Kit for detecting HER3 mutation and detection method of kit

A kit and reagent technology, applied in the field of molecular biology, can solve problems such as time-consuming, false negatives, and inability to meet requirements

Inactive Publication Date: 2018-04-06
QIAGEN SUZHOU TRANSLATIONAL MEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the detection method of HER3 mutation is mainly the direct sequencing method, which is inefficient and time-consuming from sample preparation to sequencing result interpretation, which takes about 2 days; and the detection sensitivity of direct sequencing method is low, which is suitable for the case of low mutation rate. , the method of direct sequencing is almost difficult to achieve its accurate detection, which will lead to a large number of missed detections and false negatives
Therefore, technologies such as direct sequencing are far from meeting the needs of their practical applications

Method used

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  • Kit for detecting HER3 mutation and detection method of kit
  • Kit for detecting HER3 mutation and detection method of kit
  • Kit for detecting HER3 mutation and detection method of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0129] The assembly of embodiment 1 kit

[0130] The kit includes amplification primers, sequencing primers, glycerol, betaine and conventional reagents, specifically as follows:

[0131] Amplification primers:

[0132] SEQ ID NO. 1: TATGTCCTCGTGGCCATGAAT;

[0133] SEQ ID NO. 2: AACATGACGAAGATGGCAAACT;

[0134] SEQ ID NO. 3: AGACCATCTGTGCTCCTCAGTG;

[0135] SEQ ID NO.4: GCCACATCCCAATTTCAGAAC;

[0136] SEQ ID NO.5: TTGGGATGTGGCCTTTGA;

[0137] SEQ ID NO. 6: ACTGATACTTGGTGTGGGGATTG;

[0138] SEQ ID NO. 7:AACCCAATCCCCACACCAAG;

[0139] SEQ ID NO. 8: ATCATTGTTCCTTCCCCTCAGAC;

[0140] SEQ ID NO. 9: GTGGACTCGAGCAACATTGATG;

[0141] SEQ ID NO. 10: GCTGGGGTCTAAGGAGGGAAG;

[0142] SEQ ID NO. 11: CAAGATCCCTGCCCTGGAC;

[0143] SEQ ID NO. 12: GGGCAAACTCTCTGCCACT;

[0144] SEQ ID NO. 13: TATTTGCCTCTGGGTTCTCTGCT;

[0145] SEQ ID NO. 14: GGCTTCTCTCACCTTGGCAATTT;

[0146] SEQ ID NO. 15: GGAACATGGTATGGTGCATAGAA;

[0147] SEQ ID NO. 16: ATCAGGAGGCAGCAGGTCAG;

[0148] SEQ ID NO. 1...

Embodiment 2

[0165] Firstly, the genomic DNA of the tumor tissue sample was extracted, and then using the genomic DNA extract of the patient's tumor tissue sample as a template, in a 30 μL reaction system, the HER3 gene 104, 232, 262, 284, 355, 389, 809, 846 and A total of 9 sites in 928 were amplified by PCR, and the optimal concentration of PCR primers was 0.2 μM;

[0166] The nucleotide sequences of the amplification primers are shown in SEQ ID NO.1-SEQ ID NO.18;

[0167] The nucleotide sequence of the PCR primer used for the 104-site amplification is shown in SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequence shown in SEQ ID NO.1 and SEQ ID NO.2 The sequence is as follows:

[0168] SEQ ID NO.1 (upstream primer): TATGTCCTCGTGGCCATGAAT

[0169] SEQ ID NO.2 (downstream primer): AACATGACGAAGATGGCAAACT;

[0170] The nucleotide sequence of the PCR primer used for the 232-site amplification is shown in SEQ ID NO.3 and SEQ ID NO.4, and the nucleotide sequence shown in SEQ ID NO.3 and ...

Embodiment 3

[0197] Example 3 Pyrosequencing

[0198] Experimental Materials:

[0199] Pyrosequencer and supporting reagents (adsorption buffer, denaturation buffer, annealing buffer, washing buffer, enzyme, substrate and 4 kinds of dNTPs) were purchased from QIAGEN Company, streptavidin-coated agarose Beads were purchased from GE Company.

[0200] Pyrosequencing detection steps:

[0201] (1) PCR product immobilization: Add 29 μL of ultrapure water, 40 μL of adsorption buffer and 1 μL of streptavidin-coated agarose beads to the eight-tube PCR tube, and then add 10 μL of biotin-labeled PCR To amplify the product, place the PCR eight-tube tube on a vortex shaker at room temperature for 10 minutes to immobilize the PCR product on the agarose gel beads;

[0202] (2) Preparation of sequencing primers: Dilute the sequencing primers to 0.8 μM with annealing buffer. Add 25 μl of diluted sequencing primers to each reaction well of the PyroMark Q24 24-well plate to be used;

[0203] (3) Purific...

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Abstract

The invention provides a kit for detecting HER3 mutation and a detection method of the kit. The kit mainly comprises nine pairs of PCR primers and nine sequencing primers. By means of the kit, the specific locus mutation of a target gene can be accurately analyzed, the allele frequency of a mixed cell population can be quantitatively analyzed, and the kit has the advantages of being high in sensitivity, high in specificity and low in time consumption; the detection method is quick and precise, and the operation is simple.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a kit for detecting HER3 mutation and a detection method thereof. Background technique [0002] Human epidermal growth factor receptor (Human epidermal-growth-factor receptor, HER) family includes EGFR (erbB1 / HER1), HER2, (erbB2 / neu), HER3 (erbB3) and HER4 (erbB4) four family members, belonging to I Type receptor tyrosine kinase family (Receptor tyrosine kinase, RTKs). HER is activated after binding with ligands, forming homodimers or heterodimers, regulating MAPK pathway or activating PI3K / AKT pathway to participate in cell signal transmission, transmitting signals through kinase cascades in cells, and finally regulating cell growth and development. Divide, and participate in the proliferation, invasion and angiogenesis of tumor cells. HER family proteins are closely related to the clinicopathological features and prognosis of many tumors such as breast cancer, lung cancer and...

Claims

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Application Information

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IPC IPC(8): C12Q1/6869C12Q1/6886
CPCC12Q1/6869C12Q1/6886C12Q2600/106C12Q2600/156C12Q2565/301C12Q2535/122C12Q2531/113
Inventor王洁王瑜张亚飞吴叶君申爱丽吴子侠
OwnerQIAGEN SUZHOU TRANSLATIONAL MEDICINE CO LTD