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