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TOP2A genetic detection probe and preparation method and application thereof

A gene detection and DNA probe technology, applied in the field of TOP2A gene detection probe and its preparation, can solve the problems of long hybridization time, low hybridization efficiency, and high signal background

Pending Publication Date: 2020-01-17
广州简册生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the presence of some non-specific sequences in the BAC and PCR product probes, the specificity of the fluorescent in situ hybridization probes is not high, the signal background is high, and the fragments of the existing fluorescent in situ hybridization probes are usually too large, making them hybridize Low efficiency, long hybridization time

Method used

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  • TOP2A genetic detection probe and preparation method and application thereof
  • TOP2A genetic detection probe and preparation method and application thereof
  • TOP2A genetic detection probe and preparation method and application thereof

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preparation example Construction

[0040] The invention provides a method for preparing a TOP2A gene detection probe. The prepared TOP2A gene detection probe includes a TOP2A single-strand probe for the TOP2A gene region and a control single-strand probe for the centromere region of chromosome 17.

[0041] One embodiment of the present invention provides a method for preparing a TOP2A single-stranded probe, comprising the steps of:

[0042] Step 1: Select the TOP2A gene region as the TOP2A target gene, design TOP2A single-stranded candidate probes according to the TOP2A target gene, remove the TOP2A single-stranded candidate probes that non-specifically hybridize and overlap with the TOP2A target gene to obtain the TOP2A target probe, and obtain the TOP2A target probe by screening Design TOP2A amplification primer fragments upstream and downstream of the TOP2A target probe.

[0043] In the above step 1, select the TOP2A gene region as the TOP2A target gene, and design the TOP2A single-stranded candidate probes ...

Embodiment 1

[0082] Select the TOP2A gene region as the TOP2A target gene, design a TOP2A single-stranded candidate probe every 3bp, the length of each TOP2A single-stranded candidate probe is 200nt, and design 40 TOP2A single-stranded candidate probes; through BLAST comparison, Remove TOP2A single-stranded candidate probes located in the repetitive region of the TOP2A gene, remove TOP2A single-stranded candidate probes with non-specific Tm values ​​between 75-85°C by scoring, and select non-overlapping TOP2A single-stranded candidate probes as TOP2A targets As for the probes, a total of 26 TOP2A target probes were obtained, and the sequences of the 26 TOP2A target probes are shown in SEQ ID NO.55-80. The TOP2A amplification primer fragments were designed for the upstream and downstream of the 26 TOP2A target probes screened. The sequences of TOP2A amplification primer fragments are shown in SEQ ID NO.1-52. The TOP2A amplification primer fragment and the corresponding TOP2A target probe a...

Embodiment 2

[0120] Select the centromere region of chromosome 17 as the control gene, design a single-stranded fragment with a length of 206 nt as the control target probe for the control gene, and design control amplification primer fragments for the upstream and downstream of the control target probe.

[0121] The sequences of the control amplification primer fragments are shown in SEQ ID NO.53-54, wherein the sequence shown in SEQ ID NO.53 is the upstream amplification primer, and the sequence shown in SEQ ID NO.54 is the downstream amplification primer. The sequence of the control target probe is shown in SEQ ID NO.81.

[0122] PCR is used to amplify the control target probe to obtain a PCR product.

[0123] To set up a PCR reaction:

[0124]

[0125] Purification of PCR products: Combine the PCR products, add 1ml of butanol, and mix well; then add 400μl Orange-DX, mix well; centrifuge for 2min, remove the upper organic phase, and pass the aqueous phase through the column to bind. ...

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Abstract

The invention provides a TOP2A genetic detection probe and a preparation method and application thereof. A TOP2A gene region and a No.17 chromosome centric region are selected as a target gene, according to the target gene, design and screening are performed to obtain a target probe, and the target probe is amplified to obtain an amplification product. The amplification product is subjected to transcription and reverse transcription, a single-stranded DNA probe is obtained, and the single-stranded DNA probe is subjected to fluorescence labeling to obtain a TOP2A single-stranded probe for the TOP2A gene region and a contrast single-stranded probe for the No.17 chromosome centric region. The prepared TOP2A single-stranded probe and the contrast single-stranded probe are linear single-stranded DNA, the single-stranded DNA can reduce autologous pairing of the probes, quick combination with the target sequence can be realized under the condition of guaranteeing the specificity, and the hybridization time is shortened.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a TOP2A gene detection probe and its preparation method and application. Background technique [0002] Breast cancer is one of the most common malignant tumors in women. In recent years, the incidence rate in my country has shown a linear upward trend. Clinical studies have shown that breast cancer patients with TOP2A gene abnormalities (including TOP2A gene amplification or TOP2A gene deletion) have shortened recurrence-free survival (RFS) and poor prognosis, especially breast cancer patients with TOP2A gene deletion The prognosis of patients is worse. However, patients with an abnormal TOP2A gene were more likely to receive an anthracycline chemotherapy regimen than patients with a normal TOP2A gene. Treatment of patients with TOP2A gene amplification with cyclophosphamide + epirubicin + fluorouracil (CEF) regimen can reduce the risk of recurrence by 61% and the risk of death by ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6811C12Q1/6837C12Q1/6886C12N15/11
CPCC12Q1/6811C12Q1/6837C12Q1/6886C12Q2600/118C12Q2600/106C12Q2531/113C12Q2563/107
Inventor 李怡赵浩
Owner 广州简册生物技术有限公司
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