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Lung cancer or colorectal cancer mutant gene detection primer composition and application thereof

A technology for colorectal cancer and mutated genes, applied in the field of genetic engineering, can solve problems such as inconvenience, and achieve the effects of reducing costs, removing background noise, and convenient operation

Pending Publication Date: 2020-12-04
PILLAR BIOSCI INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the differences in the principle, reagents, and sequence and length of Barcode and UID fragments of each sequencer manufacturer, the library construction reagent (hybridization capture probe library / multiple PCR primer pool) used on a sequencing platform is designed. ) are generally not suitable for use on other platforms, causing some inconvenience

Method used

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  • Lung cancer or colorectal cancer mutant gene detection primer composition and application thereof
  • Lung cancer or colorectal cancer mutant gene detection primer composition and application thereof
  • Lung cancer or colorectal cancer mutant gene detection primer composition and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The verification method adopted in the multiple PCR library construction and sequencing process of the present invention is as follows:

[0035] 1. Sample Preparation

[0036] 1.1 Sample extraction

[0037] Quantitative Multiplex Reference Standard, a commercial reference product of Horizon Company, and clinical samples from hospitals were extracted and quantified by Qubit.

[0038] 1.2 Sample quality control

[0039] For the clinical samples from the hospital, the Human Genomic DNA Quantification and QC Kit (Product No.: KK4960) from KAPA Company was used to analyze the degree of fragmentation of the samples; Bio-Rad Digital PCRAssays was used to detect the mutation frequency of some gene hotspots in the samples.

[0040] 1.3 Sample settings

[0041] 1.3.1 Reference products

[0042] Take 20ng, 5ng, and 2.5ng of the Horizon reference substance for detection respectively.

[0043] 1.3.2 Clinical samples

[0044] The clinical samples tested are shown in Table 1, an...

Embodiment 2

[0101] 1. Sample Preparation

[0102] Extract Horizon's commercial reference product Quantitative Multiplex Reference Standard, quantify it with Qubit, and take 20ng for detection. Horizon company's commercial reference product is another Tru-Q 7 (1.3% Tier) ReferenceStandard 20ng detection.

[0103] 2. Library Construction

[0104] 2.1 The first round of gene-specific PCR

[0105] See Example 1.

[0106] 2.2 Purification of gene-specific PCR products

[0107] See Example 1.

[0108] 2.3 The second round of adapter PCR

[0109] 2.3.1 Prepare the adapter PCR reaction solution as shown in Table 8 below.

[0110] Table 8

[0111]

[0112]

[0113] 2.3.2 Put the cap on the tube, vortex to mix, and then centrifuge quickly.

[0114] 2.3.3 Run the PCR reaction according to the program in Table 9 below.

[0115] Table 9

[0116]

[0117] 2.4 Library purification

[0118] See Example 1.

[0119] 3 On-machine sequencing

[0120] 3.1 Use the MGI Easy cyclization kit (...

Embodiment 3

[0133] 1. Sample Preparation

[0134] Three cases of plasma clinical samples were extracted from newly diagnosed patients with lung adenocarcinoma, and 20 ng DNA were collected for library construction and detection.

[0135] 2. Library Construction

[0136] 2.1 The first round of gene-specific PCR

[0137] 2.1.1 Prepare the gene-specific PCR reaction solution as shown in Table 12, and add the corresponding DNA sample to each tube.

[0138] Table 12

[0139]

[0140]

[0141] 2.1.2 Cover the tube cap, shake and mix well, and then centrifuge quickly.

[0142] 2.1.3 Run the PCR reaction according to the program in Table 13 below.

[0143] Table 13

[0144]

[0145] 2.1 Gene-specific PCR product purification

[0146] See Example 1.

[0147] 2.2 The second round of adapter PCR

[0148] 2.2.1 Prepare the adapter PCR reaction solution as shown in the table below.

[0149] Table 14

[0150]

[0151] 2.3.2 Put the cap on the tube, vortex to mix, and then centrifu...

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PUM

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Abstract

The invention provides a lung cancer or colorectal cancer mutant gene detection primer composition. The composition comprises 103 pairs of primers or a combination containing a part of the primers, and the forward primer sequence and the reverse primer sequence of each pair of primers comprise an amplification primer pair and other sequences connected to the 5' end of the amplification primer pair. Specific sequences in a forward primer and a reverse primer of each pair of primers are selected from sequences shown in SEQ ID NO.1-206, and preferably, the specific sequences are 18-25 base sequences in the sequences. According to the composition, 22 mutation hot spot regions of lung cancer or colorectal cancer related genes are carefully selected, a low-initial-quantity or low-quality FFPE sample can be detected, a ctDNA sample can also be detected, and general requirements of early screening, diagnosis, medication and prognosis of lung cancer and colorectal cancer are met.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a primer composition for detecting mutation genes of lung cancer or colorectal cancer and its application. Background technique [0002] Next-generation sequencing technology (Next-Generation Sequencing, NGS), also known as next-generation sequencing technology, is a high-throughput detection technology that can sequence hundreds of thousands or even hundreds of millions of DNA molecules at a time. It is used in prenatal detection, tumor mutation analysis and medication guidance, pathogenic microorganism screening and other fields. The Illumina platform is now the most commonly used NGS sequencer in China, with a relatively high market share. The Ion Torrent platform and the domestic MGI platform also have their own unique advantages, and the domestic application ratio is also growing steadily. [0003] Before NGS sequencing, it is usually necessary to enrich and am...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/118C12Q2600/156C12Q2600/16
Inventor 宋钢王朝晖
Owner PILLAR BIOSCI INC
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