Method for preparing human chromosome 7 enumeration probe and application thereof

A chromosome and probe technology, applied in biochemical equipment and methods, microbial determination/inspection, fluorescence/phosphorescence, etc., can solve the problems of time-consuming, difficult for tumor cells, lack of systematic and mature methods, etc., and achieve simple operation. , the results are reproducible

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

AI Technical Summary

Problems solved by technology

[0003] Currently commonly used karyotype analysis can determine chromosomal aneuploidy, but this requires cell culture and preparation of high-quality metaphase chromosome sheets, which is extremely difficult and time-consuming for tumor cel

Method used

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  • Method for preparing human chromosome 7 enumeration probe and application thereof
  • Method for preparing human chromosome 7 enumeration probe and application thereof
  • Method for preparing human chromosome 7 enumeration probe and application thereof

Examples

Experimental program
Comparison scheme
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Example Embodiment

[0038] Example 1: Preparation method of human chromosome 7 counting probe

[0039] (1) Primer design and synthesis: primer design and verification for PCR amplification of the centromeric region of human chromosome 7

[0040] The primer pairs are F: 5'-AGCGATTTGAGGACAATTGC-3' and R: 5'-CCACCTGAAAATGCCACAGC-3', synthesized by Sun Yat-sen University Daan Gene Co., Ltd. Prepare reaction system:

[0041] 10×Buffer 5ul

[0042] MgCl 2 (25mmol / L) 2ul

[0043] Human genomic DNA (5pg / ul) 2ul

[0044] F(10umol / L) 2ul

[0045] R(10umol / L) 2ul

[0046] d3TPs(10mmol / L) 1ul

[0047] dTTP(1mmol / L) 2ul

[0048] aa-dUTP(1mmol / L) 16ul

[0049] TaKaRa Hot Start Version(5U / ul) 0.5ul

[0050] H 2 O 17.5ul

[0051] The total volume is 50ul. Using ABI9700 PCR instrument or similar reaction equipment, set the cycle parameters as follows: 95°C for 5 minutes; (94°C for 30 seconds, 55°C for 30 seconds, 72°C for 45 seconds) × 40 cycles; 72°C for 10 minutes.

[0052] After the reaction is over, take 5ul of the product 2...

Example Embodiment

[0072] Example 2: Preparation of human chromosome 7 counting kit

[0073] Take 10 servings / box as an example.

[0074] (1) Preparation of hybridization solution

[0075] Dilute a small amount of C7 probe to 40ng / ul, and prepare the hybridization solution as follows:

[0076]

[0077] (2) DAPI counterstain preparation

[0078] Anti-fading solution: The entire operation must be protected from light. 10mg of p-phenylenediamine is dissolved in 1ml of PBS, 9ml of glycerol is added, and the mixture is shaken repeatedly. Adjust the pH to 9.0 and store at -20°C. The final solution should be colorless or slightly yellowish, if it appears yellow or orange, it needs to be discarded and reconstituted.

[0079] Prepare 1mg / ml DAPI stock solution with deionized water.

[0080] Take 2.5ul of DAPI solution (1mg / ml) and dissolve it in 1ml of anti-fading solution, shake and mix repeatedly under dark conditions, and store in an airtight seal at -20°C and dark.

[0081] (3) Finished product assembly

[0082] ...

Example Embodiment

[0083] Example 3: Using method of human chromosome 7 counting kit

[0084] Take human normal metaphase lymphocytes as an example.

[0085] (1) Human peripheral blood culture and chromosome preparation

[0086] Blood collection: After wetting the injection syringe (0.2ml) with heparin, routinely take 1-2ml of venous blood, and rotate the syringe to mix the heparin. Inoculation (aseptic operation in ultra-clean workbench): Add 0.25~0.30ml whole blood (13~15 drops of No. 7 needle) to each culture bottle (5ml of 1640 culture medium containing 20% ​​serum, pH7.2) , PHA 5mg, close the rubber stopper tightly and shake gently. Cultivation: Place the culture bottle in a 37°C constant temperature incubator for 72 hours. Two to four hours before the termination of the culture, 1-2 drops of 0.01% colchicine solution (100μg / ml) (No. 7 needle) are added to make the final concentration 0.2μg / ml culture medium. After shaking gently, put it back into the incubator and continue to culture for 2 to...

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Abstract

The invention relates to a method for preparing a human chromosome 7 enumeration probe and preparation of a human chromosome 7 enumeration kit by using the same. A human chromosome 7 can be accurately and quickly counted by applying the human chromosome 7 enumeration probe and the corresponding kit.

Description

technical field [0001] The present invention relates to a preparation method of a human chromosome 7 counting probe, and also relates to the preparation of a human chromosome 7 counting kit using the human chromosome 7 counting probe, using the human chromosome 7 counting probe of the present invention And the corresponding kit can accurately and quickly count human chromosome 7. Background technique [0002] Human chromosome 7 contains 158 million bases (approximately 5% of the entire human genome) and 1455 genes, some of which are associated with autism, cystic fibrosis, several leukemias and lymphomas . A large number of studies have shown that trisomy 7 appears in many malignant tumors, such as associated with renal cancer [Brown JA, Anderl KL, Borell TJ, etc.Simultaneous chromosome 7 and 17 gain and sex chromosome loss provide evidence that renal Metanephric adenoma is related to papillary renal cell careinoma.J Urol.1997 Aug;158(2):370-4.]; closely related to astrocy...

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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