Kit for detecting abnormity of BRCA1 and PTEN genes and detection method

An abnormal detection and kit technology, applied in the field of molecular biology, can solve the problems of increasing detection and waiting time for results, long probe length, etc., to shorten the time required for full hybridization, improve detection sensitivity, and improve detection efficiency. Effect

Inactive Publication Date: 2017-12-01
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the length of the existing FISH probes for abnormal detection of BRCA1 and PTEN genes is relatively long, resulting in a hybridization time of 12-16 hours in the detection process, and there is no parallel detection FISH kit for the two genes on the market, so it is necessary to Obtaining the abnormalities of the two genes in patients, especially prostate cancer patients, requires two FISH tests, which greatly increases the time for testing and waiting for results

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  • Kit for detecting abnormity of BRCA1 and PTEN genes and detection method
  • Kit for detecting abnormity of BRCA1 and PTEN genes and detection method
  • Kit for detecting abnormity of BRCA1 and PTEN genes and detection method

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Example 1 A BRCA1 and PTEN gene abnormality detection kit

[0042] The BRCA1 and PTEN gene abnormality detection kit described in this embodiment mainly includes:

[0043] 1. Design amplification primers

[0044] Design the first and second sets of amplification primers for the BRCA1 gene and the centromere region of human chromosome 17 respectively. The first group is the amplification primer for the BRCA1 gene, and the second group is for the centromere region of human chromosome 17 The corresponding amplification products constitute the first set of probe library and the second set of probe library; respectively, the third and fourth sets of amplification primers are designed for the PTEN gene and the centromeric region of human chromosome 10 , The third group is the amplification primers for PTEN gene, the fourth group is the amplification primers for the centromeric region of human chromosome 10, and the corresponding amplification products form the third group and the f...

Embodiment 2

[0075] Example 2 Using the BRCA1 and PTEN gene abnormality detection kit in Example 1 to detect clinical samples of prostate cancer patients

[0076] In this embodiment, the detection of abnormalities of BRCA1 and PTEN genes in circulating tumor cells of prostate cancer patients is taken as an example. The BRCA1 and PTEN gene abnormalities detection kit in Example 1 is used to examine peripheral blood samples of 20 prostate cancer patients. The steps are as follows:

[0077] 1. Sample pretreatment:

[0078] 1. Enrichment of circulating tumor cells: Peripheral blood samples are treated with commercially available red blood cell lysis to remove red blood cells. The blood sample and red blood cell lysate are mixed in a ratio of 1:3; the cell suspension after red blood cell lysis is added to the filter membrane ( The filter membrane has a pore size of 5-8um) and is subjected to suction filtration. After the suction filtration is completed, carefully remove the filter membrane with tweeze...

Embodiment 3

[0117] Example 3 The influence of different target gene selection on test results

[0118] 1. Detection of target gene selection

[0119] In order to test the influence of the selection of different target genes on the sample detection results, three experimental groups are designed in this embodiment, in which experimental group 1 uses all probes of the first and second groups to detect the BRCA1 gene; experimental group 2 uses the third , The fourth set of all probes detects PTEN gene; experimental group 3 uses all the first, second, third and fourth sets of probes to detect abnormalities of BRCA1 and PTEN genes in parallel. See Table 6 for specific test arrangements. The synthesis of the probe, the construction of the probe library, the labeling of the probe and the experimental procedures are as shown in Example 1 and Example 2.

[0120] Table 6 Selection of target genes

[0121]

[0122] 2. Sample detection

[0123] Using the kit prepared by the above design, according to the st...

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Abstract

The invention discloses a kit for detecting abnormity of BRCA1 and PTEN genes and detection method. The kit includes a first group of probes and a second group of probes, which aim to the BRCA1 gene, and/or a third group of probes and a fourth group of probes, which aim to the PTEN gene. Each group of probes is labeled with a fluorescent dye, wherein the color of the fluorescent dye on the probes in the same group is same while the color of the fluorescent dye on the probes in different groups are different. The four groups of probes respectively are amplification products by amplifying a primer with corresponding BAC cloning as a template. The FISH probes are produced through repeated comparison and selection and then utilization of optimized BAC cloning as the template and through amplification by designing a primer aiming to a non-repeated and highly-conserved sequence, so that the kit has excellent specificity and lower background noise. The kit can achieve the optimal balance between detection specificity and hybridization time length, so that not only are specificity and sensitivity of a result ensured, but also hybridization time is reduced, thereby increasing detection efficiency.

Description

Technical field [0001] The invention belongs to the field of molecular biology, and relates to medicine and biotechnology, and specifically relates to a BRCA1 and PTEN gene abnormality detection kit and a detection method. Background technique [0002] The BRCA1 (BREAST CANCER1) gene is located at 17q21, about 81kb, and has 23 exons. The N-terminal sequence of the BRCA1 encoded protein contains a ring domain, which can form a ring-2 ring heterodimer with the BRCA1-related cyclic protein. The N-terminus of BRCA1 is not only associated with RNA synthetase, but also closely related to the formation of S phase and nuclear dots. Removing the N-terminus of BRCA1 will cause nearly 98% of BRCA1 to lose contact with RNA synthetase. Therefore, it is believed that the N-terminus of BRCA1 plays an important role in regulating the function of RNA synthetase. [0003] PTEN gene (gene of phosphate and tension homology deleted on chromsometen, PTEN) is located on chromosome 10q23.3. It is compos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/44C12N15/11
CPCC12Q1/682C12Q1/6827C12Q1/6886C12Q2600/106C12Q2600/118C12Q2600/156C12Q2563/107C12Q2525/204
Inventor 廖传荣吴诗扬朱蓉
Owner SUREXAM BIO TECH
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