RU2232768C2 - Method for isolating deoxyribonucleic acids - Google Patents

Abstract

FIELD: preparative biochemistry.

SUBSTANCE: invention relates to a method for isolating deoxyribonucleic acids that involves adsorption of deoxyribonucleic acids on silicate sorbent in the presence of chaotropic agent, separation and washing out sorbent and the following elution of nucleic acids from sorbent. Small-dispersed meshed glass treated with 3.5-7.0% hydrofluoric acid solution for 2-6 h is used as sorbent. Washing out sorbent is carried out firstly twice with buffer solution containing chaotropic agent and then twice with buffer solution containing 25% of isopropyl alcohol. Elution is carried out with buffer solution containing 5-50 mM of hydrocarbonate ions at pH 8.0-10.0. Method provides simplifying a procedure, reducing time for isolating DNA, enhancing the yield of the end product and carrying out the isolation both low-molecular and high-molecular DNA fractions.

EFFECT: improved isolating method.

3 cl, 2 dwg, 3 tbl, 3 ex

Description

The invention relates to the field of biotechnology and molecular biology, and in particular to a method for the isolation and purification of deoxyribonucleic acids (DNA) using silicate sorbents from biological fluids and other samples. DNA isolation by the proposed method can be used for molecular biological studies, such as determining the concentration, composition, restriction analysis, PCR analysis of DNA, as well as in practical medicine (diagnosis of human hereditary diseases, forensic medicine, gene fingerprinting).

Obtaining highly purified DNA is an important problem in molecular biology. Methods such as CsCl ₂ gradient centrifugation, gel filtration, phenol treatment and precipitation with alcohol are used to obtain DNA from biological fluids. These methods are distinguished by their duration, multi-stage and laboriousness.

A known method of DNA extraction from clinical samples using finely dispersed (MS) mixed (standardized by size) glass (MMS) [Beld M., Sol C., Goudsmit Fractionation of nucleic acids into single-stranded and double-stranded forms. // Nucl. Acids Res. 1996. v. 24. p. 2618-2619]. The method consists in the fact that DNA is adsorbed from a buffer solution containing 4 M guanidine thiocyanate (GuSCN), 0.1 M EDTA (pH 8.0) for 10 min, then the silicate sorbent is separated by centrifugation (30 s at 1000 g), wash the silicate sorbent twice with adsorption buffer, twice with 70% ethanol and once with acetone. The silicate sorbent is dried for 10 min at 56 ° C, and then the DNA is eluted by incubation with a buffer containing 10 mM Tris-HCl, 10 mM EDTA (pH 8.0) (TE buffer) while heating to 56 ° C for 10 min.

The disadvantages of this method are the duration of DNA extraction (30-40 min), low sorption capacity of the silicate sorbent (0.5 μg of DNA is adsorbed per 1 mg of MMS). This method allows you to select DNA with a length of more than 40 TPN, while it is possible to elute from the silicate sorbent no more than 40-50% of the DNA due to the fact that 50-60% of the adsorbed DNA irreversibly binds to MMS. Thus, a DNA yield of at least 40 kbp makes up from 40 to 50%.

A known method of DNA extraction from biological fluids, which use a silicate sorbent treated with nitric acid [Short protocols in molecular biology, Wiley, 1995]. For this, MS is treated with concentrated HNO ₃ for one hour, then washed with water. The adsorption of DNA on a given glass and elution from a silicate sorbent is carried out under the conditions of the described analogue 1.

The disadvantages of this method are the duration of DNA extraction (30-40 min), low sorption capacity of the silicate sorbent (0.5 μg of DNA is adsorbed per 1 mg of MS). This method allows you to select DNA with a length of more than 40 TPN, while it is possible to elute from the silicate sorbent no more than 40-50% due to the fact that 50-60% of the adsorbed DNA irreversibly binds to MS. Thus, a DNA yield of at least 40 kbp makes up from 40 to 50%.

A known method of DNA extraction, including the adsorption of DNA on Aerosil (A-300) in a chaotropic agent, the separation of the sorbent and its associated DNA, the elution of DNA from Aerosil and purification of the target product. In this case, Aerosil is introduced into a solution containing DNA to a final concentration of 0.3-0.6%, to isolate single-stranded DNA and RNA, use a medium with a content of 0.5-2 M GuSCN, and to isolate double-stranded DNA, a medium with a content of 4 M GuSCN [RF Patent No. 2119954, cl. C 12 N 15/10, C 07 H 21/04, publ. 10/10/98].

