Primers, reagent kit and method for detecting IDH1 (isocitrate dehydrogenase 1) R132H gene variation by aid of ddPCR (droplet digital polymerase chain reaction) technologies
A technology for technical detection and gene variation, applied in the medical and biological fields, can solve the problems of difficult to detect specific types and sequences of cell-free DNA, blood-derived DNA interference, etc., and achieve the effect of guiding clinical treatment, reducing pain, and simple operation.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0043] In this example, primers suitable for the quantitative detection of R132H gene variation on the ddPCR technology platform were designed for the upstream and downstream of the target region of the human IDH1 R132H gene; the primers were designed using the Taqman probe method, and the corresponding primers were designed for the IDH1 R132H mutant and wild-type sequences Mutant and wild-type probes.
[0044] The upstream and downstream primers for amplifying the IDH1 R132H mutant gene and amplifying the wild gene:
[0045] Upstream primer SEQ NO1: 5'-ccaacatgacttacttgatcccc-3'
[0046] Downstream primer SEQ NO2: 5'-aatatcccccggcttgtgag-3'
[0047] The IDH1 R132H mutation gene detection probe includes FAM fluorescent group, mutation site binding sequence, and MGB quenching group from the 5' end to the 3' end. This probe binds to the gene mutation site during annealing ;
[0048] SEQ NO3: 5'-FAM-taagcatgatgacctatgat-MGB-3'
[0049] The wild-type detection probe includes a...
Embodiment 2
[0052] Example 2: Detection of IDH1 R132H Gene Variation in Whole Blood Samples
[0053] 1. Prepare the samples to be tested: DNA containing wild-type human IDH1 R132H gene, DNA of human IDH1 R132H mutant gene, and wild-type mutant DNA mixed samples (wherein the content ratio of mutant and wild type is 1 / 100, 1 / 100, respectively. 1000, 1 / 10000). The DNA is derived from human peripheral blood, wherein the DNA template of the human IDH1R132H mutant gene is derived from an IDH1 R132H gene mutant cell line (identified by PCR sequencing).
[0054] 2. Extraction of cfDNA: cfDNA was extracted using a kit, and cfDNA was extracted referring to the instructions of the QIAamp Circulating Nuleacid Kit kit from QIAGEN.
[0055] 3. Configure the PCR master mix
[0056] 20 μL of PCR master mix includes: 10 μL of 2× digital PCR master mix (Biorad, #1863010), 500 nM upstream primers for amplifying IDH1R132H mutant gene and wild gene, downstream primers for amplifying IDH1 R132H mutant gene a...
PUM
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com