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Library for performing high-throughput detection on circulating tumor DNA target genes, as well as detection method and application of library

A DNA-targeting and gene-targeting technology, applied in the field of high-throughput genomics sequencing, can solve problems such as low throughput, and achieve the effect of increasing detection efficiency and reducing detection costs

Inactive Publication Date: 2017-12-08
DALIAN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] ctDNA detection technology: Digital PCR is often used in the research of ctDNA. It is characterized by high sensitivity, but it can only target certain SNP sites. Personalized probes must be designed for each patient, and the throughput of simultaneous detection is relatively low. Low

Method used

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  • Library for performing high-throughput detection on circulating tumor DNA target genes, as well as detection method and application of library
  • Library for performing high-throughput detection on circulating tumor DNA target genes, as well as detection method and application of library
  • Library for performing high-throughput detection on circulating tumor DNA target genes, as well as detection method and application of library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] 1) Take the blood of stage IIIB advanced breast cancer as the biological sample, take 10ml; the blood collection tube is EDTA anticoagulant tube, heparin anticoagulant tube cannot be used, the blood is separated from the serum within 2 hours, the first centrifugal force is 3000rpm, 5min, and the serum is taken , when taking the serum for the first time, the buffy layer should not be taken by operation; the second time the centrifugal force is 14000rpm, 10min, when the plasma is taken for the second time, the bottom residue should not be taken by the operation. Extract free nucleic acid from the collected serum in time, or store it at minus 80°C. use The Circulating Nucleic Acid Kit free nucleic acid extraction kit extracts serum free nucleic acid, and the concentration of the extracted free nucleic acid is accurately quantified by Qubit 2.0.

[0045] 2) All exons of 50 targeted genes in circulating tumor DNA were captured by multiplex PCR amplification. Primers are d...

Embodiment 2

[0107] Take the pleural effusion organisms of advanced lung cancer as samples, a total of 96 samples, each sample is 10ml, the collection tube is EDTA anticoagulant tube, heparin anticoagulant tube cannot be used, pleural effusion is separated within 2 hours, the first centrifugal force is 3000rpm, 5min, Take the supernatant; the second centrifugal force is 14000rpm, 10min. When taking the supernatant for the second time, the operation should not get the bottom residue. use The Circulating Nucleic Acid Kit free nucleic acid extraction kit extracts serum free nucleic acid, and the concentration of the extracted free nucleic acid is accurately quantified by Qubit 2.0.

[0108] All exons of 50 targeted genes in circulating tumor DNA are captured by multiplex PCR amplification. The multiplex PCR reaction system is: DNA template 5ul; mixed primer (10uM) 10ul; 10X HiFi reaction solution 5ul; 2.5mM dNTPs 2.5ul ; HiFi DNA polymerase 0.5ul; deionized water 27ul; a total of 50ul react...

Embodiment 3

[0162] Example 3 Construction of high-throughput sequencing library for blood samples of tumor patients

[0163] Take advanced lung cancer blood organisms as samples, a total of 96 samples, each sample 10ml, the collection tube is EDTA anticoagulant tube, heparin anticoagulant tube cannot be used, the pleural effusion is separated within 2 hours, the first centrifugal force is 3000rpm, 5min, take it up Clear; the second centrifugal force 14000rpm, 10min, when taking the supernatant for the second time, the operation should not get the bottom residue. use The Circulating Nucleic Acid Kit free nucleic acid extraction kit extracts serum free nucleic acid, and the concentration of the extracted free nucleic acid is accurately quantified by Qubit 2.0.

[0164] All exons of 50 targeted genes in circulating tumor DNA were captured by multiplex PCR amplification. The multiplex PCR reaction system was:

[0165] DNA template 5ul; mixed primer (10uM) 10ul; 10X HiFi reaction solution 5...

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Abstract

The invention relates to the field of genomic high-throughput sequencing, in particular to a library for performing high-throughput detection on circulating tumor DNA target genes, as well as a detection method and application of the library. Circulating body fluid of a tumor patient is used as a biological sample, low-concentration free nucleic acid is extracted, the low-concentration free nucleic acid of the biological sample is used as a sample template, target gene exons of the sample template are amplified in a multiplex PCR form, then the tail ends of obtained amplicons are repaired and the 3' ends of the amplicons are connected with A basic groups, the modified amplicons are connected to Illumina sequencing adapters, and then library PCR is carried out by using an index-containing primer, and an amplified product is the library for detecting mutation diversity of the circulating tumor DNA target genes. The in-vitro circulating body fluid of the tumor patient is used as the biological sample for achieving non-invasive detection; the low-concentration free nucleic acid is used as the template for highly-sensitive detection; multiple target gene exons are amplified in a multiplex PCR form at the same time so as to improve the detection efficiency; via establishment of an amplicon library, high-throughput sequencing and data analysis, the detection cost can be greatly reduced.

Description

technical field [0001] The invention relates to the field of genomics high-throughput sequencing, in particular to a circulating tumor DNA targeted gene high-throughput detection library and its detection method and application. Background technique [0002] Circulating nucleic acid is a kind of extracellular DNA in a cell-free state, which exists in body fluids such as blood, synovial fluid, and cerebrospinal fluid. It is mainly composed of single-stranded or double-stranded DNA and a mixture of single-stranded and double-stranded DNA. Composition, exists in two forms of DNA protein complex or free DNA. [0003] Circulating tumor DNA (ctDNA) refers to the somatic DNA of tumor cells that is shed or released into the circulatory system after apoptosis. Circulating tumor DNA fragments, ctDNA is mainly fragmented genomic DNA released after the rupture of dead tumor cells. In theory, all tumors produce ctDNA. The genetic variation information that can be detected in ctDNA is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B40/08C40B50/06C12N15/10C12Q1/68
CPCC12N15/1093C12Q1/6806C40B40/08C40B50/06C12Q2531/113C12Q2537/143C12Q2525/191
Inventor 李志广马兆奎刘强陈骏吕德康陈志盛张霞张宇
Owner DALIAN MEDICAL UNIVERSITY
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