Prime group, probe group and kit for detecting Kras gene mutation
A detection kit and the technology of the kit are applied in the fields of biochemical equipment and methods, recombinant DNA technology, and the determination/inspection of microorganisms, which can solve the problems of expensive instruments, low popularization and utilization, and achieve objective interpretation of results and flexible application. , the effect of high detection sensitivity
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Embodiment 1
[0071] The design of embodiment 1 primer and probe
[0072] 1. Primer design: The 7 common mutation types of Kras gene are shown in Table 1.
[0073] Table 1 Seven common mutation types of Kras gene
[0074] Mutation type marker in the present invention
mutation name
amino acid changes
base change
34T
12CYS
G12C
c.34G>T
34A
12SER
G12S
c.34G>A
34C
12ARG
G12R
c.34G>C
35T
12VAL
G12V
c.35G>T
35A
12ASP
G12D
c.35G>A
35C
12ALA
G12A
c.35G>C
38A
13ASP
G13D
c.38G>A
[0075] In the present invention, among the primers for detecting the above seven kinds of mutations, the same reverse primers are shared, and specific forward primers are used respectively.
[0076] (1) The common reverse primer is:
[0077] Kras-Rev-Rp: GGCCTGCTGAAAATGACTG
[0078] (2) A plurality of forward primers were respectively designed for 7 different mutat...
Embodiment 2
[0094] Further optimization of embodiment 2 primers and probes
[0095] 1. Forward primer optimization
[0096] (1) The primers were further optimized using the above PCR reaction conditions.
[0097] The PCR templates are the plasmid templates of the mutant type and the wild type respectively, and the specific preparation process is as follows: refer to the reference sequences of the wild type of the Kras gene and 7 common mutant types queried in the COSMIC database, and synthesize the wild type and the wild type in sequence according to the reference sequences. Eight kinds of artificial nucleic acid sequences containing mutation sites are connected to plasmid vectors, and after screening, plasmids loaded with wild-type and seven kinds of mutant sequences are obtained. The concentration of the plasmid template is on the order of 10,000 copies. The results are shown in Table 6.
[0098] Table 6 Primer optimization results
[0099]
[0100]
[0101]
[0102] Note: "-...
Embodiment 3
[0113] The assembly of embodiment 3Kras gene mutation detection kit
[0114] 1. According to the above research results, the present invention has successfully assembled a kit for detecting Kras gene mutation with high sensitivity and high accuracy, including the following components:
[0115] (1) Primers: Forward primers are shown in Table 8:
[0116] Table 8
[0117]
[0118] Reverse primer (SEQ ID NO.8): Kras-Rev-Rp: GGCCTGCTGAAAATGACTG.
[0119] (2) probe, as shown in table 9:
[0120] Table 9
[0121]
[0122]
[0123] (3) dNTPs required for the PCR reaction, preferably 10 mM dNTPs.
[0124] (4) Enzyme; preferably Taq hot start enzyme (DNA polymerase); its concentration is preferably 5 U / μl.
[0125] (5) Reaction buffer, preferably 5*enzyme buffer.
[0126] (6)Mg 2+ , preferably 25mM Mg 2+ .
[0127] (7) Nuclease-free water.
[0128] 2. The PCR reaction system and reaction procedures when using the above kit are shown in Table 4 and Table 5 in Example 1...
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