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Compound amplification system and kit for MSI (microsatellite instability) gene mutation detection

A compound amplification system and unstable technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve problems such as difficult product development, and improve detection accuracy and sensitivity , the effect of preventing erroneous results

Pending Publication Date: 2019-02-15
GUANGZHOU FULENGEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0016] Finding quasi-monomorphic sites with high sensitivity and specificity for detecting MSI is a necessary work for establishing an MSI detection system, which has become a hot spot in the field of MSI detection in recent years, but the development of this product is not easy. So far, only Promega and several other companies have products capable of detecting single-nucleotide microsatellite gene loci. It is imperative to develop MSI detection systems with more types of loci to improve sensitivity and accuracy.

Method used

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  • Compound amplification system and kit for MSI (microsatellite instability) gene mutation detection
  • Compound amplification system and kit for MSI (microsatellite instability) gene mutation detection
  • Compound amplification system and kit for MSI (microsatellite instability) gene mutation detection

Examples

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Embodiment 1

[0093] In this embodiment, the multiple amplification system of the present invention is used to detect the microsatellite instability of colon cancer tissue and normal tissue. Colon cancer tissue and normal tissue are the first batch of samples. The detection steps are as follows:

[0094] 1. DNA extraction

[0095] Use the tissue DNA extraction kit (OMEGA) to extract genomic DNA from the patient's cancer tissue and paracancerous tissue (normal tissue) respectively, and follow the kit instructions. After the genomic DNA is extracted, use an ultraviolet spectrophotometer to quantify it and dilute the final concentration. 1ng / μl for the next step of the PCR reaction template.

[0096] 2.PCR

[0097] 2.1 Multiplex PCR Mix (5x) 2μl, MSI site primer mix (5x) 2μl, nuclease-free deionized water 4μl,

[0098] The amount of template added (0.5ng / μl-5ng / μl) was 2μl, and the total reaction volume was 10μl.

[0099]

[0100]

[0101] 2.2PCR reaction procedure:

[0102] Step 1: ...

Embodiment 2

[0116] In this example, the multiple amplification system of the present invention is used to detect the microsatellite instability of colon cancer tissue and normal tissue, and the colon cancer tissue and normal tissue are the second batch of samples, and the detection steps are the same as in Example 1.

[0117] See the result figure 2 , is the typing map obtained by detecting colon cancer tissue and normal tissue using the multiple amplification system of the present invention in Example 2 of the present invention. The results show that, using the composite amplification system of the present invention to detect microsatellite instability, there are 2 allelic locus shifts (shown by red arrows) in the 8 loci of colon cancer fine tissue, indicating that the colon cancer Tissue produces microsatellite instability (MSI), and the offset of these two sites may be misdiagnosed because it cannot be detected in the detection of 5 positions, indicating that the composite amplificati...

Embodiment 3

[0119] In this example, immunohistochemistry and the composite amplification system of the present invention are used to detect paracancerous tissues and cancer tissues respectively. The composite amplification system of the present invention is used to detect microsatellite instability in colon cancer tissues and normal tissues. The tissues are the third batch of samples. In this embodiment, the detection steps using the multiple amplification system of the present invention are the same as those in Embodiment 1.

[0120] See the result Figure 3 to Figure 5 , image 3 It shows that by immunohistochemical detection, MLH1, PMS2, MSH2 and MSH6 are positive in paracancerous tissues, MLH1 and PMS2 in cancerous tissues are negative, and MSH2 and MSH6 in cancerous tissues are positive; Figure 4 and Figure 5 It shows that microsatellite instability detection is carried out by using the complex amplification system of the present invention, and the allele locus shift (shown by th...

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Abstract

The invention belongs to the field of biotechnology and particularly relates to a compound amplification system and a kit for MSI (microsatellite instability) gene mutation detection. The compound amplification system for MSI gene mutation detection is provided and amplifies mononucleotide microsatellite gene sites and dinucleotide microsatellite gene sites simultaneously; the mononucleotide microsatellite gene sites comprise NR-21, NR-22, NR-24, NR-27, BAT-25, BAT-26 and MONO-27; the dinucleotide microsatellite gene sites comprise one or more of D2S123, D17S250, D18S34 and D5S346. The mononucleotide microsatellite sites and the dinucleotide microsatellite sites are combined for detection and analysis of MSI, the detection accuracy and sensitivity can be effectively improved, and the system can be used for quickly detecting typing of tumor to guide treatment.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a compound amplification system and a kit for detecting microsatellite instability gene mutations. This application claims the rights and interests of the Chinese patent (patent application number 201810737101.5) submitted on July 06, 2018, and the entire content of the above application is hereby incorporated by reference. Background technique [0002] A large number of studies in recent years have shown that tumorigenesis is closely related to the mismatch repair system, especially in colorectal cancer, gastric cancer, and endometrial cancer. Due to the mutation and abnormal function of the mismatch repair (MMR) gene, the replication errors of the genome cannot be repaired in time, and the frequency of replication errors continues to accumulate, thereby changing the DNA sequence of the cell genome, or causing some tumor suppressor genes to fail. Live or oncogene activa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6886C12Q2600/156C12Q2537/143C12Q2563/107C12Q2525/151
Inventor 徐学明粟龙稳黄连成常宏赏
Owner GUANGZHOU FULENGEN
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