RU2603098C1 - Method for extracting circulating dna from plasma or blood serum - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to molecular biology and diagnostic medicine. Proposed is a method for extracting circulating DNA from plasma or blood serum. Method comprises treatment of the sample with isothiocyanate guanidine of equal amount of 0.5÷1.5 M, deposition of non-target polymers by addition of equal amount of the buffer solution containing 1 M of sodium acetate, pH 6.0, and 0.5-2.5 % octanoic acid, and centrifugation. Produced supernatant is added with 1/5 of the amount of 96 % ethanol and applied on a column with a glass-fibre sorbent. Sorbent is washed from ballast biopolymers and cDNK is eluted with water.

EFFECT: invention provides high output of cDNK.

3 cl, 1 dwg, 2 tbl, 2 ex

Description

The invention relates to the field of molecular biology and diagnostic medicine and can be used to isolate circulating DNA from plasma or blood serum.

Analysis of circulating DNA (cDNA) is a promising direction in the diagnosis of various diseases, including infectious, oncological and pathologies of pregnancy. Recently, a large number of studies have been conducted aimed at introducing cDNA analysis into clinical practice. However, a number of unresolved problems remain that make it difficult to create and widely use test systems based on circulating blood nucleic acids. A great influence on the sensitivity and specificity of cDNA analysis methods is made by its features such as low concentration in biological fluids, low content of tumor DNA, as well as its fragmentation (1).

Despite a fairly large number of studies on DNA analysis in plasma or serum, there is still no single protocol for sample preparation and isolation of cDNA, which leads to a lack of convergence of results between different laboratories. Recent data from the international project SPIDIA (Standardization and improvement of pre-analytical procedures for in vitro diagnostics) indicate that the creation of a single protocol for the isolation of cDNA requires a lot of attention from researchers.

The main obstacle to the isolation of blood cDNA is the high content in the starting material of non-nucleotide biopolymers (proteins, lipoproteins, lipids and their complexes), including those that form strong complexes with DNA, the presence of enzyme inhibitors used in the subsequent analysis (for example, PCR) as well as a high content of enzymes that break down nucleic acids.

Thus, obtaining highly purified cDNA preparations suitable for subsequent analysis (PCR, microarray analysis, genome-wide sequencing) is an urgent problem in molecular biology and diagnostic medicine.

Currently, there are several fundamentally different approaches to the extraction of DNA from biological samples: the method of enzymatic cleavage of proteins, organic extraction, extraction using silicate sorbents.

The method of enzymatic cleavage of proteins consists in adding a broad spectrum serine protease (proteinase K) to the preparation, which hydrolyzes the peptide bonds located next to the carboxyl group of aliphatic and aromatic amino acids (2). However, this method does not provide sufficient purification of nucleic acid preparations from proteins and other impurities, which complicates further manipulation of the sample. In particular, the resulting solution often has a pH that is not optimal for DNA storage; contamination of the sample with products of incomplete hydrolysis often leads to DNA degradation. In addition, incomplete thermal inactivation of proteinase K can lead to inhibition of the passage of various enzymatic reactions (for example, PCR, bisulfite conversion), which can cause false negative research results.

The organic extraction method uses a mixture of phenol, chloroform and isoamyl alcohol to extract proteins and other impurities of a non-nucleotide nature from a DNA solution. When a phenol / chloroform mixture is added to the sample, the phases of the solution are separated into organic (containing proteins) and aqueous (containing nucleic acids). Subsequently, DNA is extracted from the aqueous phase by alcohol precipitation (3). The disadvantages of this method include the complexity, the long processing time of the sample, high requirements for the qualifications of personnel. In addition, when using the organic extraction method, toxic waste is generated, which must be subjected to special disposal. All this makes the method inapplicable for working with a large number of samples, and therefore, application in routine clinical practice.

Methods of isolating cDNA using silicate sorbents are based on the ability of silicate sorbents to bind nucleic acid molecules in the presence of chaotropic agents. Chaotropic agents (for example, guanidine chloride) can destroy the hydration shell of a nucleic acid, which leads to the effective binding of nucleic acids to the surface of a silicate sorbent. The process of DNA sorption on silicate carriers proceeds most actively at pH values of about 5 and a high ionic strength of the solution (4). After sorption, the DNA is washed and subsequently eluted with a solution of low ionic strength. The method based on sorption of DNA on silicate sorbents has several different formats: suspension (glass-milk method, sorbent on the surface of ferromagnetic microspheres) and in the form of a solid matrix (microcolumns).

