Kit for detecting DNA mismatch repair systems and application thereof

A technology of mismatch repair and kit, applied in the field of DNA mismatch repair system, can solve the problems of discrepancy in results, high price, inaccurate diagnosis, etc., to reduce human subjective judgment and standardization, high sensitivity and accuracy, and improve The effect of detection efficiency

Pending Publication Date: 2017-03-15
PULUOMAIGE BIOLOGICAL PRODS SHANGHAI
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  • Description
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AI Technical Summary

Problems solved by technology

[0009] At present, the methods used to detect MSI at home and abroad mainly include: (1) Immunohistochemical method (IHC), which mainly detects MLH1, MSH2, MSH6, and PMS2. This method cannot detect the biological activity of MMR protein, so false positives may occur As a result, the results

Method used

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  • Kit for detecting DNA mismatch repair systems and application thereof
  • Kit for detecting DNA mismatch repair systems and application thereof
  • Kit for detecting DNA mismatch repair systems and application thereof

Examples

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experiment example 1

[0039] Experimental example 1 PCR amplification reaction

[0040] 1. PCR reaction system

[0041] Table 1: Reaction specific components

[0042]

[0043] The dNTPs in Table 1 above include dATP, dGTP, dTTP and dCTP, and the use concentration of dATP, dGTP, dTTP and dCTP is 1 mM.

[0044] The primer pair mixture used refers to a mixture containing the microsatellite site of the present invention and its upstream primer and downstream primer, and the final concentration of each primer pair (upstream primer and downstream primer) is 0.1-1 μM. As shown in Table 2, the specific components of the two mixtures.

[0045] Table 2: Specific components of the two mixtures

[0046]

[0047]

[0048] In the above table 2, the 5'end fluorescent label JOE is 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein, FL is Fluorescein, ET-TMR is Energy Transfer-carboxy-tetramethylrhodamine, " / "Means no fluorescent labeling, and the 5'end is hydroxyl. Among them, the microsatellite sites Penta C and Penta ...

experiment example 2

[0054] Experimental example 2 Capillary electrophoresis experiment

[0055] Take the amplified PCR product, add internal standard (Promega company, internal molecular standard ILS500) and formamide, dry bath at 95 ℃ for 3 minutes, and ice water bath for 3 minutes. Load the sample on the ABI 3500Dx instrument, open the 3500Dx data collection software, edit the sample template, import the fragment analysis program, and click Run. Specific steps are as follows:

[0056] (1) Prepare the sample for loading: 1μL of the obtained PCR product, add 10μL of formamide, and 1μL of DNA molecular standard internal standard ILS500;

[0057] (2) Capillary electrophoresis: Set the capillary electrophoresis program according to the ABI3500Dx instrument manual, as follows:

[0058] A. Prepare the instrument; B. Preheat the heating furnace to 60°C; C. Prepare the sample reaction plate and load it into the instrument; D. Check the instrument status; E. Create or import a reaction plate; F. Assign the cont...

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Abstract

The invention discloses a kit for detecting DNA mismatch repair systems and application thereof; the kit comprises an amplifying system which comprises PCR (polymerase chain reaction) primer mixed liquid and PCR reaction liquid; the PCR primer mixed liquid contains microsatellite sites. The microsatellite sites in the kit are higher in sensitivity and accuracy and are suitable for detecting different cancers, such as colorectal cancer, endometrial cancer, gastric cancer, pancreatic cancer, lung cancer and breast cancer. The kit with microsatellite site for detecting DNA mismatch repair systems is better in repeatability, is suitable for detecting multiple microsatellite sites at the same time, and has improved detection efficiency.

Description

Technical field [0001] The invention belongs to the field of DNA mismatch repair systems, and specifically relates to a kit for detecting DNA mismatch repair systems and uses thereof. Background technique [0002] The human genome contains short tandem DNA repetitive sequences, also called microsatellites. These sequences are prone to replication errors due to their repetitive nature. Under normal circumstances, these errors can be carried out by the DNA mismatch repair (MMR) system Repair and correction, DNA MMR system functional defects will cause microsatellite instability (MSI), MSI is a sign of Lynch syndrome (Lynch syndrome), occurs in 90% of Lynch syndrome. [0003] Microsatellite instability is manifested in that the same microsatellite site is between different individuals and between the normal tissue and some abnormal tissues of the same individual, and the number of repetitive units of the microsatellite site is different. A large number of studies in recent years have...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q1/6886C12Q2600/156C12Q2600/166C12Q2563/107C12Q2545/101C12Q2565/125
Inventor 杰夫瑞·W·巴彻张晓丽段建涛
Owner PULUOMAIGE BIOLOGICAL PRODS SHANGHAI
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