Primer, probes, PCR reaction liquid and kit for human EGFR (epidermal growth factor receptor) gene T790M site detection
A reaction solution and kit technology, applied in the field of molecular biology, can solve problems such as poor sensitivity, and achieve the effects of reducing strength, increasing specificity and sensitivity, and improving stability and affinity
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Embodiment 2
[0047] Example 2 Probe
[0048] A kind of embodiment of the probe of the human EGFR gene T790M site detection of the present invention, the probe described in this embodiment consists of the mutant probe (combined with the mutant DNA template) and the wild type probe for the human EGFR gene T790M site. Needle (binding to wild-type DNA template) composition. The mutant probe is Mut probe1: 5'-FAM-AGCTGC+ATGA+TGAGC-MGB-NFQ-3' ("+N" indicates that the base is modified by LNA) (as shown in SEQ ID NO:3) ; The wild-type probe is Wt probe1: 5'-VIC-AGCTGC+GTGATGAGC-MGB-NFQ-3' ("+N" indicates that the base is modified by LNA) (as shown in SEQ ID NO: 4). That is: the 5' end of the mutant probe is marked with FAM, the 3' end is marked with a non-fluorescence quencher group NFQ, and at the same time it is connected with a MGB modification group, and the 7th and 11th bases of the mutant probe are carried out. Locked nucleic acid modification; the 5' end of the wild-type probe is marked w...
Embodiment 3
[0050] Embodiment 3PCR reaction solution and kit
[0051] An embodiment of the PCR reaction solution for detecting the T790M site of the human EGFR gene of the present invention, the PCR reaction solution described in this embodiment is shown in Table 1. Among them, the PCR master mix is MicroDrop from Guangdong Shunde Yongnuo Biotechnology Co., Ltd. TM The PCR premix Master Mix provided with the digital PCR instrument; the forward primer and reverse primer are as shown in Example 1; the mutant probe and wild-type probe are as shown in Example 2.
[0052] Table 1
[0053] Master Mix
10ul
Forward primer (20μm / L)
1.8μl
Reverse primer (20μm / L)
1.8μl
Mutant probe (10μm / L)
0.5μl
Wild type probe (10μm / L)
0.5μl
wxya 2 o
3.4μl
total capacity
18μl
[0054] During the experiment, the PCR reaction solution in Table 1 was mixed with 2 μl of DNA template to obtain a 20 μl PCR-specific amplification rea...
Embodiment 4
[0057] In this embodiment, the PCR reaction solution / kit described in Example 3 is used to detect the T790M site of the human EGFR gene. The specific detection method is:
[0058] (1) Genomic DNA extraction: Use Qiagen DNeasy Blood & Tissue Kit to extract normal peripheral blood genomic DNA and H1975 cell line DNA according to the kit instructions. The obtained genomic DNA was detected by NanoDrop 100 for DNA purity, gel electrophoresis for DNA integrity, qubit3.0 fluorometer for quantification, and DNA concentration ranged from 20 ng / μl to 50 ng / μl.
[0059] (2) The preparation of the PCR reaction solution for EGFR gene T790M mutation droplet digital PCR detection is as follows: PCR master mix 10 μl, 20 μm / L upstream primer 1.8 μl, 20 μm / L downstream primer 1.8 μl, 10 μm / L mutant probe 0.5μl, 10μm / L wild-type probe 0.5μl, ddH 2 O 3.4 μl, a total of 18 μl for one reaction.
[0060] (3) Preparation of PCR amplification system for EGFR gene T790M mutation droplet digital PCR d...
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