RU2539030C1 - Kit for dna recovery - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: kit consists of a lysing buffer based on 3-10 M urea solution, a binding buffer based on 2.5-5 M sodium chloride, an eluating buffer based on 10 mM Tris-HCl and 1 mM Trilon B at pH 8 and 80% ethanol as a washing buffer. As a sorbent, the kit comprises quartz sand having a particle size of 10-50 mcm.

EFFECT: kit enables the high-yield, high-purity and large molecular weight DNA recovery from biological objects.

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Description

The invention relates to medicine, namely to biochemical studies of biological objects, and can find application in the diagnosis of malignant, viral and hereditary diseases.

The currently existing commercial kits for the isolation of DNA from eukaryotic cells, including humans, are based on the reversible sorption of DNA molecules on synthetic silica gel particles in buffers containing high concentrations of sodium iodide, sodium perchlorate or guanidine thiocyanate [Proc. Natl. Acad. Sci. 1979, v. 76, 615-619, 2; Nucl. Acids Res. 1990, v. 18, 1074].

For example, sodium iodide is used as a sorption buffer in the BIO 101 ″ Geneclean ″ DNA isolation kit. This kit contains the following components: 6 M sodium iodide solution, a suspension of Glassmilk sorbent and a wash buffer consisting of sodium chloride, Tris-HCl, Trilon B, ethyl alcohol and water [The Geneclean Kit. 1989. BIO 101 Inc., Release 7]. This kit is used to isolate plasmid DNA from bacteria and agarose gels. The quality of the extracted DNA is in line with the standard. However, the presence of sodium iodide in the kit, which is capable of irreversibly binding DNA to the sorbent, leads to its loss during isolation, and the residual admixture of sodium iodide can inhibit the activity of enzymes used for subsequent DNA modification.

Bio-Rad's “Prep-A-Gene ″ DNA isolation kit is also known, which contains the following components: 6 M sodium iodide solution as binding buffer, a suspension of sorbent in a solution of 6 M sodium perchlorate and washing buffer [Prep-A -Gene DNA Purification Kit. 1990. Bio-Rad Laboratories, Bulletin 1541]. This kit is used to isolate plasmid DNA and extract DNA fragments from agarose gels. However, the value of the kit is reduced due to the use of sodium iodide and sodium perchlorate, whose residual impurities can inhibit the activity of DNA modifier enzymes.

The vast majority of commercial kits are based on the use of guanidine thiocyanate as a lysing and binding agent [Lab. Times 2005, 5: 51-58].

A common drawback of guanidine isothiocyanate-based DNA isolation kits is that the smallest residual impurities of this agent in the protocol eluted by DNA inhibit the activity of enzymes used for subsequent DNA modification, including polymerase chain reaction, which significantly reduces the value of these kits.

To eliminate this drawback, we previously developed a kit for DNA isolation [Patent No. 21116795 for the invention of “Kit for DNA isolation”. Registered on August 10, 1998, C07H 21/04] based on ingredients whose residual impurities do not inhibit enzyme activity. We have taken this kit as a prototype.

The kit consists of a lysis buffer consisting of a 3-10 M urea solution, a binding buffer consisting of 2.5-5 M sodium chloride, a wash buffer consisting of 80% ethyl alcohol, an elution buffer consisting of 10 mM Tris- HCl, 1 mm Trilon B with a pH of 8 and a sorbent consisting of a 50% aqueous suspension of silica gel.

The use of urea and sodium chloride, which have a mild denaturing effect and are easily washed from the sorbent, ensured the high quality of the obtained DNA and reduced the inhibitory effect on the DNA modification enzymes of residual impurities of the components of the buffers used. This is due to the fact that urea and sodium chloride, which are widely used in the isolation of native proteins, are physiological and do not introduce other components into the process of DNA isolation.

As in the vast majority of commercial kits, synthetic amorphous silica gel was used as a sorbent for DNA in the prototype. However, during the prolonged use of this kit for DNA isolation, as well as other commercial kits, the sorbent component of which are particles of synthetic amorphous silica gel, the following disadvantages were found: firstly, its sorption properties differ depending on the batch of amorphous silica gel used, which significantly affects on reproducibility of results, especially with regard to quantitative polymerase chain reaction; secondly, variations in the porosity of various batches of synthetic amorphous silica lead to variations in the impurities of the components of the ingredients of the set buffers in the final DNA preparation and, accordingly, to variations in the activity of DNA modification enzymes; thirdly, the spherical surface of synthetic silica gel particles has minimal sorption ability with respect to the secreted DNA molecules. This limits their use for diagnostic purposes, especially in the field of quantitative polymerase chain reaction.