The disadvantages of this method are its duration and complexity.

Closest to the claimed method, the prototype is a method for isolating DNA from blood cells [Boom R. et al. "Rapid and simple method for purification nucleic acids." J. Clin. Microbiol. 1990, v. 28, p. 495-503]. For adsorption of DNA using MMS. The method includes the following steps: DNA is adsorbed from a buffer solution containing 5.4 M GuSCN, 105 mM EDTA, 53 mM Tris-Hcl (pH 7.3), 1.12% Triton X-100 for 10 minutes. Then, the silicate sorbent is precipitated by centrifugation (15 s at 12000 g), washed twice with buffer containing 5.4 M GuSCN, 53 mm Tris-Hcl, pH 6.4, and then once with ethanol. The silicate sorbent is dried for 10 min at 56 ° C, and then adsorbed DNA is eluted by incubation with TE buffer for 10 min when heated to 56 ° C.

The disadvantages of this method is the insufficient yield of the target product due to the low sorption capacity of the silicate sorbent (0.5 μg of DNA is adsorbed per 1 mg of MMS). This method allows you to select DNA with a length of more than 50 TPN, while it is possible to elute from the silicate sorbent no more than 40-50% due to the fact that 50-60% of the adsorbed DNA irreversibly binds to MMS. Thus, a DNA yield of at least 50 kbp from 48 ± 4%, and DNA with a low molecular weight is allocated much less efficiently.

An object of the invention is to increase the yield of the target product due to more efficient adsorption of DNA, increasing the functionality of the method by providing the ability to isolate a wide range of DNA with a length of 50 bp to high molecular weight fractions and a decrease in the duration of the process.

The problem is achieved by the proposed method, which consists in incubating a biological sample with a pre-treated silicate sorbent in a solution of a chaotropic agent, washing the silicate sorbent from non-nucleotide biopolymers and subsequent elution of the DNA with a solution containing bicarbonate ions. The supernatant is a solution of purified DNA, suitable for molecular biological studies without additional stages of purification and concentration.

DNA isolation time by this method is 8-10 minutes. The method provides the selection of DNA with a length of 50 bp to high molecular weight fractions from biological samples with high efficiency and does not depend on the concentration of DNA in the sample. The method provides a quantitative elution of DNA adsorbed onto a processed finely divided glass (OMMS).

The method includes the following sequence of steps:

1) pre-treatment of MMS with 3.5-7% hydrofluoric acid (HF) for 2-6 hours, followed by washing the glass particles and drying the treated MMS (OMMS);

2) DNA extraction from biological fluids by binding DNA to OMMS in a medium containing a chaotropic agent (9 M GuSCN, 40 mm EDTA, 10 mm Tris-HCl, pH 6.4);

3) separation of OMMS and its associated DNA by centrifugation at 1000g for 10 sec;

4) washing OMMS and associated DNA twice with a buffer solution containing 4.5 M GuSCN, 20 mm EDTA, 10 mm Tris-HCl, pH 6.4, and twice with a buffer solution containing 25% isopropanol, 100 mm Nad, 10 mM Tris-HCl, pH 8.0;

5) elution of DNA with OMMS buffer solutions containing 5-50 mm bicarbonate ions (HCO ₃ ), pH 8.0-10.0.

The use of the invention allows to increase the yield of DNA (from 0.5 to 2.5 μg dc-DNA per 1 mg of silicate sorbent), isolated using silicate sorbents from biological samples, providing a quantitative selection of DNA, thereby reducing the amount of biological material used for isolation DNA for the purpose of subsequent amplification or other research. In the table. 1 presents data on the elution of P ³² -radio-labeled DNA (length from 1000 to 100 bp) with various eluents. From the table. Figure 1 shows that the use of a TE buffer for elution does not allow the quantitative elution of DNA from the surface of a silicate sorbent. The use of bicarbonate ions provides a quantitative elution of DNA of any length, regardless of the amount of adsorbed DNA.

