Reagent for detecting copy number of EGFR gene and ploidy of chromosome 7

A technology of chromosome ploidy and gene copy number, applied in the field of life science and biology, can solve the problems of high mortality, poor sensitivity, and lack of early diagnosis methods for lung cancer.

Active Publication Date: 2010-12-01
合肥艾迪康医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There are two main reasons for the high mortality rate of lung cancer: one is the lack of effective early diagnosis methods for lung cancer, and more than 70% of the patients are diagnosed at an advanced stage; the other is that advanced lung cancer (especially non-small cell lung cancer, NSCLC) is sensitive to chemotherapy. The overall effectiveness of various existing chemotherapy regimens for NSCLC is only about 30%.

Method used

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  • Reagent for detecting copy number of EGFR gene and ploidy of chromosome 7

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1: cell culture

[0055] 1) The original bacterial solution (human genome bacterial artificial chromosome (BAC) clone, purchased from Invitrogen Company) was spread on a plate and cultured at 37°C overnight.

[0056] 2) Pick a single colony, put it in 5ml LB medium (tryptone 10g / L, yeast extract 0.05g / L, sodium chloride 10g / L, pH7.0), shake the bacteria overnight at 37°C and 220 rpm .

[0057] 3) Add all 5ml of the bacterial solution into 400ml of LB medium to expand the culture, and shake the bacteria overnight at 37°C and 220 rpm.

Embodiment 2

[0058] Embodiment 2: the extraction of plasmid

[0059] 1) Transfer the incubated transformed cells to an LB plate containing the antibiotic ampicillin (100 μg / ml). When the liquid is absorbed, invert the plate and incubate at 37°C for 12-16 hours.

[0060] 2) Pick a single colony and culture it in 10ml LB medium at 37°C for 24h.

[0061] 3) Centrifuge at 8000rpm for 10-15min, and remove the supernatant.

[0062] 4) 200 μl Solution I (50 mM glucose, 10 mM EDTA pH 8.0, 10 mM Tris pH 8.0) was shaken and mixed.

[0063] 5) Shake 400 μl Solution II (0.2M NaOH, 1% SDS, ready-to-use) for 7 seconds, and stand on ice for 10 minutes.

[0064] 6) Shake 300 μl Solution III (3M potassium acetate, pH 4.8) for 5 s, and let stand on ice for 10-15 min.

[0065] 7) Centrifuge at 13000 rpm for 10 minutes, and transfer the supernatant into a 1.5ml centrifuge tube.

[0066] 8) Add an equal volume of balanced phenol, shake until milky, and centrifuge at 1000 rpm for 8 minutes.

[0067] 9) Asp...

example 3

[0077] Example 3: Labeling Probes

[0078] Use Nick translation method to mark, the process is as follows:

[0079] The 20μl reaction system contains: dATP, dCTP, dGTP, dTTP and Fluorophore-labeled dUTP (Cy3 for EGFR gene, FITC for chromosome 7 centromere), each 0.2μM, DNase I 0.5U, DNA polymerase I 2U, 0.5 ~1.0 μg plasmid DNA.

[0080] In a PCR instrument, add 1 μl of 0.5M EDTA (pH 8.0) after labeling at 15°C for 1.5-3 hours to terminate the reaction. During the process, take 5 μl of the product and use 2% agarose gel for electrophoresis detection. If the DNA fragment size is 500-600bp, stop the reaction; if the fragment is larger than this range, you need to increase the amount of DNase I and the reaction time until Fits target size. RP11-339F13 probe and RP11-144H20 probe were made.

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Abstract

The invention discloses a reagent for detecting a copy number of an EGFR gene and the ploidy of a chromosome 7. The reagent comprises a fluorescence probe RP11-339F13 used for detecting the copy number of the EGFR gene and a fluorescence probe RP11-144H20 used for detecting the ploidy of the chromosome 7, wherein the RP11-339F13 is cloned by BAC, which covers the whole EGFR gene, and marked with rhodamine fluorescein; and RP11-144H20 is cloned by specific BAC of the centromere of the chromosome 7 and marked with fluorescein isothiocyanate. The reagent can quickly, simply and conveniently detect the copy number of the EGFR gene and the ploidy of the chromosome 7, contributes to the judgment of medicament susceptibility and prognostic judgment, and has great significance for the guidance of use of molecular targeted medicament tyrosine kinase inhibitors (TKIs) of tumors, such as non-small cell lung cancer and the like.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, in particular to a reagent for detecting the gene copy number of epidermal growth factor receptor (EGFR) and chromosome 7 ploidy. Background technique [0002] In my country, lung cancer has become the leading cause of cancer death in both men and women. There are two main reasons for the high mortality rate of lung cancer: one is the lack of effective early diagnosis methods for lung cancer, and more than 70% of the patients are diagnosed at an advanced stage; the other is that advanced lung cancer (especially non-small cell lung cancer, NSCLC) is sensitive to chemotherapy. The overall effective rate of various existing chemotherapy regimens for NSCLC is only about 30%. [0003] Gefitinib (i.e., Iressa) is an aniline quinazoline compound, which is a potent and selective tyrosine kinase inhibitor (tyrosine kinase inhibitor, TKI), which can block the growth of tumor cells induced by ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 郭兴中董正伟方国伟郭尧
Owner 合肥艾迪康医学检验实验室有限公司
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