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Method for preparing human chromosome P16 gene probe and application thereof

A gene probe and chromosome technology, which is applied in the field of human chromosome P16 gene detection kits, can solve the problems of difficult, time-consuming, lack of systematic and mature methods for tumor cells, etc., and achieves good reproducibility of results, simple operation, and rapid detection. Effect

Active Publication Date: 2010-11-17
DAAN GENE CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Currently commonly used karyotype analysis can analyze chromosomal abnormalities, but this requires cell culture and preparation of high-quality metaphase chromosome sheets, which is extremely difficult and time-consuming for tumor cells
In order to solve this technical problem, one of the ideas is to screen and prepare corresponding segmental analysis gene probes, but currently there is a lack of a systematic and mature method for the preparation of chromosome segmental analysis gene probes

Method used

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  • Method for preparing human chromosome P16 gene probe and application thereof
  • Method for preparing human chromosome P16 gene probe and application thereof
  • Method for preparing human chromosome P16 gene probe and application thereof

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Experimental program
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Effect test

Embodiment 1

[0036] Embodiment 1: the preparation method of human P16 gene probe

[0037] (1) Primer design and clone screening: The P16 gene is located on the 9p21 segment of human chromosome, and the clone containing this gene was selected: RP11-149I2.

[0038](2) Cloning culture and identification: purchase the corresponding clone Invitrogen RPCI11.C, take 50ul and add it to 500ml TB culture solution (chloramphenicol resistance), and shake the bacteria in a shaker at 37°C for 24-48 hours; the bacteria solution is used STS primer pair for clonal identification. STS primer pair: upstream primer 5'-ATCCAACATGCCATAGCTCC-3' and downstream primer 5'-TTTGTCCCATGTCACTGGAA-3', PCR amplification conditions: 95°C for 5 minutes; (94°C for 30 seconds, 56°C for 30 seconds, 72°C for 45 seconds )×40 cycles; 10 minutes at 72°C. The amplified product was verified by electrophoresis, and the result was a bright band around 184bp (see attached figure 1 ).

[0039] (3) Preparation of P16 gene probe: For...

Embodiment 2

[0057] Embodiment 2: Preparation of P16 gene detection kit

[0058] Take 10 servings / box as an example.

[0059] (1) Preparation of hybridization solution

[0060] Take a small amount of P16 gene probe and dilute it to 50ng / ul, and prepare the hybridization solution according to the following table:

[0061]

[0062] (2) DAPI counterstain preparation

[0063] Anti-fading solution: The whole process must be protected from light. Dissolve 10mg of p-phenylenediamine in 1ml of PBS, add 9ml of glycerin, shake and mix repeatedly, adjust the pH to 9.0, and store at -20°C. The final solution should be colorless or slightly yellowish. If yellow or orange appears, discard and reconstitute.

[0064] Prepare a 1mg / ml DAPI stock solution in deionized water.

[0065] Dissolve 2.5ul of DAPI solution (1mg / ml) in 1ml of anti-fading solution, shake and mix repeatedly in the dark, and store at -20°C in the dark.

[0066] (3) Finished product assembly

[0067] component name

...

Embodiment 3

[0069] Embodiment 3: the usage method of P16 gene detection kit

[0070] Take human normal dividing metaphase lymphocytes as an example.

[0071] (1) Human peripheral blood culture and chromosome preparation

[0072] Blood collection: After wetting the injection syringe (0.2ml) with heparin, take 1-2ml of venous blood routinely, and turn the syringe to mix the heparin. Inoculation (aseptic operation in ultra-clean workbench): In each culture bottle (5ml of 1640 culture solution containing 20% ​​serum, pH7.2), add 0.25-0.30ml of whole blood (13-15 drops of No. 7 needle) , PHA 5mg, cover the rubber stopper tightly, and shake gently. Cultivation: Place the culture bottle in a constant temperature incubator at 37°C for 72 hours. 2-4 hours before terminating the culture, add 1-2 drops of 0.01% colchicine solution (100 μg / ml) (No. 7 needle), so that the final concentration is 0.2 μg / ml culture solution. After gently shaking, put it back into the incubator and continue culturing ...

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Abstract

The invention relates to a method for preparing a human chromosome P16 gene probe and also relates to a method for preparing a human chromosome P16 gene detection kit by using the human chromosome P16 gene probe. The human chromosome P16 gene probe and the corresponding kit can be applied to accurately and rapidly detecting human chromosome P16 genetic abnormality.

Description

technical field [0001] The present invention relates to a preparation method of a human chromosome P16 gene probe, and also relates to the preparation of a human chromosome P16 gene detection kit using the human chromosome P16 gene probe, using the human chromosome P16 gene probe and the corresponding kit of the present invention It can accurately and quickly detect the abnormality of human chromosome P16 gene. Background technique [0002] The P16 gene, also known as the MTS (multiple tumor suppressor 1) gene, is located on human chromosome 9p21, directly participates in the regulation of the cell cycle, negatively regulates cell proliferation and division, and is found to have homozygous deletions and mutations in 50% of human tumor cell lines. P16 is a new type of anti-cancer gene that is more important than P53. P16 gene has been found in lung cancer, breast cancer, brain tumor, bone tumor, skin cancer, bladder cancer, kidney cancer, ovarian cancer, lymphoma and melanom...

Claims

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Application Information

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IPC IPC(8): C12P19/34C12Q1/68G01N21/64
Inventor 李明何瑰陈娟
Owner DAAN GENE CO LTD
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