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LNA probe for detecting KRAS gene mutation and detection method

A detection method and probe technology, applied in the field of KRAS gene mutation detection, can solve the problems of time-consuming, high background of DNA sequencing, false positives or false negatives, etc., and achieve simple operation, high single-base resolution, Specificity-enhancing effect

Pending Publication Date: 2017-01-11
LINYI UNIVERSITY
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Problems solved by technology

[0007] In order to solve the above problems in the prior art that the detection of point mutations by PCR is prone to false positive or false negative results, DNA sequencing method has a high background and takes a long time, this application discloses a LNA probe for detecting KRAS gene mutations

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  • LNA probe for detecting KRAS gene mutation and detection method
  • LNA probe for detecting KRAS gene mutation and detection method
  • LNA probe for detecting KRAS gene mutation and detection method

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Embodiment 1

[0037] Embodiment 1: the establishment of detection method

[0038] 1. Design corresponding LNA probes for the three mutant sequences of the 12 and 13 codon mutations of the KRAS gene.

[0039] The KRAS mutant gene sequence is as follows:

[0040] name Sequence (5'-3') Types of 135A GTT GGA GCT GAT GGC GTA G dna 135C GTT GGA GCT GCT GGC GTA G dna 134T GTT GGA GCT TGT GGC GTA G dna

[0041] The probe sequences are as follows:

[0042] name Sequence (5'-3') Types of position in the sequence listing LNA-P 135A

ACG CCA TCA GCT C LNA sequence 1 LNA-P 135C

ACG CCA GCA GCT C LNA sequence 2 LNA-P 134T

ACG CCA CAA GCT C LNA sequence 3

[0043] 2. The LNA probes were hybridized with the corresponding three kinds of KRAS mutant genes, and three kinds of hybrids were obtained.

[0044]The LNA probe and the KRAS mutant gene were respectively made into 100 μM stock solution with ult...

Embodiment 2

[0050] Embodiment 2: Utilize LNA probe to detect KRAS mutant gene

[0051] The three kinds of probes were hybridized with the corresponding KRAS mutant genes respectively, and the three kinds of hybrids obtained (135A / LNA-P 135A , 135C / LNA-P 135C and 134T / LNA-P 134T ) were detected by nanopore sensors, respectively. As a control, the probes were hybridized with the mixture of the other two mutated genes, and detected with nanopore sensors under the same conditions (voltage: +120mV, electrolyte solution: 1M KCl (pH=7.8, 10mM Tris-HCl, 1mM EDTA)) , the result is as figure 1 shown. LNA-P 135A Hybrid with mutant gene 135A (135A / LNA-P 135A ) was injected into the detection pool, a long time blocking event was generated ( figure 1 a), the fitting value of blocking time is 452.17ms, while the probe LNA-P 135A After the mixture with the other two mutant genes was detected by the nanopore, the blocking signal was particularly short, and the blocking time was 23.55ms ( figure 1...

Embodiment 3

[0052] Example 3: Quantitative analysis of the KRAS mutant gene

[0053] Taking the KRAS mutant gene 135A as an example, different concentrations of 135A and equal concentrations of the probe LNA-P 135A After hybridization, the final concentrations after injection into the detection pool were 5nM, 25nM, 50nM, 100nM, and 500nM, and the frequency of blocking events was counted, and the relationship between the frequency and the concentration of the mutant gene 135A was established, so as to perform quantitative analysis on 135A. The result is as figure 2 as shown in a. The quantitative analysis results of 135C and 134T are shown in figure 2 b and figure 2 c. The three mutant genes showed a good linear relationship between the detection concentration of 5nM-500nM.

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Abstract

The invention relates to the technical field of gene detection, in particular to an LNA (Locked Nucleic Acid) probe, which is represented by sequences 1, 2, and 3 in a sequence table, for detecting KRAS gene mutation. A detection method for the KRAS gene mutation comprises the following steps: constructing a nanopore sensor by natural alpha-hemolysin, hybridizing the LNA probe with a gene to be detected to obtain a hybrid material, and generating a blocking signal through a nano channel of the nanopore sensor. By the hybridization of the LNA-modified probe and a KRAS mutant gene and detection of the alpha-hemolysin nanopore sensor, the specificity of target gene detection is greatly improved, and a concentration relation between the frequency of the signal and the KRAS mutant gene can be constructed to quantitatively analyze a target; the LNA probe has the advantages of simplicity, quickness and high sensitivity; DNA amplification is not needed; the operation is simple and specific; the LNA probe is high in applicability.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to an LNA probe for detecting KRAS gene mutation, and also relates to a detection method for KRAS gene mutation using the LNA probe. Background technique [0002] Malignant tumors are major diseases that seriously threaten human health and life. According to statistics from the World Health Organization, in recent years, one out of every eight deaths in the world has been caused by cancer, which is higher than the total number of deaths caused by AIDS, tuberculosis and malaria. It is reported that according to the current medical level, the 5-year survival rate of early cancer patients can exceed 80%, while the 5-year survival rate of advanced cancer patients after treatment is less than 10%. Therefore, early detection and early diagnosis of cancer are of great significance to the prevention and treatment of cancer. [0003] At present, the clinical diagnosis methods of can...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6827C12Q2565/607C12Q2565/631C12Q2525/101
Inventor 郗冬梅张书圣时鹏飞尚积祯李志
Owner LINYI UNIVERSITY
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