The technical result of the present invention is to increase the yield of DNA, the purity of DNA and the higher molecular weight of the secreted DNA.

This result is achieved in that in the known DNA isolation kit consisting of a leading buffer based on a 3-10 M urea solution, binding buffer based on 2.5-5 M sodium chloride, washing buffer based on 80% ethanol, eluting based on 10 mm Tris-HCl and 1 mm Trilon B with a pH of 8, according to the invention, crystalline quartz sand with a particle size of 10-50 μm is used as the sorbent.

In the course of many years of research and comparative analysis of the sorption ability of various forms of silicon dioxide, as well as silicon compounds, both synthetic and natural, we found that particles of the natural crystalline form of silicon dioxide - quartz sand lack all of the above disadvantages, namely: not enough high sorption capacity of DNA, insufficient bond strength of DNA with the sorbent, as well as differences in the sorption capacity of DNA of different batches of silica gels. The reason for this is the following properties of the crystalline form of silicon dioxide: 1 - a compact regular quartz crystal lattice has a minimum number of micropores and, as a result, introduces minimal contamination with the kit ingredients in the desired DNA; 2 - a higher negative charge on the surface of quartz particles compared to amorphous silica gel leads to a stronger binding of DNA molecules to particles and, accordingly, provides less DNA loss during sorption and washing of DNA on the particles; 3 - crystalline quartz with particles of irregular shape in comparison with spherical particles of amorphous silica has a large surface and, accordingly, a large capacity in relation to the secreted DNA. It should also be noted that the cost of crystalline quartz sand is many times lower than the cost of synthetic amorphous silica gel. All this leads to the fact that the use of a kit for DNA extraction based on crystalline quartz sand can be more effective in comparison with both the prototype and similar commercial kits.

The use of a urea-based lysis buffer in the kit eliminates guanidine thiocyanate and thereby prevents the inhibition of the activity of enzymes used for subsequent DNA modification.

The use of sodium chloride as a binding buffer made it possible to exclude sodium perchlorate from the kit, which, like guanidine thiocyanate, is an inhibitor of enzyme activity.

The use of crystalline quartz sand (GOST 22551-77, grade OOVS-010-B: "Quartz sand and vein quartz, enriched and premium") with a particle size of 10-50 microns as a sorbent ensures high purity of DNA released due to the fact that crystalline quartz has a higher negative surface charge compared to amorphous silica gel, which provides a higher sorption capacity with respect to DNA, and a lower porosity of quartz crystals in comparison with silica gel particles provides a higher efficiency ivnost remove residual impurities buffers used in the process, which ultimately provides a higher purity of the desired DNA product.

The composition of the kit for DNA isolation:

The kit consists of 4 buffers and a sorbent, each of which is in a separate tube and added separately to the reaction mixture in accordance with the DNA isolation procedure described below, as well as DNA isolation instructions. Test tubes with kit ingredients are located in a cardboard box with cells for their vertical arrangement.

The set has the following composition:

Lysing buffer (4 M urea solution) - 1 ml;

Binding buffer (2.5 M sodium chloride solution) - 3 ml;

Washing buffer (80% ethanol solution) - 15 ml;

Elution buffer (10 mM Tris-HCl and 1 mM Trilon B with pH 8) -1.2 ml; Sorbent (50% suspension of quartz sand GOST 22551-77, grade OOVS-01 OV: “Quartz sand and vein quartz, enriched and premium”) with a particle size of 10-50 microns in a solution of 2.5 M sodium chloride) - 1 ml .

Examples of using the kit.

Example 1

To illustrate the production of genomic DNA, we give an example No. 1 - the isolation of DNA from human blood cells using this kit.

The precipitate of white blood cells obtained from 1 ml of whole blood was dissolved in 20 μl of lysis buffer, after which 100 μl of binding buffer was added, the mixture was stirred and 30 μl of a sorbent suspension was added, mixed again and left for 10 min at room temperature, after which it was added 0.5 ml of wash buffer was mixed and centrifuged at 5000 rpm for 1 min. The supernatant was carefully taken and discarded, and 30 μl of elution buffer was added to the precipitate, the mixture was pipetted and placed in a water bath at + 60 ° C for 20 min, after which it was centrifuged at 5000 rpm for 1 min. A DNA-containing supernatant was taken and the DNA concentration was measured using a fluorimeter. The yield was usually 15-20 μg of DNA from 1 ml of blood, which corresponds to the maximum value. The degree of DNA purification from protein impurities was determined spectrophotometrically by the value of the ratio A260 / A280, which was in the range of 1.8-2.0, which corresponds to the standard. DNA nativeness and its ability to serve as a substrate for modification enzymes was controlled by electrophoresis in 0.8% agarose, DNA was stained with ethidium bromide (0.5 μg / ml), and electrophoregrams were photographed in UV light using an orange filter.

