RU2400537C2 - Method of isolation and purification of deoxyribonucleic acids - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: sample is subjected to lysis in buffer on the basis of anionic detergent (SDS), lysate is subjected to purification and deoxyribonucleic acid is precipitated with ethanol or isopropanol. Lysate purification is performed by means of detonation nanodiamond, selectively sorbing mixtures of non-nuclear nature. Detonation nanodiamond is preliminarily burnt at temperature interval 400-700°C in vacuum 10^-2-10^-3 Torr. Obtained DNA solution contains both short and long fragments. Claimed method of isolation and purification of DNA allows to avoid application of such highly toxic compounds as phenol or chloroform. Contamination of obtained DNA with chemically active substances is prevented.

EFFECT: in case of accidental contact of detonation nanodiamond with final solution, polymerase chain reaction is not blocked, it is possible to separate non-soluble of detonation nanodiamond sediment from DNA solution.

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Description

The invention relates to the field of molecular biology, and in particular to a method for the isolation and purification of deoxyribonucleic acids from plant tissue samples, as well as processed products of plant and animal origin. It is possible to use the invention in medicine, biotechnology, in the implementation of sanitary and epidemiological control.

Isolation and purification of deoxyribonucleic acid (DNA) from fragments of plant organs and products of the processing of raw materials of plant and animal origin in order to carry out polymerase chain reaction (PCR) is one of the main tasks of molecular biology. Currently, the following DNA isolation methods are used.

A known method of DNA extraction from plant tissues using a lysis buffer, followed by purification with a mixture of chloroform and isoamyl alcohol and precipitation with isopropanol [1]. The method consists in lysing 100 mg of a plant fragment in 400 μl of buffer solution (2% CTAB, 1.4 M NaCI, 20 mM EDTA, 100 mM TrisHCI), after adding 10 μl RNase, incubate at 65 ° C for 60 min. After that, the lysate was washed twice with 400 μl of a mixture of chloroform and isoamyl alcohol (92: 8) with intermediate centrifugation and transfer of the supernatant to a clean tube. Next, DNA is precipitated in the presence of 350 μl of isopropanol, centrifuged and the supernatant removed. The DNA pellet was washed with 400 μl of 70% ethanol, dried for 60 minutes at room temperature.

The disadvantage of this method is the duration of DNA extraction (more than 2 hours), the need to use highly toxic volatile substances (chloroform and isoamyl alcohol), low DNA quality with a high content of PCR blocking substances (chlorophyll, tannins), multi-stage method.

A known method for the isolation and purification of DNA, based on the adsorption of nucleic acids on a solid sorbent [2]. The sample is mixed with a choatropic agent-based buffer solution. Nucleic acid is sorbed on a fiberglass sorbent pretreated with a 0.1-7% hydrofluoric acid solution for 1-6 hours. Subsequently, the sorbent is washed from polymer components of non-nucleic nature. DNA elution is carried out with buffer solution or distilled water.

The disadvantage of this method is the dependence of the amount of extracted DNA on the volume of the sorbent (2.3 μg DNA per 1 mg of sorbent), a decrease in DNA yield as a result of irreversible adsorption and loss as a result of washing the sorbent.

A known method for the isolation and purification of DNA, based on the adsorption of nucleic acids on a solid sorbent [3]. Nucleic acid is sorbed on Aerosil (A-300) at the desired concentration of a chaotropic agent by adding Aerosil to a final concentration of 0.3 to 0.6%, the mixture is incubated for 1-2 minutes, Aerosil with sorbed NK is precipitated by centrifugation, washed with 80% isopropanol. DNA is eluted with water or a low salt buffer.

The disadvantage of this method is the complexity, DNA damage at high concentrations, associated with the need for mechanical resuspension of the sorbent during DNA washing, a decrease in DNA yield as a result of irreversible adsorption.