The defining distinguishing features of the proposed method in comparison with the prototype are:

1) MMS is used as a silicate sorbent, treated with a 3.5-7% solution of hydrofluoric acid for 2-6 hours, the sorbent is washed first twice with a buffer solution containing a chaotropic agent, and then twice with a buffer solution containing 25% isopropanol. This treatment allows you to unify the particle size of the glass, to increase the working surface and sorption capacity of the latter. Figure 1 (A, B) shows an electron micrograph (× 5000) of mixed (A) and mixed glass treated with a 3.5-7% solution of hydrofluoric acid (B). The photo shows that treating glass with hydrofluoric acid etches microcracks, destroys glass particles and produces a powder containing smaller and more homogeneous particle size glass with a more uniform surface structure and an increased surface area. Such a silicate sorbent has a higher capacity. In the table. Figure 2 shows the effect of glass processing conditions on the binding of 2.5 μg of P ³² radioactive labeled DNA to fine glass. Depending on the sorption conditions, the OMMS capacity is 1.5–2 times higher than that of the untreated one. In addition, the separation of the DNA solution from the silicate sorbent is facilitated by the formation of a uniform precipitate of glass particles, and the DNA extraction procedure is accelerated and simplified. OMMS binds both low molecular weight and high molecular weight DNA fragments. Figure 2 shows the electrophoresis of fragmented DNA in a 2% agarose gel containing 0.0003% ethidium bromide. DNA is hydrolyzed by DNAase 1 in the presence of Mn ⁺⁺ ions. The first track contains a length marker based on phage M15 (from 100 to 1000 bp) (Sibenzym, Novosibirsk), the second track contains the original high molecular weight DNA from the calf thymus, and the third track contains fragmented DNA from the calf thymus extracted from the solution adsorption of the claimed method. Figure 2 shows that the proposed method allows you to select both low molecular weight and high molecular weight DNA fragments.

2) The elution of DNA associated with a silicate sorbent is carried out by treating OMMS with buffer solutions containing 5-50 mM bicarbonate ions, pH 8.0-10.0, which allows you to quickly and quantitatively separate DNA from a silicate sorbent. In the table. 3 presents data on the extraction of P ³² -radio-labeled DNA (length from 1000 to 100 bp), introduced into human blood plasma by the proposed method. From the table. 3 shows that the inventive method for the isolation of DNA provides a quantitative isolation of DNA from biological fluids.

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

Example 1

For DNA adsorption, glass is pretreated with hydrofluoric acid. For this, MMS are incubated with 3.5% HF on a rocking chair for 4 hours, the treated glass is dried at 200 ° C for 1 hour, and then the glass cooled to room temperature is suspended in water. To isolate cellular DNA, cells are treated with a standard 0.25% trypsin solution. 0.5 ml of physiological solution and an equal volume of the mixture containing 4 mg OMMS in a buffer solution containing 9 M GuSCN, 40 mm EDTA, 10 mm Tris-HCl, pH 6.4 are added to the Petri dish with washed cells with physiological saline. After 5 min incubation on a rocking chair, the samples are centrifuged for 10 s at 1000g. The supernatant is removed, the silicate sorbent is washed twice with a buffer solution containing 4.5 M GuSCN, 20 mm EDTA, 10 mm Tris-Hcl, pH 6.4, and then twice with a buffer solution containing 25% isopropanol, 100 mm NaCl, 10 mm Tris -HCl (pH 8.0). DNA was eluted from the silicate sorbent 50 mM NaHCO ₃ at pH 10.0 for 2 minutes. After centrifugation at 10000 g for 1 min, the supernatant containing DNA was collected, the pH of the solution was neutralized with half the volume of 40 mM Tris-Hcl (pH 7.1).

To assess the completeness of DNA extraction by the claimed method, comparative experiments were conducted on DNA extraction by precipitation with trichloroacetic acid and adsorption of a water-insoluble fraction on nylon filters. For this, cells of human epidermal carcinoma A431 were grown on a medium with H ³ -thymidine. H ³ -labeled DNA was isolated from the same number (10 ⁶ ) of cells by the proposed adsorption method on OMMS and the standard method of DNA adsorption on nylon filters. To isolate DNA on the filter, an aliquot of cells (10 ⁶ ) was transferred to a 0.2 μm nylon filter, washed with 0.15 M NaCl and fixed with 5% trichloroacetic acid. The radioactivity of DNA isolated by various methods was measured on a RacBeta 1211 β-scintillation counter. It was found that the radioactivity of ³ H-labeled DNA isolated by various methods was the same. Thus, it was shown that the proposed method provides a quantitative selection of DNA. The DNA content in the sample was determined by measuring the fluorescence of the samples in complex with Hoechst 32258. The fluorescence of DNA complexes with Hoechst 32258 was measured on a Hitachi MPF4 fluorimeter at an excitation wavelength of 350-360 nm and emission of 480-495 nm [Labarca C., Paigen K. A simple, rapid and sensitive DNA assay procedure. // Anal. Biochem. 1980, v. 102, p. 344-352]. To construct the calibration curves, we used a standard solution of fragmented DNA from calf thymus at concentrations of 12.5, 25, 50, 100, 200, 400 ng / ml. In this way, 10 μg of DNA was isolated from 2 × 10 ⁶ cells of line A 431. The time of DNA extraction from samples by this method is 8-10 minutes. The glass binding capacity of DNA is up to 2.5 μg DNA per 1 mg OMMS.