Several commercial cDNA isolation kits are available on the market. The most popular among researchers are the Qiagen kits: QIAamp DNA Blood Mini Kit and QIAamp Circulating Nucleic Acid Kit (3). As a result of a large number of studies, data were collected on the significant shortcomings of these kits. For example, it was shown in (5) that the use of the QIAamp DNA Blood Mini Kit for DNA extraction from blood plasma led to a decrease in DNA extraction efficiency by a factor of two compared to phenolic extraction. Low efficiency of isolation is associated with reduced efficiency of binding of DNA fragments <200 bp by the sorbent, as well as insufficiently effective elution of the nucleic acid from the sorbent.

Effective isolation of short fragments of cDNA with a length of less than 200 bp is a separate urgent task. According to the latest data, it is this fraction of cDNA that is targeted for analysis, that is, the fraction of short DNA contains the largest number of target DNA (tumor-specific DNA, fetal DNA).

Closest to the proposed method, the prototype, is a method for the isolation of cDNA, which consists in the following. An equal volume of purification buffer (1 M NaCl, 10 mM Tris-HCl, pH 8.2, 25 mM EDTA pH 8.0, 2% SDS) was added to 200 μl of healthy donor blood plasma. The resulting mixture was stirred and incubated for 2 hours at 56 ° C. Then 200 μl of 6 M NaCl was added to the mixture, incubated for 1 hour at room temperature. The mixture was centrifuged for 15 min at 16,000 g, the supernatant was transferred to a new tube and 2 volumes of 96% ethanol were added. The tube was incubated for 20 min at -20 ° C and centrifuged for 15 min at 16.000g. The supernatant was removed, the precipitate was washed with 70% ethanol, dried in air and dissolved in 120 μl of water (6). The yield of cDNA was not more than 70%.

The disadvantages of the prototype are insufficient yield and low degree of purification of cDNA (the content of PCR inhibitors), low reproducibility, as well as the duration and need for highly qualified personnel.

The objective of the invention is to increase the efficiency of isolation of cDNA, including short fragments with a length of less than 200 bp, from plasma or blood serum.

Effect: increase the yield of the target product, reducing material and time costs for the allocation of cDNA.

The problem is achieved by the proposed method, which consists in the following.

An equal volume of a denaturing solution containing 0.5-1.5 M guanidine isothiocyanate is successively added to a blood plasma sample, then an equal volume of a precipitation buffer containing at a final concentration of 1 M sodium acetate pH 6.0, and 0.5-2.5 % octanoic acid. The mixture is stirred, incubated for 3-5 minutes at room temperature and centrifuged at 9.000-16.000g. 1/5 volume of 96% ethanol is added to the obtained supernatant and applied to a column with a biosilica fiberglass sorbent (BioSilica Ltd, Russia) by vacuum filtration or centrifugation. The sorbent is washed from impurities of non-nucleotide biopolymers, first with a buffer solution containing 0.5 M guanidine isothiocyanate, 10 mM Tris-acetate pH 6.5, 20% ethanol, and then with a buffer solution containing 10 mM Tris-HCl pH 7.5, 0.1 M NaCl 75% ethanol. Elution of DNA from the sorbent is carried out with sterile water.

The defining hallmarks of the proposed method, compared with the prototype, are:

1. DNA-containing complexes in the composition of a plasma or serum sample are denatured with a solution containing 0.5-1.5 M guanidine isothiocyanate, which allows creating conditions for the subsequent effective dissociation of DNA from nucleoprotein complexes and shortening the isolation time.

2. The precipitation of non-target biopolymers is carried out with a buffer solution containing 1 M sodium acetate pH 6.0 and 0.5-2.5% octanoic acid, followed by centrifugation, which allows additional isolation of cDNA from nucleoprotein complexes, precipitate non-target biopolymers and, as a result, increase the yield target product.

3. 1/5 volume of 96% ethanol is added to the obtained supernatant, which improves the efficiency of binding of cDNA to the sorbent, and also contributes to the denaturation of the remaining proteins and lipoproteins, additional dissociation of nucleoprotein complexes. This contributes to the enrichment of the content of small fragments of cDNA in the preparation, increases the purity and yield of the product.

