Fluorescent polarization based homogeneous phase detection method of single nucleotide polymorphism of codon118 of ERCC1 (excision repair cross-complementing 1) gene

A single nucleotide polymorphism, fluorescence polarization technology, used in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc.

Inactive Publication Date: 2012-09-19
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, there are many analysis methods for Codon118 single nucleotide polymorphism in codon 118 of ERCC1 gene, such as RFLP, allele-specific oligonucleotide hybridization, Oligonucleotide ligation analysis, allele-specific PCR, DNA sequencing, gene chip, TDI-FP and other technologies can be detected separately, but these methods involve steps such as amplification, digestion and purification, inactivation, hybridization extension and pipetting, etc. More electrophoresis and multi-step post-processing are required, which is time-consuming and labor-intensive, and the efficiency needs to be improved
Moreover, during the operation process, the reaction tube needs to be replaced, which is prone to contamination, which affects the accuracy of the test results.
Or the instruments and equipment are expensive, and it is difficult to promote and apply them on a large scale

Method used

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  • Fluorescent polarization based homogeneous phase detection method of single nucleotide polymorphism of codon118 of ERCC1 (excision repair cross-complementing 1) gene
  • Fluorescent polarization based homogeneous phase detection method of single nucleotide polymorphism of codon118 of ERCC1 (excision repair cross-complementing 1) gene
  • Fluorescent polarization based homogeneous phase detection method of single nucleotide polymorphism of codon118 of ERCC1 (excision repair cross-complementing 1) gene

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Effect test

Embodiment approach 1

[0034] Homogeneous detection of C / T single nucleotide polymorphisms at codon 118 of ERCC1 gene in peripheral blood of lung cancer patients predicts platinum resistance

[0035] 1), materials and methods

[0036] DNA was extracted from the peripheral blood of multiple healthy volunteers, and the ERCC1 gene was used to cover the Codon118 single nucleotide polymorphism. Universal primers: forward primer 5'-cagtccagaacactgggacatgac-3', reverse primer 5'-ggtcatccctattgatggcttctg-3', amplification, Amplified products were cloned into

[0037] pGEM-T-easy Vectors (Promega, USA) was used to construct standard control clones. Sequencing method was used to screen and identify the single nucleotide polymorphism of the control substance. Extract the plasmid pGEM-T-ERCC1-118T / T containing the T / T single nucleotide polymorphism on the 118th codon of the ERCC1 gene and the C / C single nucleotide polymorphism containing the 118th codon of the ERCC1 gene The specific pGEM-T-ERCC1-C / C was...

Embodiment approach 2

[0062] Homogeneous detection of C / T single nucleotide polymorphisms at codon 118 of ERCC1 gene in peripheral blood of gastric cancer patients predicts platinum resistance

[0063] 1), materials and methods

[0064] One milliliter of peripheral blood was extracted from patients D, E, and Ji respectively, and DNA was extracted from peripheral blood samples using TIANamp Genomic DNA Kit kits (Tiangen Biotech, Beijing). The extraction of genomic DNA was performed according to the instructions.

[0065] Homogeneous detection of ERCC1 gene Codon118 C single nucleotide polymorphism: Take positive and negative control standards and 5 μL of peripheral blood DNA templates of D, E, and E patients, respectively, and add 50 μL of reaction reagents for amplification: the reagents include a concentration of 50 nM The fluorescently labeled probe 5'FITC-caaattcccagggcacgttgcgcacg-3', other reagents, reaction conditions, and steps are the same as those in Embodiment 1.

[0066] Homogeneous det...

Embodiment approach 3

[0083] Biological research: Homogeneous detection of C / T single nucleotide polymorphisms on codon 118 of ERCC1 gene in normal human peripheral blood

[0084] 1), materials and methods

[0085] 1 ml of peripheral blood was drawn from blood donors 1 and 2, and DNA was extracted from peripheral blood samples using TIANamp Genomic DNA Kit kit (Tiangen Biotech, Beijing). Genomic DNA was extracted according to the instructions.

[0086] Homogeneous detection of ERCC1 gene Codon118 C single nucleotide polymorphism: Take positive and negative control standards and 5 μL of peripheral blood DNA templates of blood donors 1 and 2 respectively, add 50 μL of reaction reagents for amplification: the reagents include 50 nM The fluorescently labeled probe 5'ROX-caaattcccagggcacgttgcgcac-3', and other reagents, reaction conditions, and steps are the same as in Embodiment 1.

[0087] Homogeneous detection of ERCC1 gene Codon118 T single nucleotide polymorphism: 5 μL of positive and negative contr...

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Abstract

The invention relates to a fluorescent polarization based homogeneous phase detection method of a single nucleotide polymorphism of a codon118 of an ERCC1 (excision repair cross-complementing 1) gene. The homogeneous phase detection method solves the problems of high cost, complicated operating steps, proneness to causing contamination and influence on detection result accuracy in the prior art, and is simple in steps, easy to operate, not prone to causing contamination as operation can be completed once in a closed tube, and low in cost as any special reagent and fluorescence quenching or micro-groove binding agent are not needed. Application of a specific probe with single end marked to detection of the single nucleotide polymorphism of the codon118 of the ERCC1 gene results in changes of fluorescent polarization values and enables detection results to be more objective and accurate. Result analysis is simple as only numeric comparison is needed in result judgment, and the homogeneous phase detection method can easily realize standardization and automation, is wide in application range and can be used for detecting clinical blood or tissue specimens.

Description

Technical field: [0001] The present invention relates to the technical field of detection of Codon118 single nucleotide polymorphism at the 118th codon of ERCC1 gene Exon4, in particular to a homogeneous detection method, which monitors the presence of ERCC1 gene Exon4 in a sample by detecting the changed fluorescence polarization value Codon118 single nucleotide polymorphism in codon 118. Background technique : [0002] Single nucleotide polymorphism (SNP) refers to the presence of different bases at specific nucleic acid positions in the genome of normal individuals of a certain population, which is the difference in human DNA sequence, that is, when a single nucleotide in the genome sequence of the chromosome changes. Polymorphic changes in DNA sequences, SNPs are also known as biallelic markers. Among the 3 billion bases in the human genome, about one SNP occurs every 300 to 500 bases, and there are about 20 million SNPs in the entire human genome. SNP can occur in bo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 张菊姜英浩梁平王萌
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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