Method for extracting DNA by echinoderm living body sampling

A technology for echinoderms and living bodies, which is applied in biochemical equipment and methods, determination/inspection of microorganisms, sugar derivatives, etc., and can solve the problems of lack of practical significance of genetic information and unfavorable genetic and breeding work of echinoderms.

Inactive Publication Date: 2010-08-25
DALIAN FISHERIES UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The genetic information obtained by this sampling method has no practical significance in breeding, and is extremely unfavorable to the development of echinoderm genetics and breeding.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] a. Put live sea urchins in a container filled with a small amount of seawater. The depth of seawater should be just submerged in sea urchins. In this way, the tube feet of sea urchins can protrude radially in the water, which is convenient for sampling; cut a small amount with scissors Sea urchin podia (according to the actual situation, about 1-3mm long podia can be obtained), put them on filter paper to absorb the water; weigh the quality of the sea urchin podia, the sampling volume should not be less than 0.01g, put them in 1.5ml centrifuge tube;

[0025] b. Add 200 μl of lysate to the centrifuge tube containing sea urchin podia, then cut or homogenize the tube podia in the lysate, digest at 50°C for 45-55 minutes, and oscillate once every 10 minutes during this period to ensure Ensure that the sample tissue is fully digested; the components and final concentration of the lysate are 0.2g / l proteinase K, 0.5% SDS, 100mmol / l EDTA, 0.05g / l RNase; because the content of ...

Embodiment 2

[0038] a. Put live sea cucumbers in a container filled with a small amount of seawater. It is advisable that the depth of seawater should be just submerged in sea cucumbers. Use scissors to cut out 1 to 3 tentacles (according to the size of sea cucumbers) or a few tube feet of sea cucumbers, and place them in Blot the water on the filter paper for later use; weigh the quality of the tube feet or tentacles of the sea cucumbers taken, and the sampling volume should not be less than 0.01g, and put them into a 1.5ml centrifuge tube; in order to prevent sea cucumbers from being infected due to tentacles or tube feet Add penicillin anti-inflammatory in seawater at the dosage of / L;

[0039] DNA was extracted according to steps b-1 of Example 1.

[0040] The results of electrophoresis showed by gel imaging that the concentration and quality of the DNA of the embodiment of the present invention were consistent with the DNA extracted from traditional (dead body) sampling tissues of ech...

Embodiment 3

[0042] a. Inject fresh water into the sea cucumber to stimulate the sea cucumber. The sea cucumber will excrete viscera at the same time as the injection. You can also inject 0.35mol / L KCl2~4mL, and the internal organs will be discharged after 1~2min; Rinse with double distilled water, then put the washed sea cucumber intestines on the filter paper to absorb the water; weigh the quality of the sea cucumber intestines taken, the sampling amount should not be less than 0.01g, and put them into a 1.5ml centrifuge tube;

[0043] Operate according to steps b~f;

[0044] After step f, observe the middle layer and the lower layer. If the middle layer or the lower layer is dark in color (unclear), add 400 μl of chloroform-isoamyl alcohol (24:1) and extract again.

[0045] Then follow steps g~l to extract DNA.

[0046] The results of electrophoresis showed by gel imaging that the concentration and quality of the DNA of the embodiment of the present invention were consistent with the D...

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PUM

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Abstract

The invention discloses a method for sampling and extracting DNA from a living body of echinoderm. The tube feet of a living sea urchin or the tube feet, the tentacles or the intestines of a living sea cucumber are taken to be lysed and digested by lysis solution provided with DNA enzyme, and after the steps of extraction, centrifugal supernatant fluid taking, centrifugal deslagging (sclerite), ethyl alcohol precipitation, centrifugal separation, detection, etc., the concentration and the mass of the extracted DNA are consistent with the concentration and the mass of the DNA extracted from the traditional (dead body) sample tissue of the echinoderm. The echinoderm is not required to be killed, but is sampled from the living body to extract the DNA, so that the molecular biology technologycan really become an auxiliary mean of genetic breeding. By using the invention, the echinoderm resources can be rationally used for the works of auxiliary breeding, cultivation condition monitoring,genetic screening, genealogy identification, etc. In addition, the invention also has great significance for the protection and the research of various endangered species in the echinoderm.

Description

Technical field: [0001] The invention relates to a method for extracting DNA from echinoderms, in particular to a method for extracting DNA from living samples of echinoderms. Background technique: [0002] At present, animal DNA extraction technology has been widely used in genetic breeding, experimental research, and production testing. The basic method is to remove some tissue samples from the animal body (living body, dead body), and fully shred or homogenize the sample in the lysate. Slurry, and fully digest the sample tissue, and then go through the steps of extraction, centrifugation to get the supernatant, ethanol precipitation, centrifugation, detection and other steps, and the living sampling is more conducive to genetic breeding, breeding status monitoring, gene screening, family identification, etc. Research. Some marine animals can be sampled in vivo, such as fish, which can draw blood, cut fin rays, etc., but it is difficult to achieve for echinoderms (sea urc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12P19/34C07H21/04
Inventor 冯志纲于佳平丁君田燚常亚青
Owner DALIAN FISHERIES UNIVERSITY
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