Quality control product for detecting fragmented DNA (Deoxyribonucleic Acid) mutation and preparation method thereof

A technology of fragmentation and quality control products, which is applied in the field of clinical testing and can solve problems such as inability to evaluate the accuracy of external quality

Active Publication Date: 2017-12-15
GENOSABER BIOTECH SHANGHAI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Based on this, it is necessary to provide a method for preparing a quality control product for detecting fragmented DNA mutations in order to address the problem of inability to evaluate the accuracy of the inter-laboratory quality of gene mutation detection

Method used

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  • Quality control product for detecting fragmented DNA (Deoxyribonucleic Acid) mutation and preparation method thereof
  • Quality control product for detecting fragmented DNA (Deoxyribonucleic Acid) mutation and preparation method thereof
  • Quality control product for detecting fragmented DNA (Deoxyribonucleic Acid) mutation and preparation method thereof

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preparation example Construction

[0031] A method for preparing a quality control product for detecting fragmented DNA mutations, comprising the steps of:

[0032] S1: mutant DNA fragment;

[0033] Wherein, the specific steps for preparing mutant DNA fragments in S1 can be obtained in the following three ways;

[0034] Using the mutant genomic DNA as a template, the mutant DNA fragments are obtained through primer amplification.

[0035] Or use mutant genomic DNA as a template, amplify with primers to obtain mutant DNA fragment precursors, and connect the mutant DNA fragment precursors to plasmids to obtain mutant plasmids.

[0036] Amplify or digest with primers to obtain mutant DNA fragments.

[0037] Or use the DNA whose target gene locus is the wild-type locus as a template, and amplify with primers to obtain wild-type DNA fragments.

[0038] The wild-type DNA fragment was ligated with the plasmid to construct the wild-type plasmid.

[0039] Then use the wild-type plasmid as a template and amplify with...

Embodiment 1

[0066] Main reagents and materials

[0067] KOD Plus Mutagenesis Kit was purchased from Toyobo (Shanghai) Biotechnology Co., Ltd.

[0068] Primers used in PCR were purchased from Sangon Bioengineering (Shanghai) Co., Ltd.

[0069] Competent Escherichia coli DH5α, Universal DNA purification and recovery kit, and endotoxin-free plasmid mid-scale extraction kit were purchased from Tiangen Biochemical (Beijing) Co., Ltd.

[0070] The pMD18-T vector plasmid was purchased from Bao Biological Engineering (Dalian) Co., Ltd.

[0071] Tissue genome extraction reagent QIAamp DNA FFPE Tissue Kit and whole blood genome extraction reagent QIAampDNA blood Mini Kit were purchased from QIAGEN China (shanghai) Co., Ltd.

[0072] Plasma cell-free DNA extraction and purification kit (adsorption column method) and human EGFR gene mutation quantitative detection kit (fluorescent PCR method) were purchased from Gnosper Biotechnology Nantong Co., Ltd.

[0073] Ion AmpliSeq for library construction...

Embodiment 2

[0093] The T790M wild-type DNA of exon 20 of the human EGFR gene was extracted with a whole blood genome extraction kit (QIAamp DNA blood Mini Kit). The experimental method is basically the same as in Example 1, except that primers (SEQ ID NO: 17 to SEQ ID NO: 18 in Table 1) are used to amplify and amplify a wild-type DNA fragment with a length of 2000 bp to construct a wild-type plasmid . The primer sequences for site-directed mutagenesis are selected from SEQ ID NO:19 to SEQ ID NO:20 in Table 1; the primer sequences required for the construction of mutant DNA fragments are selected from SEQ ID NO:11 to SEQ ID NO:18 in Table 1.

[0094] The T790M mutant fragmented DNA sample of exon 20 of the human EGFR gene was mixed with the extracted healthy human plasma free DNA. Among them, the T790M mutant fragmented DNA sample of exon 20 of the human EGFR gene accounts for 0.1% of the wild-type DNA copy number in the extracted healthy human plasma free DNA. Get the quality control D1...

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Abstract

The invention discloses a quality control product for detecting fragmented DNA (Deoxyribonucleic Acid) mutation and a preparation method thereof. The preparation method comprises the following steps: taking mutant genome DNA or a constructed mutant plasmid as a template and carrying out primer amplification to obtain a mutant DNA fragment; randomly breaking the mutant DNA fragment to obtain a fragmented mutant DNA sample; mixing the fragmented mutant DNA sample with a fragmented wild genome DNA sample to prepare the quality control product. The method disclosed by the invention has the advantages that the mutant DNA fragment is randomly broken and fragment lengths, which are adaptive to the samples, can be obtained through breaking according to different detection samples. Furthermore, the method is simple to operate; compared with a synthesis method, enzyme digestion sites and protective basic groups do not need to be added at two ends of a sequence. Furthermore, according to the quality control product prepared by the method, the mutant DNA fragment is randomly broken and broken points are random; the prepared quality control product can be used for more really simulating a clinical sample.

Description

technical field [0001] The invention relates to the field of clinical testing, in particular to a quality control product for detecting fragmented DNA mutations and a preparation method thereof. Background technique [0002] At present, genetic testing technology has been widely used in individualized medicine. The emergence of high-throughput sequencing (NGS) technology has greatly reduced the cost of genetic testing. Sequencing results of the sequence. [0003] The basic principle of high-throughput sequencing is to break the DNA to be tested into small fragments, carry out library construction through steps such as end repair, linker sequence, and PCR, and finally use Illumina, Ion Torrent and other sequencers for sequencing. However, with the popularization and application of this technology, different laboratories are still blank in the application of NGS to the external quality evaluation of gene mutation detection. Therefore, it is urgent to investigate the analytic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/10
CPCC12Q1/6806C12Q1/6827C12Q2545/101
Inventor 杨国华林国旻王萍
Owner GENOSABER BIOTECH SHANGHAI
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