Fluorescence detection kit and fluorescence detection method for deletion mutation of gene

A fluorescence detection and gene deletion technology, applied in the field of chemical and biological sensing, can solve the problems of high requirements on experimental conditions, limited wide application, unsuitable for on-site use, etc., and achieves the effects of high sensitivity, simple operation and high specificity

Active Publication Date: 2018-12-07
CIXI INST OF BIOMEDICAL ENG NINGBO INST OF MATERIALS TECH & ENG CHINESE ACAD OF SCI +1
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  • Abstract
  • Description
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Problems solved by technology

qRT-PCR technology can realize the amplification of several DNA molecules, and has high sensitivity and specificity. However, the main defect is that the reaction process requires precise temperature control and high experimental conditions, which is not suitable for on-site use; the second generation gene Sequencing technology requires specialized and expensive equipment for data acquisition and analysis, which limits its wide application

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  • Fluorescence detection kit and fluorescence detection method for deletion mutation of gene
  • Fluorescence detection kit and fluorescence detection method for deletion mutation of gene
  • Fluorescence detection kit and fluorescence detection method for deletion mutation of gene

Examples

Experimental program
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Effect test

Embodiment 1

[0053] Design and synthesis of PNA capture probes, DNA probes, padlock probes, fluorescent probes and primers for rolling circle amplification

[0054] Select the 10-25 nucleotide sequence near the 3' end near the EGFR gene deletion site as the object, and design a PNA sequence that is completely complementary to it as a PNA capture probe, and the carboxyl terminal (CONH 2 ) is complementary to the 5' terminal base of the EGFR deletion mutant gene, and the amino terminal (NH 2 ) complementary base pairing with the 3' end of the EGFR deletion mutant gene, introducing 1-5 mPEGs at the carboxyl end to increase water solubility, using MBHA resin to condense one by one according to the base sequence under the conditions of HBTU and DIEA, after cleavage, purification and structural characterization For subsequent detection; DNA probes, padlock probes and rolling circle amplification primers were synthesized by Shanghai Jieli Biotechnology Co., Ltd. The detailed base sequences are sh...

Embodiment 2

[0058] Fluorescence detection of EGFR deletion mutation gene

[0059] (1) Immobilization of PNA capture probes on 96-well plate

[0060] Add 1 μL of 100 μM PNA capture probe and 100 μL NaHCO to each well 3 Fixative solution, the final concentration of PNA capture probe is 1.0μM, incubate with shaking (600-700rpm) at room temperature for 2 hours, then add 50μL of lysine shielding solution, continue shaking at room temperature (600-700rpm) for 0.5 hours, use 300μL 1× Wash four times with PBST, wash four times with 250 μL 1×PBS, store 250 μL 1×PBS for later use, remove 250 μL 1×PBS when in use, add 200 μL BLBs blocking solution, shake at room temperature (600-700 rpm) for 0.5 hours, and then remove the blocking solution , directly used in the next step of detection;

[0061] (2) Capture of EGFR deletion mutant gene

[0062] Add EGFR deletion mutant gene and 1×PBS buffer to 100 μL system to configure 9 concentration gradients (0fM, 1fM, 10fM, 100fM, 1pM, 10pM, 100pM, 1nM, 10nM)...

Embodiment 3

[0074] Negative control for fluorescence detection of EGFR deletion mutation gene

[0075] (1) Immobilization of PNA capture probes on 96-well plate

[0076] Add 1 μL of 100 μM PNA capture probe and 99 μL NaHCO to each well 3 Fixative solution, the final concentration of PNA capture probe is 1.0μM, incubate with shaking (600-700rpm) at room temperature for 2 hours, then add 50μL of lysine shielding solution, continue shaking at room temperature (600-700rpm) for 0.5 hours, use 300μL 1× Wash four times with PBST, wash four times with 250 μL 1×PBS, store 250 μL 1×PBS for later use, remove 250 μL 1×PBS when in use, add 200 μL BLBs blocking solution, shake at room temperature (600-700 rpm) for 0.5 hours, and then remove the blocking solution , directly used in the next step of detection;

[0077] (2) Capture of EGFR gene

[0078] The experiment was divided into 5 groups in a 96-well plate: a, b, c, d, e, wherein 1 μL of 1 μM EGFR deletion mutant gene was added to group a, c and ...

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Abstract

The invention provides a fluorescence detection kit for a deletion mutation gene. The kit includes: a PNA capture probe, a DNA probe, a padlock probe, a DNA polymerase, a DNA ligase, a rolling circleamplification primer, and a fluorescence probe. The invention also provides a method of using the kit to perform the fluorescence detection on the deletion mutation gene, wherein the method includes:1) immobilizing the PNA capture probe on the bottom of a pore plate, and hybridizing the probe with a target gene with a buffer solution; 2) hybridizing the target gene immobilized on the bottom of the pore plate with the DNA probe; 3) hybridizing a single chain section on the DNA probe, which is hybridized with the target gene, with the padlock probe, cyclizing the hybridized product with the DNAligase, and performing rolling circle amplification under effect of the rolling circle amplification primer and the DNA polymerase; 4) hybridizing the fluorescence probe with a rolling circle amplification product, quenching background fluorescence, and detecting the change on fluorescence intensity.

Description

technical field [0001] The application relates to a fluorescent detection kit and a fluorescent detection method for gene deletion mutations, which belong to the technical field of chemical and biological sensing. Background technique [0002] Gene sequence deletion mutation is the change of gene structure caused by the deletion of base pairs in DNA molecules. Gene deletion in exon 21 induces lung cancer, especially non-small cell lung cancer. With the occurrence and development of diseases, the deletion of mutant genes in human diseased tissues or blood has become an important physiological indicator for rapid and accurate diagnosis of clinical diseases. At present, qRT-PCR (quantitative reverse transcription polymerase chain reaction) and second-generation gene sequencing technology are the main detection methods for deletion mutant genes. qRT-PCR technology can realize the amplification of several DNA molecules, and has high sensitivity and specificity. However, the mai...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858
CPCC12Q1/6858C12Q2531/125C12Q2525/107C12Q2521/501C12Q2563/107
Inventor 徐小军赵超邢淑付盼徐梦佳徐皖星
Owner CIXI INST OF BIOMEDICAL ENG NINGBO INST OF MATERIALS TECH & ENG CHINESE ACAD OF SCI
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