检测.此方法制备的gDNA模板也适合于较大DNA片段(＞1 kb)的扩增.本方法的关键是控制好在一定溶液量中破碎合适量的叶片,以及不要加入过量的gDNA溶液,以免带入过多的杂质抑制PCR效果.这种从材料种植、制备gDNA、转移样品gDNA,到PCR都是96格式化操作的快速、高通量、低成本的方法特别适合大量植物样品的规模化基因型检测.%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 (diameter 4 mm or 3 mm) and 150 μL buffer [10 mmol L-1 Tris (pH 9.5),0.5 mmol L-1 EDTA,100 mmol L-1 KC1] 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,2-4 mg) could be broken by the tungsten beads.Small amounts (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.