Method for quantitatively detecting content of free DNA by isotope dilution mass spectrometry

A technology of isotope dilution and content, applied in the biological field, can solve the problems of improving preparation, low analysis speed and analysis throughput, etc.

Inactive Publication Date: 2021-02-09
AFFILIATED HOSPITAL OF NANTONG UNIV
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  • Abstract
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Problems solved by technology

However, the isotope dilution mass spectrometry in the prior art still has problems such as low analysis speed and low analysis throughput, and the readiness needs to be improved.

Method used

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  • Method for quantitatively detecting content of free DNA by isotope dilution mass spectrometry
  • Method for quantitatively detecting content of free DNA by isotope dilution mass spectrometry
  • Method for quantitatively detecting content of free DNA by isotope dilution mass spectrometry

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Embodiment 1

[0085] A method of isotope dilution ultra-high performance liquid chromatography-mass spectrometry for the determination of free DNA single strands in serum in this embodiment, the experimental flow chart is as follows figure 1 shown, including the following steps:

[0086] (1) Preparation of free DNA single-strand nucleic acid single-strand solution, peptide nucleic acid probe solution and isotope-labeled polypeptide internal standard solution: the amount of free DNA single-strand synthesis is 2OD value, and 380 μL DEPC water is accurately added to it to prepare a concentration of 20 μmol / Transfer L of free ssDNA solution to a 2.0 mL centrifuge tube. Take 100 μL, add 900 μL of DEPC water to dilute to a concentration of 2 μmol / L as a stock solution and dispense into 2.0 mL centrifuge tubes, each tube is dispensed with 100 μL, and place the distributed free DNA single-strand solution in- Store in refrigerator at 70°C. Before use, dilute the stock solution with DEPC water to ...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a method for quantitatively detecting the content of free DNA through isotope dilution mass spectrometry. Firstly,a peptide nucleic acid probe is disclosed, the sequence of a nucleic acid part in the peptide nucleic acid probe is shown as SEQ ID NO: 1, and the sequence of a peptide part in the peptide nucleic acid probe is shown as SEQ ID NO: 2. The invention also discloses a method for quantitatively detecting the content of the free DNA single strand by isotope dilution mass spectrometry. The method comprises the following steps: forming a free DNA-biotin-streptavidin magnetic bead compound; adding a peptide nucleic acid probe to capture the free DNA-biotin-streptavidin magnetic bead compound; performing enzymolysis; performing re-dissolving; indirectly calculating the DNA content in the sample according to the peak area ratio; correcting the DNA concentration to obtain a final concentration value.The method for quantitatively detecting the content of the free DNA single strand by the isotope dilution mass spectrometry has the advantages of high accuracy, reliable result and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for quantitatively detecting the content of free DNA by isotope dilution mass spectrometry. Background technique [0002] Currently, methods for quantitative detection of free single-strand DNA mainly include polymerase chain reaction (PCR), molecular hybridization, gene chip, and DNA-binding protein methods, among which PCR is the most widely used method. However, the PCR method first needs to amplify the free DNA single strand, and then perform gel imaging, which consumes a lot of time and labor costs, and the results are easily affected by various factors, and cannot be absolutely quantified. [0003] Isotope dilution mass spectrometry is a new method for the direct quantitative detection of free DNA single strands, and is currently recognized as an authoritative method for the accurate quantification of compounds. This method uses mass spectrometry to determine the rati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6816G01N30/88C12N15/11
CPCC12Q1/6816G01N30/88G01N2030/8827C12Q2525/107C12Q2545/101C12Q2545/113C12Q2563/131C12Q2563/143C12Q2563/149C12Q2565/627C12Q2521/537
Inventor 王峰季伙燕袁若愚袁杰吴安琪李祎鞠少卿
Owner AFFILIATED HOSPITAL OF NANTONG UNIV
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