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Primer combination for detecting instability of microsatellite, kit, detection method and application of primer combination, kit and detection method

A primer composition and instability technology, which can be used in biochemical equipment and methods, recombinant DNA technology, and the determination/inspection of microorganisms, and can solve problems such as the impact of result analysis

Inactive Publication Date: 2019-09-10
深圳海普洛斯医学检验实验室
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the 5' ends of various upstream primers are modified with the same fluorescent group, which affects the analysis of the results

Method used

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  • Primer combination for detecting instability of microsatellite, kit, detection method and application of primer combination, kit and detection method
  • Primer combination for detecting instability of microsatellite, kit, detection method and application of primer combination, kit and detection method
  • Primer combination for detecting instability of microsatellite, kit, detection method and application of primer combination, kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Embodiment 1 Primer Design and the Preparation of Primer Combination Mixture

[0098] In this example, according to the standards recommended by the National Cancer Institute of the United States and the recommendations of the "Revised Bethesda Guidelines", six single nucleotide polymorphism sites NR-21, NR-24, NR-27, BAT-25, and MONO-27 were determined , BAT-26, and three control polymorphic sites Penta D, Penta E, and Amel, using Primer Premier 5.0 to design specific primers according to the GenBank sequence information of 9 sites, and artificially synthesized according to existing methods, Obtain the primer composition shown in SEQID NO:1~18;

[0099] The 9 pairs of synthesized primers were added to nucleic acid-free water and dissolved to 100 μM: taking NR-21 as an example, take 5 μL of forward primer, 5 μL of reverse primer and 40 μL of nucleic acid-free water to prepare NR-21 primer working solution with a concentration of 10 μM; The other 8 pairs of primers were...

Embodiment 2

[0103] Example 2 kit assembly

[0104] The kit of this embodiment contains 4 components: enzyme mixture, PCR reaction solution, dNTP and primer combination mixture containing 9 polymorphic sites, the specific components are shown in Table 2:

[0105] Table 2

[0106]

[0107]

Embodiment 3

[0108] Example 3 Sample Detection

[0109] In this embodiment, taking the paraffin-embedded tumor tissue of a patient with colorectal cancer (patient number HP001) as an example, the kit of Example 2 was used to detect the state of microsatellite instability in the tumor tissue, including the following steps:

[0110] (1) Extract the DNA of the paraffin-embedded tumor tissue sample of the patient HP001 and the white blood cell gDNA of the blood sample as a control sample, and use Qubit 2.0 to detect the sample concentration. The DNA concentration of the tissue sample is 20.4 ng / μL, and the white blood cell gDNA concentration is 56.4 ng / μL;

[0111] (2) Prepare a PCR reaction system according to Table 3, and add a prepared 47 μL PCR reaction system to each detection system;

[0112] table 3

[0113] components Volume / μL Enzyme Mix 1.05 PCR reaction solution 2.1 dNTPs 8.4 primer mix mix 5.25 nuclease free water 73.5

[0114] (3) Di...

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Abstract

The invention provides a primer combination for detecting the instability of a microsatellite, a kit, a detection method and application of the primer combination, the kit and the detection method. The primer combination comprises primer pairs shown in SEQ ID NO:1-18 for NR21, NR24, NR27, BAT25, BAT26, MONO-27, Penta D, Penta E and Amel. According to the primer combination, primers are designed for six SNP loci of NR21, NR24, NR27, BAT25, BAT26 and MONO-27; according to the states of the six SNP loci, the instability of the microsatellite is evaluated; according to three contrast polymorphismloci of Penta D, Penta E and Amel, the consistency of a to-be-detected tissue sample and a contrast sample thereof is evaluated; the accuracy is high, the number of used samples is low, the amplification efficiency is high, strips are clear, and the sensitivity is high.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and relates to a primer composition, a kit, a detection method and an application thereof for detecting microsatellite instability. Background technique [0002] Microsatellite (Microsatellite) refers to the tandem repeat sequence composed of short-chain DNA of 1 to 6 bases, which widely exists in the genome. Compared with normal tissue, the length of microsatellites in tumor tissue is changed due to the insertion or deletion of repetitive sequences, forming microsatellite instability (Microsatellite Instability, MSI). Studies have shown that MSI is caused by mutations or hypermethylation modifications of DNA mismatch repair (MMR) genes such as MLH1, MSH2, and MSH6, which cause errors in the replication of genetic DNA in cells. [0003] MSI is closely related to the occurrence of tumors, and MSI positive can be detected in various cancer types such as colorectal cancer, gastric cancer, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2600/16C12Q2537/143C12Q2531/113C12Q2565/125C12Q2563/107
Inventor 张晓妮屈宏越许明炎
Owner 深圳海普洛斯医学检验实验室
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