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CDNA in situ hybridization probe of Her-2 gene, and preparation method thereof

An in situ hybridization and probe technology, which is applied in the field of Her-2 gene in situ hybridization probe and its preparation, can solve the problem of low protein accuracy and achieve the effect of detection

Active Publication Date: 2015-09-02
JIANGHAN UNIVERSITY
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  • Abstract
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Problems solved by technology

[0006] In order to solve the problem that the prior art can only detect HER-2 receptor protein by immunohistochemical method with low accuracy, the embodiment of the present invention provides a cDNA in situ hybridization probe of Her-2 gene and its preparation method

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  • CDNA in situ hybridization probe of Her-2 gene, and preparation method thereof
  • CDNA in situ hybridization probe of Her-2 gene, and preparation method thereof
  • CDNA in situ hybridization probe of Her-2 gene, and preparation method thereof

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Embodiment

[0027] 1. Primer Design

[0028] According to the Gene bank sequence of the Her-2 gene, Her-2 gene-specific primers were designed. The primer sequence is: upstream primer: 5'-AGGAGTGCGTGGAGGAAT-3', as shown in SEQ ID NO.2 in the sequence listing; downstream primer: 5'-CCAGCCCGAAGTCTGTAAT-3', as shown in SEQ ID NO.3 in the sequence listing.

[0029] 2. Preparation of a plasmid containing the cDNA in situ hybridization probe sequence of the Her-2 gene

[0030] (1) Collect fresh tumor tissues from 60 breast cancer patients with high expression of Her-2 gene (60 patients from the Sixth Hospital of Wuhan, from February 2011 to June 2013), and use RNA lysate (Trizol method) The total mRNA of the tumor tissue was extracted, and the extracted total mRNA was reverse-transcribed into cDNA using the Quanti Tect Reverse Transcription Kit;

[0031] (2) The cDNA is amplified by PCR with designed primers to obtain an amplified product, which is a Her-2 gene fragment with a length of 987bp,...

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Abstract

The invention discloses a cDNA in situ hybridization probe of a Her-2 gene, and a preparation method thereof, and belongs to the field of molecular pathology diagnosis. The preparation method of the probe comprises the following steps: collecting breast cancer patient's fresh tumor tissues highly expressing the Her-2 gene, extracting the total RNA of the tumor tissues, carrying out reverse transcription on the total mRNA to form cDNA, carrying out PCR amplification on the cDNA and a primer to obtain an amplification product, and constructing a plasmid containing the cDNA in situ hybridization probe sequence of the Her-2 gene, wherein the amplification product is a Her-2 gene fragment with the length of 987bp and also is the fragment of the probe; and carrying out probe marking with the plasmid as a template by using the primer and a DIG polymerase chain reaction probe synthesis kit to obtain the probe. The cDNA in situ hybridization probe sequence of the Her-2 gene has clear specificity to the DNA of the Her-2 gene, and realizes detection of the DNA amplification condition of the Her-2 gene.

Description

technical field [0001] The invention relates to the field of molecular pathological diagnosis, in particular to a cDNA in situ hybridization probe of Her-2 gene and a preparation method thereof. Background technique [0002] Human epidermal growth factor receptor-2 (Epidermal growth factor receptor-2, HER-2) is the second member of the epidermal growth factor receptor EGFR family, and Her-2 gene exists in about 20% to 30% of breast cancer cases And / or increased HER-2 protein level, it is an independent prognostic factor for breast cancer, and its role in targeted therapy and prognosis of breast cancer has been recognized by clinicians all over the world. Her-2 gene-positive tumors are high-risk tumors. Her-2 gene amplification indicates rapid disease progression, high risk of local recurrence, short chemotherapy remission period, disease free survival (DFS) and overall survival The period (Overall Survival, OS) is shortened, and the response to some conventional chemotherap...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/10
CPCC12Q1/6841C12Q1/6886
Inventor 茹琴李超英田香乐凯陈琳马宝苗熊琪
Owner JIANGHAN UNIVERSITY
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