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An improved co-denaturation fluorescence in situ hybridization method

A fluorescent in situ hybridization and specimen technology, applied in the field of improved co-denaturing fluorescent in situ hybridization, can solve the problems of low experimental efficiency, cumbersome steps, complex operation of classic fluorescent in situ hybridization technology, etc., to reduce the impact on the environment, reduce Experimental procedure, the effect of protecting the health

Active Publication Date: 2014-10-22
曾艳
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] The present invention aims at the disadvantages of classical fluorescent in situ hybridization technique, such as complex operation, cumbersome steps, low experimental efficiency, and the need to use toxic teratogenic reagents, and provides a method that is easy to operate, simple in steps, high in experimental efficiency, and does not use toxic teratogenic reagents. co-denaturing fluorescence in situ hybridization method

Method used

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  • An improved co-denaturation fluorescence in situ hybridization method

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Embodiment 1

[0039] An improved co-denaturation fluorescence in situ hybridization method, comprising the following steps:

[0040] Step 1 Preparation of slide specimens:

[0041] The pretreated samples are placed in EP tubes;

[0042] Add 1mL EDTA-trypsin to digest in 37°C water bath for 15min, centrifuge, and discard the supernatant;

[0043] Add 1mL of hypotonic solution to the precipitate, and bathe in water at 37°C for 15min;

[0044] Add 2mL of fixative to pre-fix and mix well, centrifuge and discard the supernatant;

[0045] After adding 2 mL of fixative to fix and mix well, fix at room temperature for 5 min, centrifuge, and discard the supernatant;

[0046] Take the remaining cell suspension, drop the slices, and bake the slices at 65°C for 15 minutes to prepare slide specimens;

[0047] Step 2: Probes covariate and hybridize with chromosomes:

[0048] Add 8 μL of probe solution dropwise to the cell area of ​​the slide specimen, add a cover slip, and drive away the air bubbles...

Embodiment 2

[0060] An improved co-denaturation fluorescence in situ hybridization method, comprising the following steps:

[0061] Step 1 Preparation of slide specimens:

[0062] The pretreated samples are placed in EP tubes;

[0063] Add 2 mL of EDTA-trypsin to digest in a 37°C water bath for 30 min, centrifuge, and discard the supernatant;

[0064] Add 2mL of hypotonic solution to the precipitate, and bathe in water at 37°C for 20min;

[0065] Add 1mL of fixative solution to pre-fix and mix well, centrifuge and discard the supernatant;

[0066] After adding 1mL of fixative to fix and mix well, fix at room temperature for 7min, centrifuge, and discard the supernatant;

[0067] Use the remaining cell suspension, drop the slices, and bake the slices at 60°C for 20 minutes to prepare slide specimens;

[0068] Step 2: Probes covariate and hybridize with chromosomes:

[0069] Add 10 μL of probe solution dropwise to the cell area of ​​the slide specimen, add a cover slip, and drive away t...

Embodiment 3

[0081] An improved co-denaturation fluorescence in situ hybridization method, comprising the following steps:

[0082] Step 1 Preparation of slide specimens:

[0083] The pretreated samples are placed in EP tubes;

[0084] Add 2 mL of EDTA-trypsin to digest in a 37°C water bath for 20 min, centrifuge, and discard the supernatant;

[0085] Add 1.5mL hypotonic solution to the precipitate, and bathe in water at 37°C for 15min;

[0086] Add 2mL of fixative to pre-fix and mix well, centrifuge and discard the supernatant;

[0087] After adding 2 mL of fixative to fix and mix well, fix at room temperature for 5 min, centrifuge, and discard the supernatant;

[0088] Use the remaining cell suspension, drop the slices, and bake the slices at 70°C for 10 minutes to prepare slide specimens;

[0089] Step 2: Probes covariate and hybridize with chromosomes:

[0090] Add 8 μL of probe solution dropwise to the cell area of ​​the slide specimen, add a cover slip, and drive away the air bu...

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Abstract

The invention relates to a fluorescence in-situ hybridization technology, and discloses a fluorescence in-situ hybridization method capable of improving covariance. The fluorescence in-situ hybridization method capable of improving covariance comprises the steps of slide specimen preparation, probe and chromosome covariance and hybridization, washing and dyeing after hybridization, and signal detection. According to the fluorescence in-situ hybridization method capable of improving covariance disclosed by the invention, a method of probe and chromosome covariance and hybridization is adopted on the basis of the prior art, the operation is simple, the reaction steps are reduced, experimental time is shortened, and a toxic and teratogenic denaturant does not need to be used, thus decreasing experimental cost, and avoiding the harm of the denaturant to experimental operating personnel and environment. The invention provides a fluorescence in-situ hybridization method capable of improving covariance, which is accurate in location, high in sensitivity, high in experimental efficiency, good in stability, simple to operate, and has environment-friendly effect.

Description

technical field [0001] The invention relates to a fluorescence in situ hybridization technique, in particular to an improved co-denaturation fluorescence in situ hybridization method. Background technique [0002] Fluorescence in situ hybridization (FISH) is an important in situ hybridization method developed in the late 1980s. It uses non-radioactive fluorescent labels instead of radioactive isotope labels to form a new molecular cytogenetic method. learn technology. As a non-radioactive detection system, FISH technology has obvious advantages compared with radioactive isotope-labeled probes: [0003] 1. Fluorescent reagents and probes are more economical and safer than radioactive isotope-labeled probes; [0004] 2. The probe is stable and can be used within two years after one marking; [0005] 3. The experimental results have high sensitivity, good specificity and accurate positioning; [0006] 4. Multicolor FISH can detect multiple sequences simultaneously by displa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 曾艳
Owner 曾艳
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