A simple and rapid plant DNA extraction method

Abstract

The invention discloses a simple and rapid plant DNA extraction method and belongs to the field of molecular biology. The method includes cutting off a small amount of leaves, rapidly grinding the leaves until uniform pulp is formed, adding an extracting liquid (including 20-50 mM of Tris (that is trihydroxymethyl aminomethane) and 13-25 mM of EDTA (that is ethylenediaminetetraacetic acid), with the pH of the liquid being 8.0), heating the extracting liquid at 70-90 DEG C for 10-15 min, and performing high-speed centrifugation for 1 min to obtain a DNA extract that can be directly used for PCR. Compared with present DNA extraction methods, the method has advantages of capability of being simple and rapid, a low cost, high stability, safety, no pollution, capability of achieving high-throughput operation, wide applicability, and the like. The method has extremely high applicability and practicality in processes of large-scale genetic mapping, molecular marker assisted breeding, crop variety identification, and the like.

Description

technical field [0001] The invention discloses a simple and rapid extraction method of plant DNA, which belongs to the field of molecular biology. The invention greatly simplifies the plant DNA extraction method, which is simple, rapid and stable, and can be widely used in extracting various plant leaf DNA required for molecular identification. Background technique [0002] With the completion of whole-genome sequencing of major crops such as rice, corn, and wheat, the difficulty of developing molecular markers for these economic crops has been significantly reduced, and the use of molecular markers for assisted breeding and variety identification has begun to be applied to actual production. However, molecular identification is different from apparent identification, and must pass through a series of procedures such as DNA extraction and identification. Therefore, when large-scale sample screening is performed, the workload is extremely high, and a method system that meets ...

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Problems solved by technology

The existing DNA extraction method cetyltrimethylammonium bromide (CTAB) method (Doyle et al.A rapid DNA isolation procedure for small quantities of frexh leaf tissue.PhytochemBull.1987,19:11-15.), needs to pass Grinding, cracking, chloroform extraction, centrifugation, precipitation and cleaning steps, the whole process takes two hours, can operate 20 samples at the same time, the average time is about six minutes, need to use detergent CTAB, toxic and harmful organic solvent chloroform And a large amount of ethanol, during which the sample needs to be transferred three times, the extraction efficiency is low, and the professionalism and carefulness of the operation are high, which is not suitable for high-throughput operation; the DNA extraction workstation currently used for high-throughput DNA extraction ( Yu Jing et al. Establishment and application of automated workstation batch extraction of DNA from cured tobacco leaves. Guizhou Agricultural Sciences. 2015, 5: 217-219.), although simple and convenient, it requires expensive equipment and special reagents, and it is difficult for experimenters to operate The requirements are relatively high; while the direct PCR method (Rogers et al. Direct PCR amplification from leaf discs. PlantSci. 1999, 143: 183-186.) can save the step of DNA extraction, but the amount of material has a significant impact on the experimental results , stability is difficult to guarantee, need to use better and more expensive enzymes and PCR enhancement reagents, and each sample can only be used for one PCR amplification

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