Method for directly converting plasma into hydrosulfite and application thereof

A technology of bisulfite and plasma, applied in the field of molecular biology research, can solve the problems of cumbersome operation steps, time-consuming, difficult automation, etc., and achieve the effect of avoiding DNA loss, simple operation, saving time and cost

Inactive Publication Date: 2018-07-10
PRO MED BEIJING TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The traditional bisulfite conversion method takes about 18 hours. The time required for the bisulfite conversion kits seen on the market is about 6 hours. The operation steps are cumbersome and time-consuming, and the current bisulfite conversion kits on the market require Extract plasma cell-free DNA, using DNA as a bisulfite-converted sample
Most of the converted DNA is recovered by column centrifugation, and the operation process needs to be completed with the help of a centrifuge, which is not easy to automate

Method used

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  • Method for directly converting plasma into hydrosulfite and application thereof
  • Method for directly converting plasma into hydrosulfite and application thereof
  • Method for directly converting plasma into hydrosulfite and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1: Direct bisulfite conversion of plasma

[0031] Test materials: healthy human plasma samples, methylation positive plasma samples

[0032] Test equipment: magnetic frame, constant temperature oscillator, vortex mixer

[0033] Main reagents: sodium bisulfite, sodium metabisulfite, magnetic beads

[0034] The specific implementation steps are as follows:

[0035] 1. Bisulfite conversion: Add 1.0mL plasma, 1.2mL sample treatment solution, 30μL proteinase K and 180μL bisulfite solution (a mixture of sodium bisulfite and sodium pyrosulfite, the final concentration is 4.5mol / L), vortex to mix, and incubate at 50°C for 40min;

[0036] 2. DNA binding: Add 25 μL of magnetic beads and 300 μL of binding solution to the above reaction solution, vortex to mix, adjust the constant temperature oscillator to rotate at 800 rpm, and incubate at 20°C for 25 minutes. remove residual liquid;

[0037] 3. For the first wash: add 800 μL of rinse solution I, vortex to mix, centr...

Embodiment 2

[0041] Example 2: Evaluation of bisulfite conversion

[0042] In this example, taking the Septin9 gene as an example, the Taqman qPCR method was used to evaluate the conversion efficiency of bisulfite.

[0043] experiment procedure:

[0044] 1. Design of primers and probes

[0045] The primers and probes for bisulfite-converted and non-converted Septin-9 genes were designed respectively; primers and probes were designed for a sequence of GAPDH internal reference gene without CpG islands.

[0046] 2. PCR reaction system

[0047] The internal reference gene and the Septin-9 gene were reacted in the same reaction tube; each probe was labeled with a different fluorescent group to distinguish the internal reference gene and the Septin-9 gene.

[0048] The Septin-9 gene positive samples and negative samples verified by Sanger sequencing were respectively used as templates for PCR amplification.

[0049] 3. Construction of standard curve

[0050] Select 6 5-fold gradient dilutio...

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Abstract

The invention relates to a method for directly converting plasma into hydrosulfite and application thereof. The method provided by the invention, in comparison with a similar hydrosulfite converted product on the market, has the greatest advantages that free DNA (Deoxyribonucleic Acid) of the plasma does not need to be extracted, the plasma can be directly used as a hydrosulfite conversion sample,and not only is the time of extracting the free DNA saved, but also the loss of the free DNA in an extraction process is avoided. Secondly, according to the method provided by the invention, in orderto protect the DNA from being influenced by miscellaneous protein, lipid and the like, a protective agent is added into a hydrosulfite conversion system. In addition, in order to further improve therecovery rate of the free DNA, the method adopts a high-sensitivity magnetic bead to carry out recovery. The entire conversion process does not exceed 2 hours; the centrifugal operation is not involved; the automation is more easily realized, and the method provided by the invention has the advantages of being low in cost, simple and convenient to operate, high in conversion ratio, high-purity inobtained DNA and the like.

Description

technical field [0001] The invention belongs to the field of molecular biology research, and relates to a nucleic acid conversion and purification method, in particular to a method and application of plasma direct bisulfite conversion. Background technique [0002] DNA methylation refers to the selective addition of a methyl group to cytosine (Cytosine, C) in the CpG island of DNA under the catalysis of methyltransferase to form 5-methylcytosine (5-methylcytosine). In eukaryotes, about 60% to 90% of the cytosines in CpG dinucleotides are methylated. [0003] The study of DNA methylation has always been regarded as the key content of epigenetic research. In recent years, studies have found that DNA methylation is closely related to embryonic development, regulation of gene expression, tumor generation and the occurrence of various diseases. With the development of science and technology, more and more detection methods are applied to the detection of DNA methylation, but bis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1013
Inventor 田茹陈勇田媛李西兰余占江陈永强
Owner PRO MED BEIJING TECH
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