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Bubble-shaped primer, reagent kit with same and application

A kit and bubbling technology are applied to bubbling primers and the kits and application fields composed of them, which can solve the problem of high operation requirements, achieve the effects of high detection sensitivity, high accuracy, and improve the sensitivity of analysis and detection

Active Publication Date: 2017-11-21
MGI TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When there are other medication needs, additional tests need to be performed again, and methods such as Fish and IHC have high requirements for experimenters

Method used

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  • Bubble-shaped primer, reagent kit with same and application
  • Bubble-shaped primer, reagent kit with same and application
  • Bubble-shaped primer, reagent kit with same and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] Example 1 Design of bubbling primers for non-small cell lung cancer-related hotspot genes

[0102] (1) Selection of target sites: read the literature and major non-small cell lung cancer treatment guidelines to determine the most relevant genes for non-small cell lung cancer medication, and use the cosmic database to find the mutation with the highest mutation rate in lung cancer patients Sites and subtypes, or important mutation sites directly related to drug resistance.

[0103] (2) Design a universal sequence: through repeated design and testing of the sequence, the nucleic acid sequence shown in SEQ ID NO.1-2 is obtained, as follows:

[0104] The general sequence of the forward primer (SEQ ID NO.1): 5'-CGACATGGCTACGATCCGACTT-3';

[0105] The general sequence of the reverse primer (SEQ ID NO.2): 5'-TAAGACCACTTGACCGTCAGCAT-3';

[0106] (3) Design bubbling primers, such as figure 1 As shown, the bubbling primer includes a forward primer and a reverse primer, both of...

Embodiment 2

[0108] The assembly of embodiment 2 kit

[0109] Specifically, according to the main reagents in the kit of the present invention include:

[0110] PCR amplification reagent: 2×PCR buffer Pfu DNA polymerase, pure water, the bubbling primer of embodiment 1 and the universal primer that are used for secondary PCR, wherein, described 2×PCR buffer is made of 10×PfuTurbo Cx buffer, dNTPs, betaine, dimethyl sulfoxide, and magnesium sulfate; the dNTPs is a dNTPs mixture with a concentration of 0.1Mm-25mM; the betaine is a betaine solution with a molar concentration of 1M-4M; Described dimethyl sulfoxide is the dimethyl sulfoxide that mass percent concentration is 100%; Described magnesium sulfate is the magnesium sulfate solution that molar concentration is 1Mm-1M; Described DNA polymerase is PfuTurbo Cx hot start DNA polymerase ;

[0111] The universal primers include a forward universal primer and a reverse universal primer, the forward universal primer is shown in SEQ ID NO.147,...

Embodiment 3

[0112] Example 3 Detection of Non-small Cell Lung Cancer Related Hotspot Gene Mutations

[0113] The method is as figure 2 shown, including the following steps:

[0114] 1) Extracting sample genomic DNA: using paraffin sample extraction kit (MagPure FFPE DNA KF Kit) for extraction, the extracted DNA is subjected to Nanodrop to detect the quality of DNA, and the D of the extracted genomic DNA is 260 / OD 230 =1.8-2.5, the extracted genomic DNA is qualified and can be used for the next step of PCR amplification.

[0115] 2) The first round of PCR reaction to capture the coding sequence of non-small cell lung cancer-related mutation hotspot genes: using the genomic DNA in step (1) as a template, and using the bubbling primers in Example 1 to perform the first round of specific multiplex PCR reaction , obtain PCR amplification product, the system and condition of described PCR reaction are as follows:

[0116] The PCR reaction system is as follows:

[0117]

[0118]

[...

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Abstract

The invention relates to a bubble-shaped primer, a reagent kit with the same and application. The bubble-shaped primer is of a nucleic acid single-strain structure and comprises four portions. The four portions are sequentially linked with one another and include a 5p arm, a universal sequence, a molecular label and a 3p arm; the 5p arm is arranged at a 5' end of the bubble-shaped primer and can be combined with a template sequence during first-round PCR (polymerase chain reaction) according to complementary base pairing principles; the universal sequence is in the shape of protruded bubble without complementation with DNA (deoxyribonucleic acid) sequences of templates during the first-round PCR, and at least one part of the universal sequence and at least one part of a sequence of a 3' end of a universal primer are identical to each other during second-round PCR; the molecular label comprises a plurality of degeneracy bases; the 3p arm is arranged at a 3' end of the bubble-shaped primer and can be combined with the template sequence according to the complementary base pairing principles. The bubble-shaped primer, the reagent kit and the application have the advantages that the analysis and detection sensitivity of sequencing data can be greatly improved by the bubble-shaped primer, a plurality of sites and a plurality of types can be simultaneously detected, and the bubble-shaped primer and the reagent kit are high in detection sensitivity, throughput and accuracy and applicable to detecting different types of tumor.

Description

technical field [0001] The invention belongs to the field of biotechnology, relates to medical molecular diagnosis and biotechnology, and relates to a bubbling primer and a kit and application thereof. Background technique [0002] In the past 10 years, the incidence of cancer has increased significantly. Among them, lung cancer is the malignant tumor with the highest morbidity and mortality in the world. The 5-year survival rate is only 16.8%, and 1.6 million new lung cancer patients are diagnosed every year. At the same time, lung cancer is also the leading cause of death of cancer patients, with 1.4 million people dying of lung cancer every year. According to the "2015 China Cancer Statistics", it is estimated that there will be about 4.292 million new cancer cases and 2.814 million cancer deaths in China in 2015. in. Lung cancer has 733,000 new cases (accounting for 17.1% of the total) and 610,000 deaths (accounting for 21.1% of the total), becoming the biggest threat ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68C40B50/06
CPCC12N15/1093C12Q1/6886C12Q2600/156C12Q2600/16C40B50/06
Inventor 江媛魏汉敏刘鹏娟黎美燕程云阳赵霞章文蔚拉多杰·德马纳克
Owner MGI TECH CO LTD
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