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Method for simultaneously measuring gene polymorphism of tacrolimus action targets of person

A technology of gene polymorphism and tacrolimus, which is applied in the field of detection of protein-coding gene polymorphisms of target signaling pathway series, can solve the problems of time-consuming, labor-intensive, high cost, and inapplicability for large-scale sample detection, etc.

Inactive Publication Date: 2016-01-13
AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, the detection methods of gene polymorphism mainly include DNA direct sequencing method and Taqman probe method, etc., said DNA direct sequencing method is currently the gold standard for mutation detection, but it has the defects of time-consuming, laborious and expensive, and is a kind of A low-throughput detection method is not suitable for a large number of samples; the Taqman is a highly specific quantitative PCR technology, which is characterized in that it is suitable for the detection of a small number of SNP sites in large samples, and is a medium-throughput SNP detection. Simultaneous detection of multiple SNP sites is expensive and not suitable

Method used

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  • Method for simultaneously measuring gene polymorphism of tacrolimus action targets of person
  • Method for simultaneously measuring gene polymorphism of tacrolimus action targets of person
  • Method for simultaneously measuring gene polymorphism of tacrolimus action targets of person

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Example 1 Simultaneous determination of human tacrolimus action target gene polymorphism

[0094] Follow the steps below:

[0095] 1) Genomic DNA extraction from human whole blood,

[0096] Extract genomic DNA from whole blood with a DNA extraction kit, and adjust the concentration to 10-100ng / ul;

[0097] 2) Multiplex PCR reaction

[0098] (1) Find the target gene sequence, design and synthesize PCR primers for the mutation site,

[0099] (2) Prepare a 384-well reaction table based on the extracted samples, and indicate the number of the DNA sample corresponding to each well, the primers used,

[0100] (3) According to the table, add 1 μL of DNA template to each well of the 384-well plate, stick to the membrane, and centrifuge at 2000 rpm / 10 seconds for later use.

[0101] (4) Prepare the PCR reaction solution according to the following table: (take 384 samples as an example)

[0102]

[0103] Note: The above 384-well reaction solution has 4% excess,

[0104] ...

Embodiment 2

[0133] Example 2: Using time-of-flight mass spectrometry to detect the genotypes of 35 SNP sites in FKBP1A, PPP3CB, and NFATC1 genes in 10 kidney transplant patients

[0134] 1. Genomic DNA extraction from human whole blood

[0135] Extract genomic DNA from whole blood with a DNA extraction kit, and adjust the concentration to 10-100ng / ul.

[0136] 2. Multiplex PCR reaction

[0137] 1) Find the target gene sequence, design and synthesize PCR primers for the mutation site

[0138] At the same time, 35 SNP sites in FKBP1A, PPP3CB, and NFATC1 genes were determined, and the designed primers are shown in Table 1.

[0139] Primers used for genotype detection of 35 SNP sites in table 1FKBP1A, PPP3CB, NFATC1 genes and single-base extensions produced

[0140]

[0141]

[0142] Note: W1, W2, W3, and W4 respectively represent 4 multiplex PCR reaction systems;

[0143] 2) Prepare a 384-well reaction table based on the extracted samples, and indicate the number of the DNA sample ...

Embodiment 3

[0177] Example 3: Detecting the genotypes of a total of 48 SNP sites in the FKBP1B, PPP3R1, and NFATC2 genes of 10 kidney transplant patients using time-of-flight mass spectrometry

[0178] 1. Genomic DNA extraction from human whole blood

[0179] Extract genomic DNA from whole blood with a DNA extraction kit, and adjust the concentration to 10-100ng / ul.

[0180] 2. Multiplex PCR reaction

[0181] 1) Find the target gene sequence, design and synthesize PCR primers for the mutation site,

[0182] Simultaneous determination of 3 genes, 48 ​​SNP sites, and designed primers (as shown in Table 3);

[0183] 2) Prepare a 384-well reaction table based on the extracted samples, and indicate the number of the DNA sample corresponding to each well and the primers used;

[0184] 3) According to the table, add 1 μL of DNA template to each well of the 384-well plate, stick to the film, and centrifuge at 2000 rpm / 10 seconds for later use;

[0185] 4) Prepare the PCR reaction solution acc...

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Abstract

The invention belongs to the technical field of molecular biology and medical examination, and relates to a matrix-assisted laser desorption ionization flight time mass spectrum method for simultaneously measuring signal channel series protein coding gene polymorphism of tacrolimus action targets of a person. The method comprises the following steps of performing multiple PCR (polymerase chain reaction) amplification after performing DNA (deoxyribonucleic acid) extraction on a sample; and performing genetic typing analysis on the sample by using a flight time mass spectrum method after single nucleotide primer extension. The method is easy and convenient to operate, a sequence specificity probe is not required, multiple detection on a plurality of samples can be implemented, 40 reactions can be realized by each system, 40 SNP (single nucleotide polymorphism) sites can be detected simultaneously, throughput is high, application range is wide, and tens of thousands of samples and dozens of to hundreds of sites can be detected simultaneously; fluorescent markers are not required, only common primers require to be synthesized, and analysis cost of a single sample is low; and flexibility is high, the amount of samples required for analysis is small, and sensitivity and detection precision are high. The method is suitable for detecting clinic conventional genes, and further provides a technical support for clinic individual rational drug use.

Description

technical field [0001] The invention belongs to the field of molecular biology technology and medical examination, and relates to a method for determining the polymorphism of the target gene of tacrolimus, in particular to a method for detecting the polymorphism of the protein coding gene of the signal pathway series of the target of tacrolimus . Background technique [0002] According to research reports, the therapeutic window of tacrolimus is narrow, and there are significant individual differences in the curative effect. At present, clinically, therapeutic drug monitoring (TDM), that is, monitoring blood drug concentration, is usually used to adjust the dosage of tacrolimus. dose. Practice has shown that traditional TDM is difficult to reflect the variation among individuals from the perspective of pharmacodynamics, and TDM can only be implemented after taking the medicine, it is impossible to predict the body's response to the drug before taking the medicine, and the i...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6888C12Q2600/106C12Q2600/156
Inventor 邱晓燕徐勤霞焦正钟明康
Owner AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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