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FISH (fluorescence in situ hybridization) probe, kit and detection method for detecting Her2 (human epidermal growth factor receptor 2) gene free from repetitive sequence

A repeat sequence and kit technology, applied in the field of FISH probes, can solve the problems of reducing the specificity and sensitivity of FISH detection

Active Publication Date: 2013-11-27
WUHAN HEALTHCHART BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, fluorescent in situ hybridization probes are mainly prepared using artificial chromosomes, which include yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) and phage artificial chromosomes (PAC), etc., due to the existence of many repetitive sequences in artificial chromosomes, These repetitive sequences appear continuously throughout the genome, resulting in the preparation of fluorescent in situ hybridization probes with many repetitive sequences, which can also produce fluorescence after the probe hybridizes with non-target DNA, resulting in non-specific fluorescent signals , greatly reducing the specificity and sensitivity of FISH detection

Method used

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  • FISH (fluorescence in situ hybridization) probe, kit and detection method for detecting Her2 (human epidermal growth factor receptor 2) gene free from repetitive sequence
  • FISH (fluorescence in situ hybridization) probe, kit and detection method for detecting Her2 (human epidermal growth factor receptor 2) gene free from repetitive sequence
  • FISH (fluorescence in situ hybridization) probe, kit and detection method for detecting Her2 (human epidermal growth factor receptor 2) gene free from repetitive sequence

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Experimental program
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Effect test

Embodiment 1

[0083] Synthesis of primer sets: through the online analysis database Repeat Masking ( http: / / www.girinst.org / ) analyze the Her2 gene to obtain the repeated and non-repeated sequences of the Her2 gene (such as figure 1 shown), import the non-repeated sequence into the primer design software Primer5 for primer design, design the amplified product of the primers to be 350-800 bp, and synthesize the following 26 pairs of primers by artificial synthesis:

[0084] 1 F: 5' GGTTGGAATGAGTAAGAAGTAGC 3'

[0085] 1 R: 5' TAGGAATTTTCTAAAGGGGAGAC 3'

[0086] 2 F: 5' AGGTTAATGGTGGAAGTGGG 3'

[0087] 2 R: 5'ACACTCTACTATTAACATATGC 3'

[0088] 3 F: 5' TTTACTTTGGCTGCTGTTGC 3'

[0089] 3 R: 5' ATATTTTCGGATTTGGTATAGG 3'

[0090] 4 F: 5' TGGCTGTAGGTCAGAGTGGC 3'

[0091] 4 R: 5' AAGGATAAGGTGGAGTTTTGT 3'

[0092] 5 F: 5' TGGGTGATGGAATGATCTGG 3'

[0093] 5 R: 5' ACGAGTCTCCGAACAAAAGG 3'

[0094] 6 F: 5'AAAAGAAAACCAATGGGACAG 3'

[0095] 6 R: 5' CCCACGCCAACATAAATACAC 3'

[0096] 7 F: 5'TCCT...

Embodiment 2

[0141] Her2 gene BAC clone construction, the steps are as follows:

[0142] ① Genomic DNA preparation: DNA from breast cancer tissue was extracted using a commercially available DNA extraction kit;

[0143] ② Enzymatic digestion of genomic DNA: select restriction endonuclease HindⅢ to digest genomic DNA, and perform DNA electrophoresis on the digested products. After electrophoresis, select and recover DNA fragments of 200-300kb fragments;

[0144] ③ Ligation of large fragment DNA and BAC clone: ​​Use commercially available pIndigoBAC-5 as the carrier, and construct the connection system according to the mass ratio of vector to large fragment DNA at a ratio of 1:10:

[0145]

[0146] Ligation at 16°C for 16 hours; treatment at 65°C for 20 minutes to inactivate T4 ligase; the ligation product was dialyzed with 0.025um Millipore membrane for 3 hours, the dialyzed ligation product was divided into 0.5ml centrifuge tubes, transformed into DH10B cells and cultured filter;

[0...

Embodiment 3

[0149] Centromeric STR sequence plasmid construction, the steps are as follows:

[0150] ① Due to the short centromere STR sequence (>700bp), the STR sequence was obtained by chemical synthesis;

[0151] ② Ligation of STR sequences with commercially available plasmids: Use commercially available T vector plasmids as carriers, and construct a ligation system with a 10ul system:

[0152]

[0153] ③ After shaking the above mixture gently, centrifuge at 12,000rpm for 30 seconds, and then keep it at 14°C overnight (12-16h);

[0154] ④ The connected product should be stored in a refrigerator at 4°C for later use or at -85°C for long-term storage.

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Abstract

The invention belongs to the field of biotechnology, and discloses a FISH (fluorescence in situ hybridization) probe, a kit and a detection method for detecting Her2 (human epidermal growth factor receptor 2) gene free from repetitive sequence. The Her2 gene is used as a template to perform polymerase chain reaction on non-repetitive sequence in the Her2 gene, the amplification product is DNA (deoxyribonucleic acid) fragments with 350-800 different base pairs, the repetitive sequence in the Her2 gene is eliminated so as to form the product free from repetitive sequence; after the product is in fluorescence labeling, the Her2 gene free from repetitive FISH is obtained for the Her2 gene FISH detection. The Her2 gene FISH probe obtained by the invention can eliminate the repetitive sequence, the non-specific background signal of the Her2 gene FISH probe can be obviously reduced, and the specificity of the FISH probe is improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a FISH probe, a kit and a detection method for detecting Her2 gene without repetitive sequences. Background technique [0002] Breast cancer is the most common malignant tumor in women. In 2011, the latest statistical data published by CA: A Cancer Journal for Clinicians in the United States showed that in 2011 in the United States, 230,480 women were expected to suffer from breast cancer, accounting for 30% of new malignant tumors in women, ranking first in the incidence of female malignant tumors . The statistics in Beijing, Shanghai, Tianjin and other big cities in my country show that breast cancer is also the most common malignant tumor among women in my country, and the incidence rate is increasing year by year. The etiology of breast cancer has not been fully elucidated, but many research data show that the occurrence of breast cancer is related to the long-term stimulation of...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 叶伦薛力泉李雪梅付金玲程弘夏陈刚
Owner WUHAN HEALTHCHART BIOLOGICAL TECH
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