Method for quantitatively detecting content of free DNA (deoxyribonucleic acid) by isotope dilution mass spectrometry

An isotope dilution and content technology, applied in the biological field, can solve the problems of poor preparation, low analysis speed and analysis throughput, achieve accurate and reliable DNA quantitative results, and avoid the effect of amplification efficiency

Pending Publication Date: 2021-11-23
AFFILIATED HOSPITAL OF NANTONG UNIV
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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 (deoxyribonucleic acid) by isotope dilution mass spectrometry
  • Method for quantitatively detecting content of free DNA (deoxyribonucleic acid) by isotope dilution mass spectrometry
  • Method for quantitatively detecting content of free DNA (deoxyribonucleic acid) by isotope dilution mass spectrometry

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Embodiment 1

[0084] In this embodiment, the isotope dilution ultra-high performance liquid chromatography spectrum of the free DNA single strand in serum is used, and the experimental flow chart is figure 1 As shown, including the following steps:

[0085] (1) Free DNA Single-stranded nucleic acid single chain solution, peptide nucleic acid probe solution and isotope labeled polypeptide endonuclear solution:

[0086]The free DNA single chain synthesis is 2od value, and 380 μl of DEPC water is added to 2.0 ml of a free DNA single chain solution to 2.0 mL of a concentration of 20 μmol / L. 100 μl was taken, and 900 μl of DEPC water was added thereto to a concentration of 2 μmol / L as a storage solution and displaced into a 2.0 ml of centrifuge tube, 100 μl of the centrifuge tube was dispensed, and the dispensed free DNA single chain solution is placed in- 70 ° C refrigerator storage. Before use, dilute the storage solution into a series concentration with DEPC water to establish a standard curv...

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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 by isotope dilution mass spectrometry. Firstly, a peptide nucleic acid probe is disclosed, wherein 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 the method for quantitatively detecting the content of the free DNA single chain by the isotope dilution mass spectrometry. The method comprises the following steps of forming a free DNA-biotin-streptavidin magnetic bead compound; adding the peptide nucleic acid probe to capture the free DNA-biotin-streptavidin magnetic bead compound; performing enzymolysis; performing redissolving; indirectly calculating the DNA content in the sample according to the peak area ratio; and correcting the DNA concentration to obtain a final concentration value, and the like. The method for quantitatively detecting the content of the free DNA single chain by the isotope dilution mass spectrometry has the advantages of high accuracy, reliable result and the like.

Description

Technical field [0001] The present invention belongs to the biotechnology field, and more particularly to a method of detecting free DNA content in an isotope dilution mass spectrometry. Background technique [0002] At present, the free DNA single-stranded quantitative detection method mainly includes a polymerase chain reaction method (PCR method), molecular hybridization method, gene chip method, DNA binding protein method, etc. The most widely used PCR method. However, the PCR method first needs to amplify the free DNA single strand, then the gel imaging, it takes a lot of time and human cost, and the results are susceptible to various factors, and cannot be absolutely quantified. [0003] Isotope dilution mass spectrometry is a new free DNA single-strand direct quantitative detection method, which is the current authoritative method of accurate quantitative amount of compounds. This method uses the mass spectrometry to add the same-to-homed sample to the sample to be teste...

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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