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The method of re-hybridization of fish production

A technique of fluorescence in situ hybridization and coverslipping, which is applied in the field of molecular cytogenetics, can solve the problems of easily polluted environment, difficult to obtain, and difficult to wash off hybridization signals, and achieves the effect of reducing environmental pollution and saving experimental costs.

Inactive Publication Date: 2016-07-06
INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Films capable of fluorescence in situ hybridization are often not easy to obtain, and a large amount of experimental materials need to be consumed
Under normal circumstances, because the hybridization signal is difficult to wash off after the first hybridization of the slides, and it is not known how to correctly handle the reagents left on the surface of the slides after observation without affecting the integrity of the chromosomes on the slides, the slides It is often discarded after being used once, which causes a huge waste of resources and easily pollutes the environment

Method used

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  • The method of re-hybridization of fish production
  • The method of re-hybridization of fish production
  • The method of re-hybridization of fish production

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1 takes Raymond's cotton No. 1 chromosome-specific BAC clone as probe, Raymond's cotton (D 5 ) The mitotic metaphase chromosome was used as the target chromosome, and the FISH preparation was used for rehybridization experiments.

[0018] 1 Materials and methods

[0019] 1.1 Experimental materials

[0020] The experimental material is Raymond cotton, which comes from the Cotton Research Institute of the Chinese Academy of Agricultural Sciences. The BAC used was the specific BAC clone of chromosome 1 of Raymond cotton (WangK, 2007). Experimental reagents: cellulase OnazukaR-10 and pectinase PectolyaseY-23 were purchased from Solarbio Company, Digoxigenin Probe Labeling Kit was purchased from Roche Company, and other domestic reagents were of analytical grade.

[0021] 1.2 Experimental method

[0022] 1) Material collection and pretreatment: Soak the seeds of Raymond cotton in warm water for about 12 hours, and then cultivate them in a light incubator. When...

Embodiment 2

[0039] Example 2 Taking the mitotic metaphase chromosome 12 of Upland Cotton Cotton Center as the target chromosome, a rehybridization experiment of FISH preparation was carried out.

[0040] 1 Materials and methods

[0041] 1.1 Experimental materials

[0042] The experimental material is upland cotton Zhongmiansuo No. 12, which comes from the Cotton Research Institute of the Chinese Academy of Agricultural Sciences. Experimental reagents: cellulase OnazukaR-10 and pectinase PectolyaseY-23 were purchased from Solarbio Company, Digoxigenin Probe Labeling Kit was purchased from Roche Company, and other domestic reagents were of analytical grade.

[0043] 1.2 Experimental method

[0044] At step 9, extend the time in 70% deionized formamide at 78°C to 5 minutes. Other steps are the same as above.

[0045] 2 Experimental results

[0046] The experimental results found that when the processing time in step 9 was extended, the chromosome morphology was obviously expanded ( im...

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Abstract

The invention belongs to the field of molecular cytogenetics, and in particular relates to a method for re-hybridizing a FISH film. The method mainly comprises the following steps: soaking the film into 2*SSC to wash away a cover plate after first fluorescence in situ hybridization (FISH), and then soaking the treated film in 2*SSC for 3*5min; putting the film into 2*SSC of 90 DEG C for 10min while shaking continuously; putting the film into 70% deionized formamide of 78 DEG C for 4min while shaking continuously; and quickly putting into 70% alcohol of -20 DEG C. A hybridization signal can be washed away by adopting the method, and then the hybridization signal cannot be detected by observing the film. When the treated film is reused for a FISH experiment by adopting the same probe, the hybridization signal can be detected again at the same position of a chromosome. By adopting the method, the film subjected to in situ hybridization can be reused, so that resources are effectively saved, the cost is reduced and the environmental pollution is reduced.

Description

technical field [0001] The invention belongs to the field of molecular cytogenetics, and specifically relates to a method for rehybridization of FISH slices. Background technique [0002] Fluorescence in situ hybridization (FISH) technology began in the late 1960s and is an effective means of positioning DNA sequences on chromosomes, interphase nuclei and DNA fibers using the principle of complementary base pairing. Fluorescence in situ hybridization initially used DNA repeat sequences and multi-copy gene families as probes. Later, with the continuous improvement and perfection of the technology, single low-copy sequences were gradually used as probes to improve the resolution and sensitivity of detection. have been greatly improved. At present, fluorescence in situ hybridization technology is widely used in the physical location of genes, chromosome identification, physical map construction, kinship research and transgenic detection, etc., and plays a pivotal role in the f...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6841C12Q2543/10
Inventor 崔兴雷刘玉玲刘方王星星蔡小彦彭仁海周忠丽王春英王玉红王坤波
Owner INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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