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Kit and method for constructing DNA nanospheres by one-step method

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

Active Publication Date: 2021-08-13
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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  • Kit and method for constructing DNA nanospheres by one-step method
  • Kit and method for constructing DNA nanospheres by one-step method
  • Kit and method for constructing DNA nanospheres by one-step method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] The actual sample of the clinical detection macro-based due group sequencing (MNGS) is an experimental material, first extracting the nucleic acid (DNA and / or RNA) of the sample, and then the establishment of the library, and finally constructs the DNB that can be used in the MGI platform.

[0053] The specific experimental steps are as follows:

[0054] 1. Sample nucleic acid extraction: DNA extraction kit used in this example is Nanjing Practice Medical Inspection Co., Ltd. Sample Microbial Genome DNA Extraction Kit (Item No. PMD101), sputum / nasal secretion sample microbial genomic DNA extraction kit (Item No. PMD102) and Nasopharyngeal Swipe / Anal Sweex Sample Microbiological Genome DNA Extraction Kit (Item No. PMD103); RNA Extraction Kit is Nanjing Practice Medical Inspection Co., Ltd. body fluid sample microbial RNA extraction kit (Item No. PMD201) and nose Throew Water / Anal Sweex Sample Microbial RNA Extraction Kit (Item No. PMD203).

[0055] 1.1DNA extraction ...

Embodiment 2

[0235] Sample: Another actual sample of the clinical detection macro base group sequencing (MNGS).

[0236] Unlike the examples, the DNB prepared buffer components during DNB preparation were divided into 80 mM potassium acetate, magnesium acetate, 50 mmtris-acetic acid, 0.3 mg / mlbsa, 10 mmdtt, 5 μm oligonucleotide 1,5 μM oligomer Nucleotide 2,1 μm oligonucleotide 3,5 μm oligonucleotide 4;

[0237] The DNB polymerase buffer is divided into 10 mM potassium acetate, magnesium acetate, 40 mmtris-acetic acid, 0.3 mg / mlbsa, 5mmdt, 5 mmatp and 5 mmdNTP;

[0238] DNB polymerase mixed solutions have a concentration of 1 U / μL29DnApolymerase, 0.5u / μlt4 dnaligase, 50 ng / μL pyrophosphate, 0.5u / mlt4gene32Protein;

[0239] The concentration of EDTA in DNB terminating buffer is 500 mm;

[0240] The rest are the same as the embodiment, and each sample information is as follows:

[0241] Sample number Sample type Nucleic acid type Nucleic acid concentration Ng / μL Library...

Embodiment 3

[0249] Sample: Another actual sample of the clinical detection macro base group sequencing (MNGS).

[0250]In fact, the DNB prepared buffer components in the DNB preparation process are 60 mM potassium acetate, magnesium 15 mM, 30 mmtris-acetic acid, 0.2 mg / mlbsa, 4mmdtt, 2.5 μm oligonucleotide 1 2,0.03 μm oligonucleotide 3, 2 μm oligonucleotide 4;

[0251] The DNB polymerase buffer is divided into 5 mM potassium acetate, 10 mM magnesium acetate, 20 mmtris-acetic acid, 0.2 mg / mlbsa, 4mmdt, 2mmatp and 2 mmdNTP;

[0252] The components of the DNB polymerase mixture were 0.2u / μlphi29dnapolymerase, 0.3u / μlt4 dnaligase, 10 ng / μL pyrophosphate, 0.2u / mlt4gene32Protein and 0.2u / mle.colissb.

[0253] The EDTA concentration in DNB terminating buffer is 250 mm;

[0254] The rest are the same as the first, second, and each sample information is as follows:

[0255] Sample number Sample type Nucleic acid type Nucleic acid concentration Ng / μL Library concentration N...

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Abstract

The invention discloses a kit and a method for constructing DNA nanospheres by a one-step method. The kit comprises R1, R2 and R3, the R1 comprises a DNB preparation buffer solution, the R2 comprises a DNB polymerase buffer solution and a DNB polymerase mixed solution, and the R3 comprises a DNB termination buffer solution. The method comprises the following steps: uniformly mixing a dsDNA library and the DNB preparation buffer solution, and then carrying out high-temperature denaturation and low-temperature annealing; uniformly mixing an annealing product with the DNB polymerase buffer solution and the DNB polymerase mixed solution, and carrying out a reaction at low temperature; and terminating the reaction using the DNB termination buffer solution. According to the kit and the method, single-chain cyclization and rolling circle amplification processes are creatively performed in one step, without the need of enzyme digestion and purification, thus greatly saving operation steps and time.

Description

Technical field [0001] The present invention belongs to the field of high-throughput sequencing technology, and in particular, a kit and method for constructing DNA nanospheres. Background technique [0002] In recent years, NGS techniques have not rely on known nucleic acid sequences, without special probe design, it can directly detect unknown pathogenic microorganisms, breaking the limitations of traditional microbiological inspections, showing broad prospects in the clinical microbiology . [0003] At present, the incidence of global infectious diseases has risen, and the pathogenic microorganism presents a diversified and complicated development trend. Intimate Acute Respiratory Syndrome (SARS), Novel Coronary Virus Pneumonia (NCP), new variant Kaya, H7N9 avian influenza, etc. New and infectious diseases continue to appear. HIV, multiple or broad-spectrum resistant tuberculosis, classical infectious diseases such as Hoshocyptoplasma, and new disease-resistant characteristics...

Claims

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Application Information

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Patent Type & Authority Applications(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