Non-toxic, fast and efficient DNA extraction method for recalcitrant plants and application of DNA extraction method

Abstract

The present invention discloses a non-toxic, fast and efficient DNA extraction method for recalcitrant plants. The DNA extraction method comprises the following steps: using an extraction lysis buffercontaining cross-linked polyvinylpyrrolidone and activated carbon to lyse plant cells at 65 DEG C to isolate chromosomes, denature proteins and release nucleic acid, at the same time, removing impurities, then after adding acetic acid and potassium acetate to remove some proteins and polysaccharides, using PEG / high salt solution and ethanol to precipitate DNA twice continuously to further removethe remaining impurities, and finally obtaining the high-purity DNA. The used method can extract a large amount of high-purity genomic DNA from young or mature leaves of the recalcitrant plants, is non-toxic to operators, little in environmental pollution, low in cost and short in time, and can be widely applied to extraction of the genomic DNA of the recalcitrant plants.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to the extraction of plant DNA, in particular to a non-toxic, fast and efficient DNA extraction method and application for recalcitrant plants. Background technique [0002] The extraction of DNA is the basis for carrying out plant molecular biology research. Generally, the DNA extraction method should meet the following main conditions: (1) The obtained DNA should have ideal purity; (2) The DNA should be complete enough and the degree of damage should be small, so as to ensure that the electrophoresis is easy to obtain accurate and repeatable results. Migration mode; (3) As much DNA as possible should be extracted from a batch of samples, so as to ensure that there is no difference between DNA samples in a complete experiment; (4) The extraction method should be as simple and time-saving as possible , low cost, etc., if possible, avoid the use of dangerous chemicals. [0003] Compared w...

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Problems solved by technology

[0003] Compared with the traditional SDS method, the traditional CTAB method has high cost, high toxicity, cumbersome operation and low yield, while the yield of DNA extracted by the SDS method is nearly twice that of the CTAB method

For common grasses and leguminous plants, the extraction of genomic DNA is generally relatively easy, but for recalcitrant plants, the high-quality DNA isolation is seriously affected due to the large amount of polysaccharides, polyphenols, tannins, and pigments in their bodies. secondary metabolites, so conventional DNA extraction methods are difficult to meet the above requirements at the same time

Studies have shown that if these secondary metabolites are not handled properly, they will irreversibly combine with proteins and nucleic acids, which not only reduces the purity of DNA, but also seriously inhibits the effects of endonucleases and DNA polymerases, resulting in DNA solution stickiness. It is thick and cannot be digested by endonucleases, nor can it be used as a PCR template for further analysis. If it is detected by a spectrophotometer, the D of the extracted genomic DNA 260nm / D 280nm The value drops to between 1.2 and 1.4

[0004] Secondly, if various purification methods (such as multiple extractions with phenol-chloroform) are used, although the purity of the final DNA may reach the required standard, the DNA will be damaged due to the continuous mechanical shearing during the extraction. are degraded into small fragments, and the amount obtained is also greatly reduced

Third, due to the repeated extraction in the extraction process, on the one hand, it will inevitably take up a lot of time; It is highly toxic, which is not only harmful to the health of experimenters, but also easily causes water and air pollution. At the same time, the treatment of experimental waste also requires special attention

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