4. The washing of the sorbent from unbound biopolymers is carried out sequentially first with a buffer solution containing 0.5 M guanidine isothiocyanate, 10 mM Tris-acetate, pH 6.5, 20% ethanol, and then with a buffer solution containing 10 mM Tris-HCl pH 7.5, 0, 1 M NaCl, 75% ethanol, which ensures the effective removal of non-nucleotide impurities from the surface of the sorbent and increases the purity of the resulting DNA preparation.

Thus, the proposed method allows you to quickly and efficiently isolate cDNA from plasma or blood serum.

The invention is illustrated by the following examples of specific performance of the method.

Example 1. Isolation of cDNA from blood plasma.

Isolation of cDNA from blood plasma of 10 healthy donors was performed using the prototype method and the proposed method. According to the method of the prototype method, an equal volume of purification buffer (1 M NaCl, 10 mM Tris-HCl, pH 8.2, 25 mm EDTA pH 8.0, 2% SDS) was added to 200 μl of healthy donor blood plasma. The resulting mixture was stirred and incubated for 2 hours at 56 ° C. Then 200 μl of 6 M NaCl was added to the mixture, incubated for 1 hour at room temperature. The mixture was centrifuged for 15 min at 16,000 g, the supernatant was transferred to a new tube and 2 volumes of 96% ethanol were added. The tube was incubated for 20 min at -20 ° C and centrifuged for 15 min at 16.000g. The supernatant was removed, the precipitate was washed with 70% ethanol, dried in air and dissolved in 120 μl of water.

According to the proposed method, 200 μl of a denaturing solution containing 0.5 M guanidine isothiocyanate was added to 200 μl of blood plasma of healthy donors. Then 200 μl of precipitation buffer was added, containing at a final concentration of 1 M sodium acetate pH 6.0 and 0.5% octanoic acid. After adding each reagent, the solution was mixed on a shaker for 3-5 seconds, the resulting solution was incubated for 5 minutes at room temperature, and the resulting precipitate was precipitated by centrifugation at 16.000g (Thermo Scientific, USA) for 3 minutes. 1/5 volume of 96% ethanol was added to the obtained supernatant. The supernatant was applied to a column manufactured by BioSilika LLC, which is a cylindrical-shaped plastic container with a lid and a hole in the conical bottom, on which 10-15 mg of a fiberglass sorbent consisting of alkali glass fibers with a diameter of 0.4-1 micron. A centrifuge (Thermo Scientific, USA) at 400 g was used to apply the supernatant. The sorbent was washed with 300 μl of buffer containing 0.5 M guanidine isothiocyanate, 10 mm Tris-acetate pH 6.5, 20% ethanol. Then the sorbent was washed with 300 μl of buffer containing 10 mM Tris-HCl pH 7.5, 0.1 M NaCl, 75% ethanol and the DNA was eluted by adding 120 μl of water (centrifugation at 400g, 1 min, 20 sec at 16.200g).

The concentration of cDNA isolated by different methods was determined using quantitative PCR on L1 elements (7).

The results of the study are presented in table 1.

As can be seen from table 1, the use of the proposed method for the isolation of cDNA from blood plasma can increase the yield of cDNA by an average of 31%. At the same time, saving time is about 3 hours. This example illustrates the possibility of using the developed method in clinical laboratories using in-line methods with high reproducibility.

Example 2. Isolation of cDNA from blood serum.

To evaluate the efficiency of isolation of short fragments of cDNA, DNA was extracted from the serum of 10 healthy donors using the proposed method, prototype method, and commercial QIAamp Circulating Nucleic Acid Kit (Qiagen).

To this end, 1 ml of a blood serum was added with 1 ml of a denaturing solution containing 1.5 M guanidine isothiocyanate. Then, 1 ml of precipitation buffer was added, containing at a final concentration of 1 M sodium acetate pH 6.0 and 2.5% octanoic acid. After adding each reagent, the solution was mixed on a shaker for 3-5 seconds, the resulting solution was incubated for 3 minutes at room temperature, and the resulting precipitate was precipitated by centrifugation at 9.000g for 3 minutes. 1/5 volume of 96% ethanol was added to the obtained supernatant. The supernatant was applied to a column manufactured by BioSilica LLC using a centrifuge (Thermo Scientific, USA) at 400g. Washing of the sorbent and elution of the DNA was carried out as described in example 1.

