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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 cells
In order to solve this technical problem, one of the ideas is to screen and prepare corresponding chromosome counting probes, but there is currently a lack of a systematic and mature method for the preparation of chromosome counting probes

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
Effect test

Embodiment 1

[0038] Embodiment 1: the preparation method of counting probe of human chromosome 7

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

[0040] The primer pairs are F: 5'-AGCGATTTGAGGACAATTGC-3' and R: 5'-CCACCTGAAAATGCCACAGC-3', which were synthesized by Daan Gene Co., Ltd. of Sun Yat-sen University. 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. Use ABI9700 PCR instrument or similar reaction equipment, set cycle parameters as: 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] Af...

Embodiment 2

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

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

[0074] (1) Preparation of hybridization solution

[0075] Take a small amount of C7 probe and dilute it to 40ng / ul, and prepare the hybridization solution according to the following table:

[0076]

[0077] (2) DAPI counterstain preparation

[0078] 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.

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

[0080] 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.

[0081] (3) Finished product assembly

[0082] component name ...

Embodiment 3

[0083] Embodiment 3: the usage method of human chromosome 7 counting kit

[0084] Take human normal dividing 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, 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 c...

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PUM

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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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