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Detection method of molecular tag nucleic acid

A technology of labeling nucleic acid and detection method, applied in the field of DNA nucleic acid detection, can solve the problems of inability to estimate the content, difficult qualitative and quantitative, PCR error, etc., and achieve the effects of convenient implementation, short sequencing time, and short library length.

Pending Publication Date: 2017-05-31
HANGZHOU GENE META MEDICAL DEVICE CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the overall error rate of large-scale sequencing is still relatively high, and the error rate of mainstream high-throughput sequencers on the market is basically 1-2%.
The error rate mainly comes from certain errors in the sequencing process itself. At the same time, during the PCR amplification process, there is also the possibility of PCR errors. There is also a certain specific template that is amplified in large quantities. There is no way to determine the true content of this site in the original template. It is estimated that only a qualitative result can be obtained in the end, and it is difficult to perform accurate qualitative and quantitative results by directly performing high-throughput sequencing with conventional library construction methods

Method used

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  • Detection method of molecular tag nucleic acid

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Effect test

Embodiment 1

[0038] This example is used to illustrate the method for constructing a free DNA sequencing library in the peripheral blood of pregnant women for non-invasive detection of monogenic diseases.

[0039] 1. 2ml of pregnant women's plasma was used to extract plasma free DNA through QIAamp Circulating Nucleic Acid Kit.

[0040] 2. Add 8 units / ul end-filling enzyme (T4 DNA polymerase or Klenowexo-), 490uM dNTPs, 55mM Tris buffer, and the reaction conditions are 37°C for 20 minutes.

[0041] 3. Add 1ul CAP directly to the test tube, denature at 37°C for 1 minute, and 75°C for 5 minutes.

[0042] 4. Add DNA ligation reagent directly into the test tube: 100 units / ul of DNA ligase, 3.5mM of ATP, 55mM of Tris buffer, 30mM of dithiothreitol and DNA linker, the linker sequence is composed of multiple bands Mixture of adapters with sequence tags.

[0043] 5. The reaction condition is 20°C for 20 minutes. After the reaction is completed, it is directly purified by silica gel column or magn...

Embodiment 2

[0053] This example is used to illustrate the method for constructing a plasma cell-free DNA sequencing library of tumor patients for liquid biopsy.

[0054] 1. 4ml of tumor patient plasma was used to extract plasma free DNA through QIAamp Circulating Nucleic Acid Kit.

[0055] 2. Add 8 units / ul end-filling enzyme (T4 DNA polymerase or Klenowexo-), 490uM dNTPs, 55mM Tris buffer, and the reaction conditions are 37°C for 20 minutes.

[0056] 3. Add 1ul CAP directly to the test tube, denature at 37°C for 1 minute, and 75°C for 5 minutes.

[0057] 4. Add DNA ligation reagent directly into the test tube: 100 units / ul of DNA ligase, 3.5mM of ATP, 55mM of Tris buffer, 30mM of dithiothreitol and DNA linker, the linker sequence is composed of multiple bands Mixture of adapters with sequence tags.

[0058] 5. The reaction condition is 20°C for 20 minutes. After the reaction is completed, it is directly purified by silica gel column or magnetic particles

[0059] 6. 10ul product, add ...

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Abstract

The invention discloses a detection method of molecular tag nucleic acid, which comprises the following steps of (1) performing tail end repair and A increase on to-be-detected DNA (Deoxyribonucleic Acid), (2) connecting a product with a DNA joint with a plurality of molecular tags, (3) extending the connected product by a universal primer, (4) hybridizing the extended product with a capture probe, and (5) extending the capture product by a molecular tag primer with a sample identification effect and detecting the final product by high-throughput sequencing. An analysis method comprises the step of homogenizing a sequencing result by comprehending double correction factors of initial positions of the molecular tags and DNA fragments, so that an actual qualitative and quantitative analysis result on mutation and single nucleotide polymorphyism (SNP) can be obtained. The detection method can be used for a sequencing library detected by a high-throughput sequencer, and can qualitatively and quantitatively detect mutation and SNP conditions in nucleic acid fragments.

Description

technical field [0001] The invention belongs to the field of DNA nucleic acid detection, in particular to a qualitative and quantitative method for detecting specific base mutations and SNPs in deoxyribonucleic acid. Background technique [0002] High-throughput sequencing is through massively parallel sequencing, which detects tens of millions of sequences each time, and can perform DNA detection quickly and cheaply. However, the overall error rate of large-scale sequencing is still relatively high, and the error rate of mainstream high-throughput sequencers on the market is basically 1-2%. The error rate mainly comes from certain errors in the sequencing process itself. At the same time, during the PCR amplification process, there is also the possibility of PCR errors. There is also a certain specific template that is amplified in large quantities. There is no way to determine the true content of this site in the original template. It is estimated that only a qualitative ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6869C12Q2531/113C12Q2525/191C12Q2535/122
Inventor 祝云英
Owner HANGZHOU GENE META MEDICAL DEVICE CO LTD