Isolation of cDNA using the prototype method was carried out analogously to example 1.

All cDNA isolation operations with the commercial QIAamp Circulating Nucleic Acid Kit were performed as recommended by the manufacturer.

The concentration of cDNA isolated by different methods was determined using quantitative PCR on L1 elements (7). The length distribution of fragments of the extracted DNA was determined using the electrophoretic analysis of nucleic acids on Bioanalyzer 2100 microarrays (Agilent).

The results of comparing the efficiency of isolation of cDNA using different methods are presented in table 2.

As can be seen from table 2, the use of the proposed method for the isolation of cDNA from blood serum can increase the yield of cDNA by an average of 28%.

The results of studying the distribution of the lengths of cDNA fragments isolated by different methods are presented in FIG. 1. From the obtained data it follows that the developed method allows more efficient to isolate low molecular weight DNA (short DNA fragments with a length of less than 200 bp), compared with the QIAamp Circulating Nucleic Acid Kit.

Using the proposed method will improve the efficiency of isolation of cDNA from plasma or blood serum, increase the yield of cDNA on average by 28-31%, reduce the time of isolation of cDNA by 3 hours.

Information sources

1. Fleischhacker, M., Schmidt, B., Circulating nucleic acids (CNAs) and cancer-a survey. Biochim. Biophys. Acta, 2007.1775 (1): p. 181-232.

M., Ponce L.K., Salvador I., Vivar

N. Improved method for extraction and detection of Helicobacter pylori DNA in formalin-fixed paraffin embedded gastric biopsies using laser micro-dissection. MethodsX. 2014. p. 1-7.2. Loayza MF, Villavicencio FX, Santander SC,

M., Ponce LK, Salvador I., Vivar

N. Improved method for extraction and detection of Helicobacter pylori DNA in formalin-fixed paraffin embedded gastric biopsies using laser micro-dissection. MethodsX. 2014. p. 1-7.

3. Xue X., Teare M.D., Holen I., Zhu Y.M., Woll P.J. Optimizing the yield and utility of circulating cell-free DNA from plasma and serum. Clin Chim Acta. 2009. 404. P. 100-104.

4. Parida, S.K., et al., Adsorption of organic molecules on silica surface. Adv. Colloid Interface Sri., 2006.121 (1-3): p. 77-110.

5. Schmidt, B., et al., Improved method for isolating cell-free DNA. Clin Chem, 2005.51 (8): p. 1561-1563.

6. Davoudi, A., et al., Evaluation of two DNA extraction methods from maternal plasma for using in non-invasive bovine fetus gender determination. Iran J Reprod Med, 2012.10 (6): p. 523-530.

7. Morozkin E.S., Babochkina T.I., Vlassov V.V., Laktionov P.P. The effect of protein transport inhibitors on the production of extracellular DNA. // Ann N.Y. Acad. Sci. - 2008. - V. 1137. - P. 31-35.

Claims (3)

1. A method of isolating cDNA from plasma or blood serum, including treating the sample with denaturing buffer solution, precipitating non-targeted biopolymers, followed by incubating and centrifuging the mixture and separating the supernatant, adding alcohol to the latter, followed by isolation and purification of the target product, characterized in that the sample is treated equal volume of denaturing buffer solution containing 0.5 ÷ 1.5 M guanidine isothiocyanate, non-target biopolymers are precipitated by adding an equal volume of buffer solution containing 1 M sodium acetate, pH 6.0, and 0.5-2.5% octanoic acid, 1/5 volume of 96% ethanol is added to the obtained supernatant and applied to a column with a fiberglass sorbent, and the sorbent is washed from unbound biopolymers first with buffer a solution containing 0.5 M guanidine isothiocyanate, 10 mm Tris-acetate pH 6.5, 20% ethanol, and then a buffer solution containing 10 mm Tris-HCl pH 7.5, 0.1 M NaCl, 75% ethanol. 2. The method according to p. 1, characterized in that for the isolation of cDNA using columns with fiberglass sorbent produced by LLC BioSilica. 3. The method according to p. 1, characterized in that the mixture is incubated at room temperature for 3-5 minutes and centrifuged at 9000-16000g.

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