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TOP2A gene abnormality detection probe, kit and method

An abnormality detection and kit technology, applied in the field of molecular biology, can solve problems such as result judgment and interpretation variation, and achieve the effects of high accuracy, avoiding cross-reaction, and simple operation.

Inactive Publication Date: 2015-04-29
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with FISH, IHC is cost-effective and simple, but it is easily affected by tissue processing methods, fixation time, etc., and there are large variations in the judgment and interpretation of results, while FISH has a high degree of repeatability and reliability.

Method used

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  • TOP2A gene abnormality detection probe, kit and method
  • TOP2A gene abnormality detection probe, kit and method
  • TOP2A gene abnormality detection probe, kit and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The detection TOP2A gene abnormality (amplification / deletion) probe and kit described in the present embodiment are designed as follows:

[0032] 1. Design the amplification primers

[0033] Two sets of amplification primers were designed respectively for the TOP2A gene and the centromere region of human chromosome 17, wherein the first set was for TOP2A, and the second set was for the amplification primers for the centromere region of human chromosome 17. The corresponding amplification products respectively constitute the first group of probe libraries and the second group of probe libraries. The amplification products obtained by each set of amplification primers can be used as rearranged FISH probes, do not contain repetitive sequences, can avoid cross-reactions, and perform specific recognition and hybridization with the TOP2A target detection fragment in the chromosome.

[0034] The following primers were artificially synthesized, and the amplification primers an...

Embodiment 2

[0087] Example 2 Detection of clinical samples using the TOP2A gene abnormality detection kit in Example 1:

[0088] 20 paraffin-embedded tissues provided by the hospital were carried out using the TOP2A gene abnormality detection kit (including SEQ NO.41-50 and SEQ NO.51-60, a total of 20 probes) in Example 1, and the steps were as follows.

[0089] 1. Sample pretreatment process:

[0090] Preparations: Turn on the slicer (45°C-60°C); turn on the water bath (37±1°C); preheat the pepsin buffer in a 37±1°C water bath, use pepsin and preheated pepsin Prepare a working solution of pepsin with a concentration of 1.0 mg / mL in the buffer solution, and place the working solution in a water bath at 37±1°C to preheat (preheat for at least 1 hour).

[0091] Section dewaxing: Submerge the section in xylene at room temperature, dewax for 10 min, replace with fresh xylene and repeat dewaxing twice (a total of 3 dewaxing). The xylene-treated slices were submerged in absolute ethanol for 5...

Embodiment 3

[0118] The influence of the selection of embodiment 3 primer pair quantity on sample detection result

[0119] A probe library was constructed for the TOP2A gene and the centromere region of human chromosome 17, and different numbers of primer pairs were selected for amplification of the TOP2A gene (the first group) and the centromere region of human chromosome 17 (the second group). 4 groups of experiments, in order to study whether the detection effect of the corresponding probe library composed of different numbers of amplification products (i.e., probes) is consistent, wherein the experimental group 1 is to choose 1 pair of amplification primers; the experiment 2 is to choose 3 pairs of amplification primers. pair of amplification primers; in experiment 3, 5 pairs of amplification primers were selected; in experiment 4, 10 pairs of amplification primers were selected. The specific test arrangements are shown in Table 10. The synthesis of probes, the construction of probe l...

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Abstract

The invention discloses a TOP2A gene abnormality detection probe, a kit and a method. The detection probe comprises a first group of probes for labeling fluorescent dye and targeted to a TOP2A gene, and a second group of probes targeted to the centromere region of human chromosome 7, wherein the first group of the probes are selected from at least one of probes as shown in SEQ ID NO.41-SEQ ID NO.50; the second group of the probes are selected from at least one of probes as shown in SEQ ID NO.51-SEQ ID NO.60; and the fluorescent dyes labeled by the two groups of the probes are different in color. A FISH probe in the TOP2A gene abnormality detection kit provided by the invention is free from a repeated sequence, and capable of avoiding a cross reaction and undergoes hybridization recognition with a TOP2A target detection fragment in the chromosome; and the probe has the advantages of high accuracy, good specificity and low false positive rate.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, and in particular relates to a TOP2A gene abnormal detection probe, kit and method. Background technique [0002] The TOP2A gene encodes DNA topoisomerase Ⅱα (TOPIIα). DNA topoisomerase has important functions such as mediating DNA unknotting or unwinding, and directly participates in important life activities such as DNA replication, repair, and transcription. It is an anthracycline drug. and an important target of the topoisomerase inhibitor etoposide. The amplification frequency of TOP2A gene in breast cancer is about 10%, and the deletion frequency is about 3%-12%. [0003] Clinical studies have shown that patients with TOP2A gene abnormality have shortened recurrence-free survival (RFS) and poor prognosis, especially those with TOP2A gene deletion. Patients with TOP2A gene abnormalities receive anthracycline chemotherapy regimens better than those wit...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/118C12Q2600/156
Inventor 胡文晖吴诗扬廖传荣黄萌
Owner SUREXAM BIO TECH
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