Of the known technical solutions, the closest (prototype) in purpose and technical essence to the claimed object is the SDS DNA extraction method [4] using a lysing buffer based on sodium dodecyl sulfate (SDS). Approximately 2 cm ² of plant leaves were placed in a 1.5 ml tube, 400 μl of extraction buffer ((200 mm Tris-HCl, 250 mm NaCl, 25 mm EDTA, 0.5% SDS, pH 7.5) 10 μl RNase was added and incubated for 15 min at a temperature of 65 ° C. Next, 200 μl of 5M potassium acetate was added, mixed and put in ice water for 10 min. After 15 minutes centrifugation at maximum acceleration, 500 μl of the upper phase was transferred to a new tube. 400 μl of cleaning solution was added (92 % chloroform and 8% isoamyl alcohol), centrifuged for 1 min at 12000 g.The upper phase was transferred to a clean tube and repeated washing was carried out with a solution based on chloroform and isoamyl alcohol.DNA was precipitated with 500 μl of isopropanol, centrifuged at 12,000 g for 10 min, the supernatant was washed, the precipitate was washed in 70% ethanol, ethanol was removed, the DNA precipitate was dried for 1 hour at room temperature and dissolved in 100 μl of distilled water or TE buffer.

The prototype has the following disadvantages eliminated by the claimed invention.

The multi-stage method, the need to use 4 tubes in each selection and, accordingly, 3-fold transfer of the DNA solution, resulting in mechanical damage to the molecules when passing through the tip of the pipette.

The need to work with toxic substances - chloroform and isoamyl alcohol - is harmful to the health of the researcher and imposes a limit on the widespread use of the method.

It is often impossible to completely purify DNA from chlorophyll and other pigments, resulting in inhibition of PCR, and the shelf life of the extracted DNA is reduced.

The essence of the method of DNA isolation and purification is that, unlike the prototype, the biological solution is purified after lysis of the sample in a buffer based on an anionic detergent (for example, sodium dodecyl sulfate - SDS) with a suspension of detonation nanodiamond, which is a hydrophilic sorbent, previously annealed in vacuum with a weak positive charge and a highly developed specific surface. As a result, impurities that inhibit the polymerase chain reaction — peptides, pigments, tannins, and lipids — are removed from the solution. Subsequent centrifugation of the solution makes it possible to separate the detonation nanodiamond with adsorbed impurities and the supernatant containing DNA. Next, the DNA is precipitated with alcohol (isopropanol or ethanol) and dissolved in distilled water.

The implementation of the method of isolation and purification of DNA is achieved as follows.

A suspension of 30 mg of annealed for 1-2 hours in vacuum (10 ^-2 - 10 ^-3 Torr) is preliminarily prepared at a temperature of 300-700 ° C of detonation nanodiamond in 100 μl of buffer solution (200 mM Tris-HCl, 250 mM NaCl, 25 mM EDTA, 0.5% SDS, pH 7.5).

Lysis A 50 mg sample of plant tissue or a product based on raw materials of plant or animal origin is carried out in 400-500 μl of a buffer solution with the addition of 10 μl of ribonuclease for 15-20 minutes at a temperature of 65 ° C in a 1.5 ml tube. The resulting lysate is centrifuged at an acceleration of 12,000 g for 2 minutes. The supernatant in a volume of 300-400 μl (depending on the humidity of the tissue sample) is transferred to a clean 1.5 ml tube.

Purification of 300-400 μl of the obtained biological solution is carried out by adding a suspension to a final concentration of detonation nanodiamonds of 5-10% and stirred for 1 min. After that, 200 μl of 5M potassium or sodium acetate are added, stirred for 1 minute and cooled to 0 ° C for 10 minutes.

Separation of the mixture is carried out by centrifugation for 5-10 minutes at 12000 g. The supernatant containing the DNA was transferred to a clean 1.5 ml tube. As a result, impurities inhibiting the polymerase chain reaction — peptides, pigments, tannins, and low molecular weight compounds — are removed from the solution.

From the prepared DNA solution, 1 ml of 96% ethanol or 400 μl of isopropanol is precipitated, centrifuged for 2–3 min at an acceleration of 12,000 g, and the supernatant is removed.

The precipitate was washed in 70% ethanol, dried in an open tube for 2 min at 65 ° C and dissolved in 100 μl bidistilled water.

