Reagent and method for detecting KRAS mutation

The technology of a reagent and a kit is applied in the field of reagents for detecting KRAS mutation

Active Publication Date: 2012-11-28
山东国九堂制药集团股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, clinical trials have shown that these targeted drugs only have significant curative effects on some tumor patients

Method used

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  • Reagent and method for detecting KRAS mutation
  • Reagent and method for detecting KRAS mutation
  • Reagent and method for detecting KRAS mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1. Design of primers

[0057] The primers were designed according to the mutation sites of KRAS codons 12, 13, and 61. In view of the location of each mutation site in the gene structure and the characteristics of the mutation, the sequence length and position of the primers are all considered as factors. After repeated comparison and optimization, the inventors obtained primers with the following sequences:

[0058] For KRAS codon 12, the primer sequence is:

[0059] Positive:

[0060] Wild type WF1: 5'-cttgtggtagttggagctg (SEQ ID NO: 1);

[0061] Wild type WF2: 5'-cttgtggtagttggagctgg (SEQ ID NO: 2);

[0062] Mutant MF1: 5'-cttgtggtagttggagct(a / c / t) (SEQ ID NO: 3 / 4 / 5);

[0063] Mutant MF2: 5'-cttgtggtagttggagctg(a / c / t) (SEQ ID NO: 6 / 7 / 8);

[0064] Reverse:

[0065] R1: 5'-tgcacagagagtgaacatc (SEQ ID NO: 9).

[0066] Note: The above MF1 and MF2 are mixed primers, that is, a / c / t is generated at the end base at the same time during synthesis.

[0067] For KRAS codon 13, the pr...

Embodiment 2

[0086] Example 2. Detection of primer combinations

[0087] The detection was performed by the primer combinations listed in Table 1.

[0088] Table 1

[0089]

[0090]

[0091] The PCR reaction system is shown in Table 2.

[0092] Table 2

[0093] Reagent

Final concentration

Volume 20μl

1) Milli Q water

8.875μl

2) 10×Taq buffer (containing Mg 2+ )

1×

2μl

3) 2.5mM dNTP mix

0.25mM each

2μl

4) Forward primer (10μM)

0.5μM

1μl

5) Reverse primer (10μM)

0.5μM

1μl

6) Template DNA

5-10ng

5μl

7) Taq DNA polymerase (5U / ul)

0.0725u / μl

0.125μl

[0094] Except for Taq DNA polymerase, all the above reagents are thawed on ice and operated on crushed ice. Finally, Taq DNA polymerase is taken out from -20°C and added to the reaction system. The above template DNA is mutation positive, mutation negative and tested genomic DNA.

[0095] The reaction conditions are shown in Table 3.

[0096] table 3

[0097]

[0098] The above reaction was performed on the ABI 9700 PCR mach...

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Abstract

The invention relates to a reagent and a method for detecting KRAS mutation. The invention discloses a primer which is particularly suitable for amplifying and identifying the gene mutations at codon 12, 13 and 61 positions of KRAS, wherein the primer is obtained through reasonable design and preference, good in specificity when being used for PCR (Polymerase Chain Reaction) amplification, high in amplification efficiency, capable of rapidly identifying the mutations of different basic groups of a same codon and capable of rapidly identifying the mutations of different codons of a same gene.

Description

Technical field [0001] The present invention belongs to the field of biotechnology; more specifically, the present invention relates to reagents and methods for detecting KRAS mutations. Background technique [0002] At present, targeted therapy has become an important means of clinical treatment of tumors. Epidermal growth factor receptor (EGFR) is the main target of targeted therapy. EGFR targeted drugs include EGFR monoclonal antibody drugs cetuximab, nimotuzumab, panitumumab, and EGFR tyrosine kinase inhibitors gefitinib and erlotinib. These drugs can block the signal transduction of tumor cells by inhibiting the epidermal growth factor receptor tyrosine kinase that is necessary for tumor occurrence and development, thereby inhibiting tumor cell proliferation, invasion, metastasis, angiogenesis and promoting tumor cell apoptosis . However, clinical trials have shown that these targeted drugs only have significant effects on some cancer patients. Further studies have found...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 王明
Owner 山东国九堂制药集团股份有限公司
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