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Method for preparing FISH probe of EGFR gene

A probe and gene technology, applied in the field of FISH probe preparation, can solve the problems of long detection time and mutual interference.

Active Publication Date: 2019-04-12
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It overcomes the problems of mutual interference and long detection time in the combined detection of multi-site fluorescence in situ hybridization

Method used

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  • Method for preparing FISH probe of EGFR gene
  • Method for preparing FISH probe of EGFR gene
  • Method for preparing FISH probe of EGFR gene

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0119] A method for preparing a FISH probe library of EGFR gene, comprising the following steps:

[0120] (1) Prepare probe 1A and probe 1B;

[0121] (2) After digesting and breaking probe 1A and probe 1B obtained in step (1), respectively, use Taq DNA polymerase to complete the end and add the dA tail at the 3' end to obtain probe 2A and 2B respectively;

[0122] The enzyme cutting system is:

[0123] Reagent

Amount added

10×CutSmart Buffer

5μL

restriction endonuclease mix

2μL

Probe 1A or 1B (1ng / μL)

40μL

Taq DNA polymerase (5U / μL)

0.5μL

10mM dNTPs

1μL

wxya 2 o

1.5μL

[0124] The reaction conditions for enzyme digestion and interruption are: 37°C for 30 minutes, 72°C for 20 minutes, and 80°C for 20 minutes;

[0125] (3) Add adapters to the probes 2A and 2B obtained in step (2) to obtain probes 3A and 3B respectively;

[0126] The system of the ligation reaction is:

[0127]

[0128] ...

Embodiment 8

[0133] The condition of the connection reaction of embodiment 8 is:

[0134] temperature

time

16℃

60min

65℃

10min

4℃

∞

[0135] The ligation product was purified using the purification kit HiPure Nucleotide Remove Kit, the product was eluted with 25 μl of sterile ultrapure water, and 20 μl was taken for subsequent steps.

[0136] (4) performing asymmetric amplification on the probes 3A and 3B obtained in step (3) respectively to obtain a FISH probe library;

[0137] Asymmetric amplification reaction system:

[0138] Reagent

Amount added

5X EpiMark Hot Start Taq Reaction Buffer

10μL

dNTP Mixture (containing aminoallyl-dUTP)

3μL

Epimark Hot strat Taq DNA polymerase

0.25 μL

Nuclease-free water

14.75 μL

Primer F (1μM)

1μL

Primer R (20μM)

1μL

Probe 3A or 3B

20 μL

[0139] Primer F and Primer R are:

[0140] name

Base sequence ...

Embodiment 1-10

[0153] Embodiment 1-10 is that on the basis of the basic embodiment, various parameters are adjusted to obtain the following embodiments:

[0154]

[0155]

[0156]

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Abstract

The invention belongs to the field of biochemistry, in particular to the field of measurement or detection related to biochemistry. More particularly, the invention relates to a method for preparing aFISH probe of an EGFR gene, and a probe prepared by the method. The method of the invention uses BAC cloning, enzyme digestion interruption and asymmetric amplification to prepare a probe library, thereby effectively improving product sensitivity, specificity and signal strength.

Description

technical field [0001] The invention belongs to the field of biochemistry, in particular to the field of determination or detection related to biochemistry; more specifically, it relates to a method for preparing a FISH probe of EGFR gene, and the probe prepared by the method. Background technique [0002] Epidermal growth factor receptor (EGFR) is a member of the cell surface receptor tyrosine kinase HER / ErbB family, widely distributed on the cell membrane of various normal human tissues, and plays an important role in cell proliferation and migration. effect. EGFR has a molecular weight of 170KDa, is located on the surface of the cell membrane, and belongs to the tyrosine kinase receptor. [0003] After being activated by ligands, EGFR initiates intracellular signal transduction, and through the cascade reaction of adapter proteins and enzymes in the cytoplasm, it regulates the transcription of transcription factor-activated genes and guides cell migration, adhesion, prol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C40B40/08C12Q1/6886
CPCC12Q1/6886C12Q2600/156C40B40/08C40B50/06
Inventor 许嘉森吴诗扬刘志明朱蓉
Owner SUREXAM BIO TECH
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