Real-time fluorescence PCR detection kit and detection method for DAB2IP genes

A detection kit and real-time fluorescence technology, which is applied in the direction of fluorescence/phosphorescence, microbial measurement/inspection, biochemical equipment and methods, etc., can solve difficult measurement problems, achieve improved sensitivity, high sensitivity, and reduce false positive interference Effect

Inactive Publication Date: 2011-04-06
ZHEJIANG UNIV
View PDF3 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the research on DAB2IP is in the ascendant, and the methods for conventional detection of DAB2IP expression are still mainly based on immunohistochemistry and semi-quantitative PCR. The former is difficult to detect in peripheral blood, and the latter can only play a qualitative role.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Real-time fluorescence PCR detection kit and detection method for DAB2IP genes
  • Real-time fluorescence PCR detection kit and detection method for DAB2IP genes
  • Real-time fluorescence PCR detection kit and detection method for DAB2IP genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Specific primers, fluorescent probes

[0045] 1. Materials:

[0046] RNA extraction reagents, reverse transcription reagents, and PCR reaction reagents were all purchased from Dalian Bao Bioengineering Co., Ltd.; Taq DNA polymerase was purchased from Promega Company of the United States, and MX3000P quantitative PCR instrument was a product of Agilent-Stratagene Company of the United States.

[0047] 2. Primer and probe design and synthesis:

[0048] Using the DAB2IP gene sequence (registration number NM 032552.2) as a template, use PrimerExpress TM (V2.0, American ABI Company) software analyzes TaqMan primer and probe site, selects the optimal combination therefrom.

[0049] The sequences of PCR primers and probes for detection are as follows:

[0050] Upstream primer: 5′-TGTGAAGTGGACCCAAGCAA-3′

[0051] Downstream primer: 5′-ACGCAGTAGGAGTTGATGATTTTG-3′

[0052] Fluorescent probe: 5′-FAM-CCCGAGCACCAGGGCAACCTC-TAMRA-3′

[0053] Among them, FAM is a fluo...

Embodiment 2

[0055] Embodiment 2: Fluorescent quantitative PCR method detects DAB2IP gene

[0056] 1. Clinical sample testing:

[0057] 5 cases of clinical tumor tissue specimens, the total RNA was extracted with RNA extraction reagent RNAi Plus (Dalian Bao Biology, D19108A), and 1.0ug was taken for reverse transcription reaction, 2×RT buffer, 10.0μL; dNTP (10mM), 1μL; Oligo dT (10uM), 1μL; RNA inhibitor, 0.5μL; RTase, 1μL; total RNA 1μg, made up to 20μL with water. Reverse transcription conditions: 30°C, 10 minutes; 42°C, 30 minutes; 95°C, 5 minutes, take 1ul cDNA as a template, and perform PCR amplification on an Agilent-Stratagene MX3000P quantitative PCR instrument with upstream and downstream primers for detection.

[0058] The composition of the PCR reaction solution is as follows:

[0059] 2×PCR buffer 10.0μL

[0060] Detection upstream primer (10μM) 1μL

[0061] Downstream primer for detection (10μM) 1μL

[0062] Fluorescent probe for detection (10μM) 0.5μL

[0063] DNA polym...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention provides a real-time fluorescence PCR detection kit for DAB2IP genes, which mainly comprises a specific primer, a fluorescence probe, PCR buffer solution, a deoxynucleotide triphosphate mixture and DNA polymerase, wherein in the sequences of the specific primer and the fluorescence probe, the upstream primer is 5'-TGTGAAGTGGACCCAAGCAA-3', the downstream primer is 5'-ACGCAGTAGGAGTTGATGATTTTG-3' and the fluorescence probe is 5'-FAM-CCCGAGCACCAGGGCAACCTC-TAMRA-3', wherein the FAM is a fluorescence report group, and the TAMRA is a fluorescence quenching group. The kit has the main advantages that: a method of the invention has high detection sensitivity of DAB21IP genes and good specificity, and reduces the false positive rate of conventional PCR amplification; and the kit can realize quick, accurate and specific detection and analysis of the DAB2IP genes, also can be added with a standard substance for quantitative detection, and can be added with housekeeping genes (such as beta-actin genes and the like) for relative quantitative detection at the same time.

Description

(1) Technical field [0001] The invention relates to a real-time fluorescent PCR detection kit of DAB2IP gene and a detection method thereof. (2) Background technology [0002] Adaptive and self-accelerating growth is the only way for the occurrence and development of malignant tumors. As an important expression network regulator, DAB2IP belongs to the RAS-GTPase activating protein family and has the activity of inhibiting tumor cell proliferation. pathway to inhibit tumor growth and metastasis. DAB2IP can attenuate the expression of oncogenes such as H-RAS, R-RAS and TC21, and is associated with TNF-a-mediated tumor cell apoptosis. As a target gene of EZH2, DAB2IP may be involved in EZH2-involved tumor epigenetic silencing regulation. In addition, DAB2IP can mediate the inactivation of the PI3K-Akt pathway, which is an important tumor cell signaling pathway that regulates tumor proliferation, survival and metastasis. The latest study found that DAB2IP can also block tumor...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 方维佳林才照徐农
Owner ZHEJIANG UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap