阅读说明：本技术 一种核酸助沉剂及改良的循环肿瘤dna的提取方法 (Nucleic acid precipitation promoter and improved method for extracting circulating tumor DNA ) 是由 蒋国平 麻锦程 曾丽 贾俊玲 于 2020-05-18 设计创作，主要内容包括：本发明公开了一种核酸助沉剂及改良的循环肿瘤DNA的提取方法,属于生物技术领域。本发明公开的核酸助沉剂,由Carrier RNA、Acryl Carrier、PolyA、糖原中的至少一种与醋酸钠组合而成。本发明公开的改良的循环肿瘤DNA的提取方法,通过在血浆样本中添加助沉剂,以提高样本中游离DNA的检测得率。本发明在提高样本中游离DNA提取得率的同时,提高了对肿瘤突变基因的检出率,有助于临床对肿瘤诊断的辅助作用。(The invention discloses a nucleic acid precipitation aid and an improved method for extracting circulating tumor DNA, and belongs to the technical field of biology. The invention discloses a nucleic acid precipitation aid, which is prepared by combining at least one of Carrier RNA, Acryl Carrier, polyA and glycogen with sodium acetate. The improved method for extracting the circulating tumor DNA disclosed by the invention has the advantage that the detection yield of free DNA in a sample is improved by adding the settling agent into a plasma sample. The invention improves the extraction yield of free DNA in a sample, improves the detection rate of tumor mutant genes and is beneficial to the clinical auxiliary effect on tumor diagnosis.)

1. A nucleic acid deposition aid for extracting circulating tumor DNA is characterized by being prepared by combining 0.5-1.5 mu g/mu l of Carrier rRNA, l-3 mu g/mu l of Acryl Carrier, 1-5 mu g/mu l of PolyA and 2-6 mu g/mu l of glycogen with 0.1-0.6 mol/mu l of sodium acetate.

2. The nucleic acid precipitation aid of claim 1, wherein the nucleic acid precipitation aid is prepared from 1.0 μ g/μ l of carrierna, 3 μ g/μ l of glycogen and 0.2mol/μ l of sodium acetate.

3. The nucleic acid precipitation aid of claim 1, wherein the nucleic acid precipitation aid comprises 1.5 μ g/μ l Acryl Carrier, 4.0 μ g/μ l PolyA, and 0.3mol/μ l sodium acetate.

4. An improved method for extracting circulating tumor DNA is characterized by comprising the following specific steps:

(1) separating the plasma sample;

(2) adding the nucleic acid precipitation aid of any one of claims 1-3 into the plasma sample separated in the step (1), adding 200 mu L protease K, and extracting free DNA according to a free DNA kit by a paramagnetic particle method;

(3) and (3) carrying out PCR detection on the free DNA extracted in the step (2).

Technical Field

The invention relates to the technical field of biology, in particular to a nucleic acid precipitation promoter and an improved method for extracting circulating tumor DNA.

Background

Circulating tumor gene (ctDNA), an extracellular DNA in a cell-free state, is derived from somatic mutation DNA fragments of dead tumor cells, and exists in body fluids such as blood, synovial fluid, and cerebrospinal fluid as free DNA. The tumor marker has wide application prospect, high sensitivity and high specificity, plays an important role in the aspects of tumor diagnosis, treatment, prognosis detection and the like, and can be applied to liquid biopsy of tumors for early screening and diagnosis of the tumors. The content of ctDNA is very low, so that the efficient extraction of ctDNA becomes one of the keys of the tumor liquid biopsy technology, and the detection rate of ctDNA mutation can be improved by improving the extraction rate of ctDNA, so that the accuracy of early tumor screening is improved.

The method for separating and purifying nucleic acid generally comprises the following main steps of cell lysis, enzyme treatment, separation of nucleic acid from other biomacromolecules, purification of nucleic acid and the like. The separation and purification of nucleic acid is a key step, the high-charge phosphate skeleton of nucleic acid makes it more hydrophilic than other biomacromolecule substances of protein, polysaccharide and fat, etc., and according to the difference of their physicochemical properties, the nucleic acid can be separated and purified by using the methods of selective precipitation, chromatography and density gradient centrifugation, etc. The nucleic acid extraction effect obtained by different methods is different.

The separation and purification methods of free DNA (cfDNA) mainly comprise a phenol-chloroform method, a silica gel membrane adsorption column method and a magnetic bead method. The traditional phenol-chloroform method has low extraction efficiency, poor repeatability, large dosage of toxic solvent, complex operation and easy pollution. The silica gel membrane adsorption column method realizes the adsorption of nucleic acid by combining specific buffer solution, accurate pH and salt concentration. The method has the advantages of high speed, high nucleic acid purity and good reproducibility, and has the main defects of low extraction efficiency of small fragment nucleic acid compared with a magnetic bead method, common chaotropic salt condition and complex and long subsequent salt washing. A serious problem of the column extraction method is that the shearing force caused by high-speed centrifugation is easy to degrade DNA, and the integrity of the DNA is influenced. The major products for extracting free nucleic acid by a silica gel membrane adsorption column method in the current market are kits of Qiagen companies, the price is expensive, and the recovery rate is only about 40%. The magnetic bead method nucleic acid purification technology adopts nanometer magnetic bead microbeads, the surfaces of which are marked with a functional group which can perform adsorption reaction with nucleic acid. The magnetic bead extraction technology can be used for batch processing of various samples, is one of the best choices of automation and high flux, is simple and convenient to operate, and consumes short time, but most of commercially available extraction kits have low extraction rate of free DNA, are not suitable for extracting the free DNA with extremely low content such as tumor circulating DNA, and cannot be well applied to the tumor liquid biopsy technology.

Therefore, it is an urgent need to solve the problems of the art to provide a nucleic acid precipitation promoter and an improved method for extracting circulating tumor DNA.

Disclosure of Invention

In view of the above, the invention provides a nucleic acid precipitation aid and an improved method for extracting circulating tumor DNA, which improves the existing method for extracting free DNA by a magnetic bead method, improves the extraction yield of the free DNA by adding the nucleic acid precipitation aid into a sample, has good effect on extracting the circulating tumor DNA, and can be applied to the liquid biopsy of the tumor.

In order to achieve the purpose, the invention adopts the following technical scheme:

a nucleic acid deposition aid for extracting circulating tumor DNA is prepared from at least one of 0.5-1.5 μ g/μ l carrier RNA, l-3 μ g/μ l acrylic carrier, 1-5 μ g/μ l polyA, and 2-6 μ g/μ l glycogen and 0.1-0.6mol/μ l sodium acetate.

Further, a nucleic acid precipitation aid, which is composed of 1.0. mu.g/. mu.l of CarrierRNA, 3. mu.g/. mu.l of glycogen and 0.2 mol/. mu.l of sodium acetate.

Further, a nucleic acid precipitation aid, which is composed of 1.5. mu.g/. mu.l of acrylic Carrier, 4.0. mu.g/. mu.l of PolyA and 0.3 mol/. mu.l of sodium acetate.

Further, an improved method for extracting circulating tumor DNA comprises the following specific steps:

(1) separating the plasma sample;

(2) adding the nucleic acid precipitation aid into the plasma sample separated in the step (1), adding 200 mu L ProteinaseK, and extracting free DNA according to a free DNA kit by a paramagnetic particle method;

(3) and (3) carrying out PCR detection on the free DNA extracted in the step (2).

According to the technical scheme, compared with the prior art, the invention discloses the nucleic acid precipitation aid and the improved extraction method of the circulating tumor DNA, and aims at the situation that most of the existing extraction kits are not high in free DNA extraction rate and are not suitable for extracting free DNA with extremely low content such as tumor circulating DNA. The invention improves the extraction yield of free DNA in a sample, improves the detection rate of tumor mutant genes and is beneficial to the clinical auxiliary effect on tumor diagnosis.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a diagram showing the result of fluorescent quantitative PCR verification according to the present invention;

wherein, A: no nucleic acid precipitating agent is added; b: adding a nucleic acid precipitation aid;

FIG. 2 is a graph showing the detection rate of the mutation positivity of RNF43 gene according to the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The free DNA kit by the magnetic bead method is purchased from Jiangsu Mule Biotech GmbH; glycogen is purchased from Hefei Qufeng Biotech limited; polyA was purchased from jejie biotechnology limited, shanghai.