Plant single chromosome laser microdissection separation method

Abstract

The invention provides a plant single chromosome laser microdissection separation method. According to the method, firstly, a plant organ requiring single chromosome separation is put into nitrous oxide gas to be pretreated; then, the pre-treated plant organ is subjected to immobilization treatment and enzymolysis treatment; the plant organ subjected to enzymolysis is crushed and is prepared into cell suspension; the cell suspension is dripped onto a glass slide or a coverslip to obtain a chromosome mounting chip; dye liquid is dripped into the prepared chromosome mounting chip, and then, drying is performed; microscope is used for observing the dried chromosome to determine the target chromosome to be separated; a laser capture micro cutting process is used for separating the target chromosome. The chromosome dispersion degree of the prepared chromosome mounting chip is high, so that the later stage laser cutting operation difficulty is reduced; the damage to the chromosome in the ultraviolet laser cutting and infrared laser adhesion process is reduced; the single chromosome structure obtained after the separation is not damaged; the later-stage experiment study requirements are met.

Description

technical field [0001] The invention belongs to the technical field of single chromosome separation, and in particular relates to a laser microdissection separation method for plant single chromosome. Background technique [0002] Chromosomal micro-segregation is a specific gene cloning technology that was born with the development of molecular biology and the combination of molecular biology and cytogenetics techniques. It can isolate any chromosome or fragment according to the experimental needs, and then create a DNA library, and Completion of relevant molecular biology research. Chromosome microdissection refers to the technology of cutting and separating chromosome segments or target chromosomes from specimens under a microscope. It is a bridge combining cytogenetics and molecular genetics. , Chromosome evolution, comparative genome and single chromosome sequencing are widely used in research, especially with the development of the third generation sequencing technolog...

AI Technical Summary

Problems solved by technology

[0003] Aiming at the above-mentioned deficiencies existing in the prior art, the technical problem to be solved by the present invention is: the separation degree of chromosomes in the chromosome loading sheet prepared in the prior art is not enough, which increases the difficulty for later stage ultraviolet microdissection, and the existing laser display The operating parameters of the micro-cutting method are not suitable for the separation of plant single chromosomes, and the technical problem that it is easy to cause damage to plant single chromosomes, and provide a plant that is suitable for plant single chromosome separation, does not cause damage to plant single chromosomes, and is less difficult to operate. Laser Microdissection Separation Method for Single Chromosome

Aiming at the technical problem that the separation degree of the chromosome on the chromosome sheet obtained by using the existing single chromosome separation method is not enough, and the chromosome is easily damaged when the single chromosome is separated by the ultraviolet cutting method in the later stage, and the separation is difficult, the present invention adopts the inventive method The study proposes that nitrous oxide is used to pretreat plant organs in the early stage of film production. Plant cells treated with this pretreatment method are more likely to disperse chromosomes after enzymatic hydrolysis. Plant cells can be ruptured by using the gravity when dropping, and the dispersion effect between individual chromosomes in plant chromosomes is better, and this drop method will not cause damage to chromosomes, and can obtain complete, uniform distribution, and good dispersion Chromosomes, which reduce the difficulty of separation in the later stage of laser cutting, and avoid the problem that the target chromosome is damaged by ultraviolet laser or polluted by other chromosomes due to the close distance between chromosomes during laser cutting. Separation of the target chromosomes on the chromosome plate prepared by the above method reduces the difficulty of operation, and the shape of the obtained single chromosome is not damaged

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