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Method for extracting DNA from wood targeted cells

A technology targeting cells and extraction methods, applied in the direction of DNA preparation, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problem of incomplete extraction of wood DNA, restricting the development of wood DNA identification methods, and inability to effectively remove exogenous DNA pollution and other problems, to achieve the effect of shortening the experimental period, wide application range, and removing foreign DNA contamination

Active Publication Date: 2019-11-05
INST OF WOOD INDUDTRY CHINESE ACAD OF FORESTRY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, the existing wood DNA extraction methods cannot effectively remove exogenous DNA contamination, and the operation cycle is long (about 2 working days), which greatly restricts the development of wood DNA identification methods
[0005] Overall, existing methods still cannot fully address the effective extraction of wood DNA

Method used

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  • Method for extracting DNA from wood targeted cells
  • Method for extracting DNA from wood targeted cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: A method for precise extraction of DNA from air-dried wood

[0048] (1) Select the air-dried wood sample, firstly undergo laser radiation purification (1000W, 30Hz), scan speed 1cm / s, repeat 2 cycles; use 3% sodium hypochlorite to soak for 30min; then, cut the outer surface of the sample with a thickness of about 3mm to remove exogenous pollution;

[0049] (2) The wood sample obtained in step (1) was cut with a slide-away microtome to cut wood radial slices, placed under an inverted microscope to accurately separate the wood-targeted cells, and collected in a 50ml centrifuge tube;

[0050] (3) In a sterilized ultra-clean working environment, add 4.5ml of DNA lysate (3% (w / v) CTAB, 100mM Tris-HCl (PH 8.0), 1.4M NaCl to the wood sample obtained in step (2) , 20mM EDTA, 1% (w / v) Boric Acid, 5% (w / v) PVP, 2% (v / v) DTT), vortex and mix; 55 ℃ water bath for 300min, during which the centrifuge tube was turned several times; Cool for 2 minutes, add an equal volume ...

Embodiment 2

[0056] A method for extracting DNA from wood-targeted cells, which removes exogenous pollution on the wood surface, uses cytochemical methods combined with microscopic techniques to locate and separate DNA-enriched target cells, and adds DNA lysate, removes impurities, precipitates, dissolves, Purification and nested PCR amplification for efficient extraction of wood DNA.

Embodiment 3

[0058] A method for extracting DNA from wood-targeted cells, which removes exogenous pollution on the wood surface, uses cytochemical methods combined with microscopic techniques to locate and separate DNA-enriched target cells, and adds DNA lysate, removes impurities, precipitates, dissolves, Purification and nested PCR amplification for efficient extraction of wood DNA;

[0059] Removing exogenous contamination of the wood surface includes scanning the surface of the wood sample with laser radiation, then soaking the wood sample in sodium hypochlorite, and then using a scalpel to cut off the outer surface of the wood sample;

[0060] Using cytochemical methods combined with microscopic techniques to locate and isolate DNA-enriched target cells, place wood radial section sections on glass slides, stain wood radial section sections with DAPI solution, and then wash with phosphate buffer; Wood-targeted cells were isolated and collected in centrifuge tubes for DNA extraction.

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Abstract

The invention discloses a method for accurately extracting DNA from wood targeted cells; through removal of exogenous pollution on a wood surface, in-situ detection of wood DNA, radial section of woodtissues, precise separation of the wood targeted cells enriched in DNA under an inverted microscope, cracking of wood cell walls, removal of a wood extract, DNA precipitation and adsorption, dissolution, purification, PCR amplification and other steps, DNA is extracted accurately from the wood tissues. The method aims at the wood targeted cells to carry out DNA accurate extraction, the quantity and purity of DNA extracted from the wood tissues are improved, and the interference of other types of cells of wood on DNA extraction is reduced. The method can be directly applied to study of molecular biology and provide a new technical support for identification of wood species.

Description

technical field [0001] The invention relates to a method for extracting DNA from wood tissue, in particular to a method for precisely extracting DNA from targeted wood cells. Background technique [0002] With the continuous and rapid growth of my country's economy and the complete cessation of logging of natural forests, my country's demand for imported timber has shown an expansionary development trend, and China has become the world's largest importer of tropical timber. In this context, my country's timber trade, especially tropical timber import trade represented by CITES regulated timber, has received extensive attention from the international community. Therefore, the realization of scientific and accurate identification of wood will provide important technical support for my country's wood import and export supervision, protection and sustainable use of global wood resources. [0003] Wood is the stem part of trees. If the identification is not combined with roots, ...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/6806
CPCC12N15/1003C12Q1/6806C12Q2523/32C12Q2523/313C12Q2531/113
Inventor 焦立超陆杨殷亚方何拓李姗
Owner INST OF WOOD INDUDTRY CHINESE ACAD OF FORESTRY
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