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Human chromosome P16 gene detection kit and application thereof

A detection kit and gene technology, applied in the field of human chromosome P16 gene detection kits, can solve the problems of high probe background, affecting the accuracy of detection results, etc., and achieve the effect of bright detection signal, rapid detection, and long probe length

Inactive Publication Date: 2012-06-27
DAAN GENE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As we all know, the length of the probe will directly affect the strength of the hybridization signal, but similarly, too long a probe will also cause high background and affect the accuracy of the detection results

Method used

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  • Human chromosome P16 gene detection kit and application thereof
  • Human chromosome P16 gene detection kit and application thereof
  • Human chromosome P16 gene detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1: the preparation method of human P16 gene probe

[0050] (1) Cloning screening: The P16 gene is located on the 9p21 segment of human chromosome, and the clone combination containing this gene is selected. Take RP11-149I2 as an example. For the probe set site pattern map, see figure 1 .

[0051] (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.

[0...

Embodiment 2

[0061] Embodiment 2: the preparation method of CSP9 (internal control) probe

[0062] In order to ensure the hybridization effect and avoid false positives, the human P16 gene detection kit includes a CSP9 probe. For the probe set site pattern map, see figure 1 .

[0063] (1) Primer design and synthesis

[0064] Design and verification of primers for PCR amplification of the repetitive region of human chromosome 9, the primer pairs are respectively, F:

[0065] 5'-GAAATAAGGGCACCTACCCCATA-3' and R: 5'-CTACACCTTGGCTCCTTTTAGCA-3' were synthesized by Sun Yat-sen University Daan Gene Co., Ltd. Prepare reaction system:

[0066]

[0067] The total volume is 50ul. Use ABI9700 PCR instrument or similar reaction equipment, set cycle parameters as: 95°C for 5 minutes; (94°C for 30 seconds, 60°C for 30 seconds, 72°C for 45 seconds) × 5 cycles (94°C for 30 seconds, 55°C for 30 seconds, 72°C 45 seconds) x 30 cycles; 10 minutes at 72°C.

[0068] After the reaction, 5 ul of the prod...

Embodiment 3

[0076] Embodiment 3: Preparation of P16 gene detection kit

[0077] Take 10 servings / box as an example.

[0078] (1) Preparation of hybridization solution

[0079] Take a small amount of P16 gene probe and dilute it to 50ng / ul, and prepare the hybridization solution according to the following table:

[0080]

[0081] (2) DAPI counterstain preparation

[0082] 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.

[0083] Prepare a 1mg / ml DAPI stock solution in deionized water.

[0084] 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.

[0085] (3) Finished product assembly

[0086] component name

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Abstract

The invention relates to a human chromosome P16 gene detection kit. Particularly, the detection kit comprises a probe used for detecting a P16 gene and a hybridization buffer. The human chromosome P16 gene detection kit can be used for accurately and quickly detecting the abnormity of the human chromosome P16 gene.

Description

technical field [0001] The invention relates to a human chromosome P16 gene detection kit. Specifically, the detection kit includes probes and hybridization buffer for P16 gene detection. The human chromosome P16 gene detection kit of the invention can be used to accurately and quickly detect the abnormality of the human chromosome P16 gene. Background technique [0002] The P16 gene is also called MTS1 (multiple tumor suppressor 1, also known as multiple tumor suppressor gene) or CDKN2A gene. It was identified in 1994 and located on human chromosome 9p21.3. The product encoded is a cell protein-dependent kinase (CDKS) Inhibitory protein, which maintains the orderly proliferation of cells in the body through the p16 INK4αcyclin D1-PRb pathway, directly participates in the regulation of the cell cycle, and negatively regulates cell proliferation and division. The homozygous deletion of P16 gene often leads to malignant transformation of cells, [0003] P16 homozygous delet...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 李明何瑰陈娟
Owner DAAN GENE CO LTD
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