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Microsatellite instability detection method

A technology of microsatellite instability and microsatellite sites, applied in the field of molecular diagnosis, can solve problems such as low sensitivity and accuracy, difficult detection, cumbersome operation, etc., achieve high sensitivity and accuracy, improve reliability, and improve The effect of precision

Inactive Publication Date: 2017-12-15
生工生物工程(上海)股份有限公司
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Problems solved by technology

However, both of the above detection techniques have their own drawbacks.
Polyacrylamide gel electrophoresis technology has many defects such as cumbersome operation, long time-consuming, low throughput and low sensitivity.
The direct sequencing method has the disadvantages of high price, high experimental conditions, and low throughput.
In addition, the sequence copy number of microsatellite loci to be detected in the genome is low, making it difficult to be detected directly
In addition, according to the two mononucleotide repeat units and three dinucleotide repeat units of the five microsatellite sites to be tested, the change in the length of the microsatellite is relatively small, and the change in the length of the microsatellite is basically less than 25bp, while the traditional method based on The sensitivity and accuracy of the MSI detection method of the PCR method are relatively low, and the detection of five sites requires multiple PCR reactions and subsequent corresponding experimental procedures to complete the detection of one sample

Method used

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  • Microsatellite instability detection method
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Examples

Experimental program
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Embodiment 1

[0058] In this example, the method for detecting colorectal cancer microsatellite instability using the constructed wild-type plasmid and mutant plasmid is described in this example, as follows.

[0059] 1 Wild-type plasmid construction

[0060] Using human blood sample genomic DNA as a template, non-fluorescence-labeled primers (the first primer shown in SEQ ID NO. Pair, the second primer pair shown in SEQ ID NO.3-4, the third primer pair shown in SEQ ID NO.5-6, the fourth primer pair shown in SEQ ID NO.7-8, SEQ ID NO The fifth primer pair shown in .9-10 and the sixth primer pair shown in SEQ ID NO.11-12) carry out PCR amplification respectively, and amplified product adopts PMD18-T vector Cloning Kit (numbering: 6011, TaKaRa) Carry out TA cloning, then pick the positive clones identified by colony PCR as correct for expansion, plasmid extraction, and the extracted positive plasmids are sent to the sequencing department of Sangon Bioengineering (Shanghai) Co., Ltd. for seque...

Embodiment 2

[0132] In this embodiment, paraffin-embedded tissue sections from 5 patients with colorectal cancer are used as samples to be tested, and the method for detecting microsatellite instability of colorectal cancer provided in this embodiment is described, as follows.

[0133] The numbers of paraffin-embedded tissue sections of five colorectal cancer patients were: SA-1, SA-2, SA-3, SA-4, SA-5, and the MSI analysis system v1.2 of Promega Company was used as a control. And operate in strict accordance with its instructions.

[0134] 1 DNA extraction

[0135] Paraffin-embedded tissue DNA rapid extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) was used to extract genomic DNA from paraffin-embedded paraffin-embedded tissue sections of colorectal cancer and paracancerous tissues of five patients. According to the instructions, the extracted genomic DNA was quantified by a micro-spectrophotometer, and a part was drawn and diluted to 5 ng / μl.

[0136] 2MSI detection r...

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Abstract

The invention discloses a microsatellite instability detection method, and belongs to the field of molecular diagnosis. The method used for detecting colorectal cancer microsatellite instability is invented based on multiplex fluorescent PCR technology and capillary electrophoresis technology. According to the method, a first primer pair represented by SEQ ID NO.1-2, a second primer pair represented by SEQ ID NO.3-4, a third primer pair represented by SEQ ID NO.5-6, a fourth primer pair represented by SEQ ID NO.7-8, and a fifth primer pair represented by SEQ ID NO.9-10 are used for multiplex fluorescent PCR and detection of five microsatellite points BAT-25, BAT-26, D2S123, D5S346, and D17S250. The sensitivity and the precision are higher; detection of internal reference microsatellite point Penta C is introduced to eliminate foreign DNA pollution in samples to be detected, and the credibility of obtained results is increased.

Description

technical field [0001] The invention relates to the field of molecular diagnosis, in particular to a method for detecting microsatellite instability. Background technique [0002] Colorectal cancer (carcinoma of colon and rectum, CRC) is a common malignant tumor that seriously endangers human health. With the development of human society, people's lifestyle and living environment have changed, the pressure of life is increasing day by day, and the pollution of living environment is becoming more and more serious. rejuvenation trend. Hereditary colorectal cancer accounts for an important proportion in the colorectal cancer system. It has the characteristics of heredity, high incidence, multiple, and multi-organ tumorigenesis. Clinically, based on the presence or absence of multiple polyposis, hereditary colorectal cancer Cancer is divided into two categories: hereditary polyposis and hereditary nonpolyposis colorectal cancer (HNPCC). Both types of hereditary colorectal canc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 张云静李威张中娜
Owner 生工生物工程(上海)股份有限公司
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