Example 1

To identify the optimal conditions for the preliminary preparation of the sorbent, 6 samples of detonation nanodiamonds are calcined (TU 84-112-87, manufactured by Altai Federal Research and Production Center) in vacuum (10 ^-2 Torr) at a temperature of 300 ° C to 700 ° C for 1 hour.

DNA is extracted from 6 samples of 50 mg of living tissue from Allium sativum L. leaf. A suspension of each sample of 30 mg of detonation nanodiamond in 100 μl of buffer solution (200 mM Tris-HCl, 250 mM NaCl, 25 mM EDTA, 0, 5% SDS, pH 7.5).

Lysis of 6 identical samples of plant tissues is carried out in 400 μl of buffer solution with the addition of 10 μl of RNase for 15 min at a temperature of 65 ° C in 1.5 ml tubes. The resulting lysate is centrifuged for 2 min at 12000 g. The supernatant was transferred to clean 1.5 ml tubes.

Purification of the resulting biological solution is carried out by introducing a suspension of detonation nanodiamond and then stirred for 1 min by inverting the tubes. Samples are cleaned with variously prepared detonation nanodiamonds in the following combination:

sample No. 1 - preliminary annealing of nanodiamonds at 300 ° C;

sample No. 2 - preliminary annealing of nanodiamonds at 400 ° C;

sample No. 3 - preliminary annealing of nanodiamonds at 500 ° C;

sample No. 4 - preliminary annealing of nanodiamonds at 600 ° C;

sample No. 5 - preliminary annealing of nanodiamonds at 700 ° C.

After that, 200 μl of 5M sodium acetate was added and cooled to 0 ° C over 10 minutes.

Separation of the mixture is carried out by centrifugation for 10 min at 12000 g. The supernatant containing the DNA was transferred to a clean 1.5 ml tube. 400 μl of isopropanol is precipitated from the DNA solution.

The precipitate was washed in 70% ethanol and dissolved in 100 μl bidistilled water.

As a result, 6 samples of DNA solution were obtained, the quality of which was checked spectrophotometrically, the measurement results are presented in table 1.

The ratio of extinction at the wavelengths of DNA and protein (OD ₂₆₀ / OD ₂₈₀ ) for very pure DNA has a range from 1.7-1.8 and higher. Thus, the maximum DNA isolation (OD ₂₆₀ / OD ₂₈₀ = 2.1) is obtained by calcining a detonation nanodiamond at 500 ° C for 1 hour; the best quality is obtained by calcining a detonation nanodiamond in a temperature range from 400 to 700 ° C for 1 hours.

Example 2

To assess the quality of DNA, Hedysarum neglectum Ledeb leaves are extracted from dried and stored for 5 years leaves. with the preparation of 6 samples of detonation nanodiamonds analogously to example 1, except that the lysis is carried out for 40 minutes The result is 6 DNA samples, depending on the temperature of preparation of detonation diamond. The length of the obtained DNA fragments was evaluated by electrophoresis on 1% agarose gel for 3 hours at a voltage of 2.5 V / cm, using a molecular weight marker to estimate the length of the reference 100 bp (base nucleotide pairs). After staining the gel with ethidium bromide solution, DNA is photographed in UV radiation of 312 nm (Figure 1). The stability of the length of fragments of the extracted DNA is revealed, regardless of the conditions for the preparation of detonation nanodiamonds. The highest DNA content is detected by luminescence for sample No. 3, which corresponds to a detonation nanodiamond preparation temperature of 500 ° C.

Example 3

To select RAPD primers and identify species-specific markers, DNA is extracted from 30 mg of leaves of Papaver somniferum L. and Papaver orientale Bieb.

Lysis of two tissue samples of different plant species is carried out in 500 μl of buffer solution with the addition of 10 μl of RNase for 30 min at a temperature of 65 ° C in 1.5 ml tubes. The resulting lysate is centrifuged for 2 min at 12000 g. The supernatant was transferred to clean 1.5 ml tubes.

