A kit and method for constructing DNA nanospheres in one step

A nanosphere and kit technology, applied in the field of high-throughput sequencing, can solve the problems of many processes and long time, and achieve the effect of saving operation time, saving operation steps, and improving the efficiency of circularization

Active Publication Date: 2021-10-29
南京实践医学检验有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In view of the urgent need for timeliness in clinical practice, it is still necessary to improve the existing technology to improve the problems of many existing processes and long time

Method used

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  • A kit and method for constructing DNA nanospheres in one step
  • A kit and method for constructing DNA nanospheres in one step
  • A kit and method for constructing DNA nanospheres in one step

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Using a batch of actual samples submitted for clinical metagenomic sequencing (mNGS) as experimental materials, first extract the nucleic acid (DNA and / or RNA) of the samples, then build a library, and finally construct a DNB that can be used for sequencing on the MGI platform.

[0053] The specific experimental steps are as follows:

[0054] 1. Sample nucleic acid extraction: The DNA extraction kits used in this example are the Microbial Genomic DNA Extraction Kit (Cat. No. PMD101) from Nanjing Practice Medical Laboratory Co., Ltd., and the Microbial Genomic DNA Extraction Kit from Sputum / Nasal Secretion Samples. (Cat. No. PMD102) and Nasopharyngeal Swab / Anal Swab Sample Microorganism Genomic DNA Extraction Kit (Cat. No. PMD103); Pharyngeal swab / anal swab sample microbial RNA extraction kit (Cat. No. PMD203).

[0055] 1.1 The specific experimental process of DNA extraction is as follows:

[0056] (1) Sample processing:

[0057] A. Blood: Centrifuge at 2100rpm for 5m...

Embodiment 2

[0208] Sample: Another batch of actual samples submitted for metagenomic sequencing (mNGS) for clinical examination.

[0209] The difference from Example 1 is that the DNB preparation buffer components in the DNB preparation process are 80mM potassium acetate, 20mM magnesium acetate, 50mM Tris-acetic acid, 0.3mg / mlBSA, 10mMDTT, 5μM oligonucleotide 1, 5μM oligonucleotide Nucleotide 2, 1 μM oligonucleotide 3, 5 μM oligonucleotide 4;

[0210] DNB polymerase buffer components are 10mM potassium acetate, 20mM magnesium acetate, 40mM Tris-acetic acid, 0.3mg / mlBSA, 5mMDTT, 5mMATP and 5mMdNTP;

[0211] The working concentration of each component of the DNB polymerase mixture is 1U / μl Phi29DNAPolymerase, 0.5U / μl T4DNALigase, 50ng / μl pyrophosphatase, 0.5U / mlT4Gene32Protein;

[0212] EDTA concentration in DNB stop buffer is 500mM;

[0213] sample number sample type nucleic acid type Nucleic acid concentration ng / μl Library concentration ng / μl total library ng 1 ...

Embodiment 3

[0219] Sample: Another batch of actual samples submitted for metagenomic sequencing (mNGS) for clinical examination.

[0220] Different from Example 1 and Example 2, the DNB preparation buffer components in the DNB preparation process are 60mM potassium acetate, 15mM magnesium acetate, 30mM Tris-acetic acid, 0.2mg / mlBSA, 4mMDTT, 2.5μM oligonucleotide 1 , 2 μM oligonucleotide 2, 0.03 μM oligonucleotide 3, 2 μM oligonucleotide 4;

[0221] DNB polymerase buffer components are 5mM potassium acetate, 10mM magnesium acetate, 20mM Tris-acetic acid, 0.2mg / mlBSA, 4mMDTT, 2mMATP and 2mMdNTP;

[0222] The working concentration of each component of the DNB polymerase mixture is 0.2U / μl Phi29DNAPolymerase, 0.3U / μl T4DNALigase, 10ng / μl pyrophosphatase, 0.2U / mlT4Gene32Protein and 0.2U / mlE.coliSSB.

[0223] The concentration of EDTA in DNB stop buffer is 250mM;

[0224] sample number sample type nucleic acid type Nucleic acid concentration ng / μl Library concentration ng / μl...

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Abstract

The invention discloses a kit and method for constructing DNA nanospheres in one step. The kit includes R1, R2 and R3, R1 includes DNB preparation buffer, R2 includes DNB polymerase buffer, DNB polymerase mixed solution, R3 Includes DNB stop buffer. The method comprises the steps of mixing the dsDNA library with the DNB preparation buffer, denaturing at high temperature and annealing at low temperature; mixing the annealed product with the DNB polymerase buffer and the DNB polymerase mixture, and reacting at low temperature; and terminating the reaction with the DNB termination buffer. The kit and method creatively put single-strand circularization and rolling circle amplification into one step for reaction, without enzyme digestion and purification in the middle, greatly saving operation steps and time.

Description

technical field [0001] The invention belongs to the technical field of high-throughput sequencing, and in particular relates to a kit and a method for constructing DNA nanospheres in one step. Background technique [0002] The rapid development of NGS technology in recent years, because it does not depend on known nucleic acid sequences and does not require special probe design, can directly detect unknown pathogenic microorganisms, breaking the limitations of traditional microbial testing and showing broad prospects in the field of clinical microbiology . [0003] At present, the incidence of infectious diseases in the world has increased, and pathogenic microorganisms are showing a trend of diversification and complexity. Severe acute respiratory syndrome (SARS), novel coronavirus pneumonia, new mutant Creutzfeldt-Jakob disease, H7N9 avian influenza and other new infectious diseases continue to emerge. The pathogenic microorganisms of classic infectious diseases such as ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6869
CPCC12Q1/6869C12Q2531/125C12Q2563/155C12Q2522/101C12Q2521/501C12Q2521/101C12Q2521/525C12Q2535/122
Inventor 谢珍曾志鹏张鹏邢宽
Owner 南京实践医学检验有限公司
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