Example 2

For DNA adsorption, glass is pretreated with hydrofluoric acid. For this, MMS are incubated with 7% HF on a rocking chair for 2 hours, the treated glass is dried at 200 ° C for 1 hour, and then the glass cooled to room temperature is suspended in water. To isolate cellular DNA, cells are treated with a standard 0.25% trypsin solution. 0.5 ml of physiological saline and an equal volume of the mixture containing 4 mg OMMS in a buffer solution containing 9M GuSCN, 40 mM EDTA, 10 mM Tris-HCl, pH 6.4 are added to a Petri dish with washed saline cells. After 5 min incubation on a shaker, the samples are centrifuged for 10 s at 1000g. The supernatant is removed, the glass is washed twice with a buffer solution containing 4.5 M GuSCN, 20 mm EDTA, 10 mm Tris-HCl, pH 6.4, and then twice with a buffer solution containing 25% isopropanol, 100 mm NaCl, 10 mm Tris- HCl, pH 8.0. DNA was eluted from the silicate sorbent 5 mM NaHCO ₃ at pH 8.0 for 2 minutes. After centrifugation at 10000 g for 1 min, the supernatant containing DNA was collected, the pH of the solution was neutralized with half the volume of 40 mM Tris-HCl (pH 7.1).

The time of DNA extraction from samples by this method is 8-10 minutes. The glass binding capacity of DNA is up to 2.5 μg DNA per 1 mg OMMS.

Example 3

To 0.5 ml of human blood plasma add an equal volume of the mixture containing 3 mg OMMS obtained analogously to example 1 in a buffer solution containing: 9 M GuSCN, 40 mm EDTA, 10 mm Tris-HCl, pH 6.4. The procedure for isolation and determination of DNA concentration using fluorescence of DNA complexes with Hoechst 32258 was carried out analogously to examples 1 and 2. The concentration of DNA in the blood plasma of healthy donors was 20 ng / ml plasma.

Using the proposed method will allow, in comparison with the prototype:

 - reduce the consumption of fine glass;

 - increase the efficiency of DNA extraction in 1.5-2 times due to quantitative elution of DNA adsorbed on a silicate sorbent;

 - to increase the functionality of the method by providing the ability to isolate both low molecular weight and high molecular weight DNA fragments;

 - increase the sensitivity of PCR diagnostic systems due to the binding of low molecular weight DNA fragments;

 - use the proposed method for the quantitative determination of DNA concentration in biological fluids;

 - reduce the amount of biological material used to isolate DNA for subsequent amplification or other research;

reduce the time of the isolation procedure to 8-10 minutes.

Claims (3)

1. A method of isolating deoxyribonucleic acids, including adsorption of deoxyribonucleic acids on a silicate sorbent in the presence of a chaotropic agent, separating and washing the sorbent, followed by elution of the nucleic acids from the sorbent, characterized in that finely dispersed mixed glass treated with 3.5-7 is used as the sorbent With a 0% hydrofluoric acid solution for 2-6 hours, the sorbent is washed first twice with a buffer solution containing a chaotropic agent, and then twice with a buffer solution containing 25% isopropanol, elution is carried out with a buffer solution containing 5-50 mm bicarbonate ions at a pH of 8.0-10.0. 2. The method according to claim 1, characterized in that the adsorption of nucleic acids on the treated sorbent is carried out in a buffer solution containing 9 M GuSCN, 40 mm EDTA, 10 mm Tris-HCl, pH 6.4 for 3 minutes 3. The method according to claim 1, characterized in that the first washing of the sorbent is carried out with a buffer solution containing 4.5 M GuSCN, 20 mm EDTA, 10 mm Tris-HCl, pH 6.4, and the second washing of the sorbent is carried out with a buffer solution containing 25% isopropanol, 100 mM NaCl, 10 mM Tris-HCl, pH 8.0.

Images