Primer, probes and detection kit used for full RAS mutation detection

A mutation detection and probe technology, applied in the direction of DNA/RNA fragment, recombinant DNA technology, microbial determination/inspection, etc., can solve the problems of few detection sites and inability to meet clinical detection and other problems

Inactive Publication Date: 2015-07-08
张道允 +1
View PDF4 Cites 12 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the clinically available RAS detection products only include the detection of the 12th and 13th codon sites o

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
  • Primer, probes and detection kit used for full RAS mutation detection
  • Primer, probes and detection kit used for full RAS mutation detection
  • Primer, probes and detection kit used for full RAS mutation detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0236] Applying the system of the present invention to detect plasmids, plasmid templates for experiments (taking the KRAS p.12 / 13 wild-type plasmid as an example), the method for detecting the full RAS mutation using the above-mentioned fluorescent PCR detection is as follows:

[0237] (1) Treatment and extraction of plasmids:

[0238] The plasmid was extracted using the plasmid extraction kit of TIANGEN (HighPure Plasmid kid, DP116), and the specific extraction steps were performed according to the instructions of the kit. The extracted DNA was dissolved in Tris-HCl (10mM, pH 8.0), and the quality of the extraction was detected by a UV spectrophotometer to determine its concentration, and then the DNA concentration was adjusted to 20ng / ul with Tris-HCl (10mM, pH 8.0) solution as a diluted mother solution .

[0239] According to formula C 拷贝浓度 =(C 拷贝浓度 *6.02*10 23 ) / MW DNA Dilute wild-type plasmid to 10 6 copies / microliter.

[0240] Use the wild-type plasmid at the K34...

Embodiment 2

[0255] Using the present invention to detect clinical samples, detect 120 patients with metastatic colorectal cancer sent to our company for detection. Among them, there were 72 males and 48 females, aged 39-75 years, with an average age of 57 years and a median age of 54 years. The test results were compared with the traditional ARMS-QPCR method.

[0256] (1) Sample processing and DNA extraction

[0257] The FFPE DNA extraction kit of Kangwei Century was used to extract the DNA in the paraffin section tumor tissue, and the specific steps were as follows:

[0258] a Take about 4-5 slices and place them in a centrifuge tube, add 1ml of xylene, cover the tube tightly, and vortex for 10 seconds;

[0259] b After centrifuging at 12,00rpm for 2 minutes, discard the supernatant, then add 1ml of absolute ethanol, and vortex evenly. Centrifuge at 12,000rpm for 2 minutes, discard the supernatant;

[0260] c Open the cap of the tube and incubate at room temperature or up to 37 degree...

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 discloses a primer and probes used for full RAS mutation detection. The primer and probes comprise a full RAS specific primer and the probes, and the full RAS specific primer and the probes comprise twenty seven mutation types of KRAS 2,3,4 exons and twenty three mutation types of NRAS 2,3,4 exons. A detection kit used for full RAS mutation detection extracts a DNA sample and performs a PCR reaction. The specific MGB Blocker probe, the NRAS specific probe and the KRAS specific probe and the primer are adopted and can detect RAS mutation in DNA in tumor tissue; MGB Blocker is used for retarding nonspecific augmentation of NRAS and KRAS, and sensitivity is improved and higher than 1% of ARMS; RAS mutation can be detected in circular tumor DNA. The primer and the probes are easy to operate, low in detection cost, capable of being popularized in a large-scale mode and high in detection speed, and the detection process can be finished after about 2 hours.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a primer, a probe and a detection kit for detecting the full RAS mutation. Background technique [0002] The RAS gene family is a proto-oncogene, including KRAS, NRAS and HRAS. The KRAS gene is located on human chromosome 12 and plays an important role in the development of human tumors. KRAS is like a molecular switch: it can control and regulate the path of cell growth in normal conditions; when abnormalities such as KRAS gene mutations occur, the gene is permanently activated, disrupting intracellular signal transduction, causing cells to continue to grow, and preventing cell apoptosis from occurring cancerous. The mutation of KRAS gene occurs in the early stage of tumor, and the KRAS gene of primary tumor and metastases is highly consistent. It is generally believed that KRAS gene status will not change with treatment. The KRAS gene is mutated in a variety of tumors, and it...

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
IPC IPC(8): C12Q1/68C12N15/11
Inventor 张道允巩子英
Owner 张道允
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