Purification of the resulting biological solution is carried out by introducing into each sample a suspension of 100 μl of buffer and 30 mg of detonation nanodiamond pre-annealed at 450 ° C for 2 hours in vacuum (10 ^-3 Torr). After that, 200 μl of 5M sodium acetate was added and cooled in the freezer for 10 minutes.

Separation of the mixture is carried out by centrifugation for 5 min at 12000 g. The supernatant containing the DNA was transferred to a clean 1.5 ml tube. 800 μl of 96% ethanol are precipitated from the DNA solution. After centrifugation, the supernatant was drained and, after drying, the DNA pellet was dissolved in 100 μl bidistilled water.

Amplification was performed with commercial RAPD primers. The amplified DNA is dispersed on an agarose 1.5% gel at 5 V / cm for 2.5 hours in parallel with a molecular weight marker. The gel is stained with ethidium bromide solution and visualized in UV radiation (Figure 2). According to the photograph of the gel, differences in the patterns of S and O samples are revealed, which corresponds to the difference in the genomes of the two species.

This example confirms the possibility of using DNA extracted using the developed method to solve typification and molecular systematics of plants using the RAPD method.

Example 4

DNA is isolated from freshly dried leaves of Hedysamm theinum Krasnob, similarly to Example 3, the obtained DNA is used to amplify the ITS 1 fragment in the BioRad thermal cycle using the Medigen PCR kit. Used primers:

ITS-1 (5'-TCCGTAGGTGAACCTGCGG-3 '),

ITS-2 (5'-GCTGCGTTCTTCATCGATGC-3 ').

The amplified DNA is dispersed on an agarose 1.5% gel at 5 V / cm for 2.5 hours in parallel with a molecular weight marker and a control sample. The gel is stained with ethidium bromide solution and visualized in UV radiation (Figure 3). An amplified DNA region (patterns No. 1 and No. 3) of about 700 bp in length is detected. Subsequently, the amplified region was purified from the gel using a NucleoSpin kit and sequenced. The decrypted sequence of the amplified region is compared with the ITS sequence 1 fragment of other species of the genus Hedysarum taken from freely available Internet databases. Thus, the nucleotide sequence of DNA, in the case of isolation in this way, is not violated and the DNA molecule is not modified. This example confirms the possibility of using the DNA obtained using the developed method to decipher its sequence by sequencing.

The proposed method for the isolation and purification of DNA avoids the use of highly toxic compounds such as phenol and chloroform. The DNA solution obtained in this way contains both short and long fragments. Contamination of the obtained DNA with chemically active substances is prevented. Due to the characteristic black color of detonation nanodiamonds, the process of fraction separation is facilitated - after centrifugation, the phase separation boundary is clearly visible. If the detonation nanodiamond accidentally enters the final solution, the polymerase chain reaction is not blocked, and it is possible to separate the insoluble precipitate of detonation nanodiamond from the DNA solution. In view of the constancy of the sizes of detonation nanodiamond nanoparticles, regardless of the synthesis conditions, the adsorption capacity of the cleaning suspension is stable.

Literature

1. Doyle J.J. and J. L. Doyle. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. // Phytochemical Bulletin, 1987 .-- T.19. - P.11-15.

2. RF patent No. 2272072, cl. C12N 15/10, publ. 03/20/2006.

3. RF patent No. 2119954, cl. C12N 15/10, С07Н 21/00, С07Н 21/02, С07Н 21/04, publ. 10/10/1998.

4. Friesen N. Molecular methods used in plant taxonomy. - Barnaul: Alphabet, 2007 .-- 64 p.

Claims (1)

The method of isolation and purification of deoxyribonucleic acids, which consists in the fact that after lysis of the sample in a buffer based on an anionic detergent, the solution is purified and the DNA is precipitated with alcohol, characterized in that a suspension of detonation nanodiamond to a final concentration of 5 to 10% at pH is used to clean the solution 7.5, pre-annealed at a temperature from + 400 ° to + 700 ° C in a vacuum of 10 ^-2 to 10 ^-3 torr, then add potassium or sodium acetate, is stirred, the mixture was cooled at 0 ° C, centrifuged and separated detonation nano ointments, non-nucleic sorbing material nature, and the supernatant containing DNA.

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