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ALK gene rearrangement detection probe, kit and method

A detection kit and gene rearrangement technology, applied in the field of molecular biology, can solve the problems of inability to detect fusion type, reduce the specificity and sensitivity of FISH detection, and achieve the effects of fast detection speed, high accuracy and low false positives

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

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Problems solved by technology

However, there are still some deficiencies in the above-mentioned techniques. For example, fluorescent in situ hybridization probes are mainly prepared using artificial chromosomes. Due to the repeated sequences in the artificial chromosomes appearing continuously throughout the genome, non-specific fluorescent signals are caused, which greatly reduces the specificity of FISH detection. The determination of positive criteria for routine IHC has not yet been unified; although RT-PCR method for detecting ALK fusion gene is fast, simple and easy, and can clarify the type of known fusion variants of ALK at the same time, it cannot detect unknown fusion types. biggest shortcoming

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  • ALK gene rearrangement detection probe, kit and method
  • ALK gene rearrangement detection probe, kit and method
  • ALK gene rearrangement detection probe, kit and method

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Embodiment 1

[0033] The detection probe and kit for detecting ALK gene rearrangement described in this embodiment are designed as follows:

[0034] 1. Design the amplification primers

[0035] The ALK gene is located on chromosome 2 (position 2p23), and the breakpoint of the ALK gene rearrangement is located between exons 19 and 20. According to the characteristics of the ALK gene, two sets of amplification primers were designed respectively: for the breakpoint The first group of amplification primers designed upstream, the second group of amplification primers designed downstream of the breakpoint, and the corresponding amplification products respectively constitute the first group of probe libraries and the second group of probe libraries. For each set of amplification primers, the obtained amplification product can be used as a rearranged FISH probe, does not contain repeated sequences, can avoid cross-reaction, and can specifically recognize and hybridize with the ALK target detection ...

Embodiment 2

[0088] Example 2 Detection of clinical samples using the ALK gene rearrangement detection kit in Example 1

[0089] 20 paraffin-embedded tissues provided by the hospital were performed using the ALK gene rearrangement 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. 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. The xylene-treated slices were submerged in absolute ethanol for 5 min, and repeated with fresh absolu...

Embodiment 3

[0123] The influence of the selection of embodiment 3 primer pair quantity on sample detection result

[0124] For the upstream and downstream of the ALK gene rearrangement breakpoint, a probe library was constructed, and the first group and the second group of amplification primer pairs of different numbers were respectively selected to design 4 groups of experiments to study different numbers of amplification products (i.e., probe Needle) to determine whether the detection effect of the corresponding probe library is consistent, in which the test group 1 selected 1 pair of amplification primers; test 2 selected 3 pairs of amplification primers; test 3 selected 5 pairs of amplification primers Right; in experiment 4, 10 pairs of amplification primers were selected for each. The specific test arrangements are shown in Table 10. The synthesis of probes, the construction of probe libraries, the labeling of probes and the experimental steps are shown in Example 1 and Example 2. ...

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Abstract

The invention discloses an ALK gene rearrangement detection probe, a kit and a method. The detection probe comprises a first group of probes for labeling fluorescent dye and targeted to the upstream side of a breaking point, and a second group of probes targeted to the downstream side of the breaking point, 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 ALK gene rearrangement detection kit provided by the invention is free from a repeated sequence, and capable of avoiding a cross reaction and undergoes hybridization recognition with an ALK target detection fragment in a 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 detection probe, a kit and a method for ALK gene rearrangement. Background technique [0002] The anaplastic lymphoma kinase (ALK) encoded by the ALK gene was first discovered in anaplastic large cell lymphoma and is one of the important targets of targeted therapy. The ALK gene is located on chromosome 2 (position 2p23), the length is 728kb, the full-length cDNA contains 29 exons, the full-length ALK protein contains 1620 amino acids, the molecular weight is 177 kilodaltons (kDa), and 254 amino acids The kinase domain consists of amino acid residues 1123 to 1376, preceded by a short transmembrane region of amino acids. The breakpoint of ALK gene rearrangement is located between exons 19 and 20. After rearrangement of ALK gene, most of them have biological functions. The expression product is a chimeric tyrosine kinase, which ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q2600/136
Inventor 胡文晖吴诗扬廖传荣黄萌
Owner SUREXAM BIO TECH
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