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Treatment method of chromosome preparation used for fluorescence in situ hybridization of hyacinthus plant root tip chromosomes

A technology of fluorescence in situ hybridization and chromosome preparation, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., and can solve problems such as inability to obtain clear hybridization signals

Active Publication Date: 2019-04-05
NANJING FORESTRY UNIV
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Problems solved by technology

FISH technology has been successfully applied to many plants. However, bulbous flowers contain more secondary metabolites such as polysaccharides and polyphenols, and there are more impurities in the root tip meristematic zone. In situ hybridization research is relatively mature, but hyacinth plants contain a lot of impurities in water-cultured root tips, and chromosome preparation can not be directly used in fluorescence in situ hybridization to obtain clear hybridization signals

Method used

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  • Treatment method of chromosome preparation used for fluorescence in situ hybridization of hyacinthus plant root tip chromosomes
  • Treatment method of chromosome preparation used for fluorescence in situ hybridization of hyacinthus plant root tip chromosomes
  • Treatment method of chromosome preparation used for fluorescence in situ hybridization of hyacinthus plant root tip chromosomes

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Embodiment 1

[0042] A processing method for fluorescent in situ hybridization chromosome preparation of root tip chromosomes of plants of the genus Hyacinth, other operations are the same, and the difference from Comparative Example 1 is:

[0043] Treatment solution preparation:

[0044] (1) RNase A: Concentration 25mg / mL, dissolve with 1×TE, store at -20°C, dilute with 2×SSC before use, add 60 µl RNase A stock solution to 30ml 2×SSC;

[0045] (2) 4% pepsin: dissolve with 0.1 mol / L HCl, store at 4°C, dilute with 0.1 mol / L HCl before use, add 30 µl 4% pepsin stock solution to 30 ml 0.1N HCl;

[0046] (3) 4% paraformaldehyde: ddH2 O 900µl and 1mol / L NaOH 500µl heated to 65-70°C, added 40g of paraformaldehyde, heated and stirred until fully dissolved, cooled to room temperature, added 100µl 10×PBS, added HCl to adjust the pH to 7.3, after filtration and sterilization Aliquot and store in the dark at 4°C.

[0047] Chromosome preparation treatment: place the chromosome preparation in 2×SSC so...

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Abstract

The invention discloses a treatment method of a chromosome preparation used for fluorescence in situ hybridization of hyacinthus plant root tip chromosomes. The method comprises the steps of hyacinthus chromosome preparation and chromosome preparation treatment, and emphatically overcomes the defect that clear hybridization signals of hyacinthus can hardly be detected by means of existing fluorescence in situ hybridization technologies. Hyacinthus plant root tips contain secondary metabolites of polysaccharide, polyphenol and the like, the root diameter of hydroponic hyacinthus is about 1.5-2.0 mm, mitogenetic regions are covered by thick epidermic cells on middle portions of the root tips, therefore, during squashing, many impurities exist, when the obtained chromosome preparation is directly used for FISH, clear hybridization signals are hard to detect, and according to the treatment method of the chromosome preparation used for fluorescence in situ hybridization of the hyacinthus plant root tip chromosomes, by conducting FISH after treating the chromosome preparation with enzymes, small but clear signals can be captured. The hyacinthus root tip chromosome preparation treated through the method is suitable for FISH of 45S rDNA probes, 5S rDNA probes, 18S rDNA probes, telomere probes and the like.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a treatment method for the fluorescence in situ hybridization chromosome preparation of root tip chromosomes of plants of the genus Hyacinth. Background technique [0002] Fluorescence in situ hybridization (FISH) is a non-radioactive molecular cytogenetic technique developed on the basis of radioactive in situ hybridization in the late 1980s. It is formed by replacing isotope labels with fluorescent labels. A new in situ hybridization method, which can intuitively locate the DNA sequence complementary to the probe on the chromosome, and the oligocopy probe can also be anchored on a single chromosome, which not only improves the sensitivity of the experiment, but also greatly The experimental period is shortened. At present, this method has been widely used in genetic mapping, transgene localization, chromosome aberration detection, genome structure and species e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12Q1/6841
CPCC12Q1/6806C12Q1/6841C12Q2521/327C12Q2521/537
Inventor 胡凤荣苏晓倩张鸽香丁彦芬
Owner NANJING FORESTRY UNIV
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