The quality of the desired DNA is illustrated by electrophoregrams of the isolated genomic DNA and its cleavage products by restriction enzymes, which are widely used in molecular diagnostics. Figure 1 shows the results of DNA extraction from blood cells using a kit based on amorphous silica (prototype) (lane 1) and this kit (lane 2). Lambda phage DNA (lane M) and a yeast DNA ladder (lane M ′) were used as molecular weight markers. As can be seen from Fig. 1, the yield of genomic DNA is higher when using a kit based on crystalline quartz (lane 2) than when using a similar set based on amorphous silica (lane 1), as evidenced by a comparison of the signal intensities of ethidium bromide stained DNA bands on an electrophoregram . Figure 1 also shows that the molecular weight of DNA obtained using a kit based on crystalline quartz (lane 2) is 2 times larger than when using a similar set based on amorphous silica (lane 1), as evidenced by the lower electrophoretic the mobility of the desired DNA relative to molecular weight markers (lanes M and M ′).

A quantitative analysis of the yield of the desired DNA in three independent DNA isolations from blood samples with fluorescence measurement of the amount of DNA on a DyNa Quant 200 instrument (GE company) showed that the DNA yield using a kit based on crystalline quartz was 30-40% higher than in the case of the use of a similar set based on amorphous silica.

The key physicochemical parameter that determines the size and strength of DNA binding to the sorbent is the surface charge of the particles. Therefore, we compared the surface charge of quartz and silica particles. The surface charge of the quartz particles included in this kit and amorphous silica gel, which is a component of commercial DNA isolation kits, was determined as the value of the zetta potential using a Zetasizer Nano ZS device (Malvern). The zeta potential of quartz particles was -50.9 mW, and for three batches of amorphous silica this value was -13.6, -14.8, and -16.5 mW, respectively, i.e., the zeta potential of the surface of crystalline silica particles was more than three times higher than that of amorphous silica, which determines the higher sorption ability of quartz particles in relation to DNA.

The most important criterion for the purity obtained using a DNA kit is its ability to serve as a substrate for DNA modification enzymes used in molecular diagnostics. As a quality control of the desired DNA, its enzymatic hydrolysis with three restriction endanicleases was used. Figure 2 shows the results of DNA hydrolysis with the following endonucleases - EcoRI (lane 1), BamHI (lane 2) and HindIII (lane 3). Each enzyme was added in an amount of 1 unit per 1 μg of DNA, incubation was carried out at + 37 ° C for 12 h, analysis of DNA hydrolysates was carried out in 0.8% agarose, DNA staining was carried out with ethidium bromide (0.5 μg / ml), Electrophoregrams were photographed in UV light using an orange filter. A DNA ladder with a step of 1000 nucleotide pairs (lanes M) was used as a molecular weight marker. As shown by enzymatic hydrolysis of genomic DNA by three restriction endanucleases, the desired DNA is an excellent substrate for endonucleases.

As can be seen from the above examples, the proposed kit provides a high DNA yield, 30-40% higher than the prototype and other commercial kits, as well as a high degree of DNA purity, which allows its use in medical practice for diagnostic purposes.

The proposed set, in comparison with the known, has a number of significant advantages.

1. The use of crystalline quartz provides a higher yield of DNA preparations in comparison with the prototype.

2. The use of crystalline quartz provides a higher quality DNA preparations with a higher molecular weight compared to the prototype.

3. The components of the kit are physiological, have a mild denaturing effect, are easily washed from the sorbent, and thereby ensure high quality of the DNA obtained and the absence of residual impurities of the buffers used, which cannot be provided by any of the known kits.

4. The kit consists of readily available, non-toxic components and is the cheapest known kit for these purposes.

The kit was developed by the author in the laboratory of genetic engineering of the Federal State Budgetary Institution “Russian Scientific Center for Radiology and Surgical Technologies” of the Ministry of Health of the Russian Federation, underwent laboratory research at the Research Institute for Special Biological Drugs (St. Petersburg), Obstetrics and Gynecology Institute named after Otto (St. Petersburg), Research Institute of Virology. DI. Ivanitsky (Moscow) and is ready for implementation in wide medical practice.

Claims (1)

DNA isolation kit consisting of a lysing buffer based on a 3-10 M urea solution, binding buffer based on 2.5-5 M sodium chloride, washing buffer based on 80% ethanol, eluting buffer based on 10 mM Tris-HCl and 1 mm Trilon B with pH 8, and a sorbent, characterized in that as a DNA sorbent it contains crystalline quartz sand with a particle size of 10-50 microns.

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