RU2595816C1 - Method of preparing suspension of human lymphocytes in ethanol-acetic retainer for dna extraction - Google Patents

Abstract

FIELD: biology.

SUBSTANCE: invention relates to molecular biology and can be used in biomedical research. Disclosed is a method of preparing a suspension of human lymphocytes in ethanol-acetic retainer for DNA recovery, which includes centrifugation of said suspension and removal of supernatant. Then obtained preparation is dried for 5 minutes at temperature of 55 °C, followed by adding physiological solution.

EFFECT: method ensures preservation of qualitative and quantitative characteristics of sample, acceptable for further polymerase chain reaction.

1 cl, 3 dwg, 1 ex

Description

The invention relates to molecular biology and can be used in biomedical research.

DNA analysis as a carrier of genetic information plays a key role in a number of biological and medical studies. In the practice of biomedical research, a variety of sources of nucleic acids (NK) are used: from epithelial scrapings to dry blood stains and permanent preparations of human tissues [1]. Moreover, DNA extraction from fixed tissue samples is difficult. Thus, when cells are treated with ethanol-acetic or methanol-acetic fixative, the biological material is dehydrated, macromolecules are denatured and their solubility decreases, which makes it impossible or very difficult to transfer nucleic acids to aqueous solutions.

Despite the wide variety of protocols for NK isolation, there is still no unified scheme for obtaining DNA preparations from fixed human lymphocytes. However, this type of biological material in some cases is the only potential source of DNA. So, in the practical activities of cytogenetic laboratories, both scientific and clinical, fixed lymphocytes are used to prepare chromosome preparations and are collection material [2]. If retrospective analysis is necessary, samples of such a collection can be used to obtain and further analyze nucleic acids.

As an analog, a method for isolating DNA from human liquid media was adopted [3]. The method is based on the lysis of cellular elements by the chaotropic agent guanidine thiocyanate (GuSCN) with the simultaneous binding of NK released by a sorbent based on silica or diatomite. Subsequently, the sorbent containing DNA is freed from impurities during successive washes using a buffer with GuSCN, 70% ethanol solution, and finally acetone. The sorbent is dried, and in the last step, the bound DNA elutes into the TE buffer.

The most technologically closest to this analogue and the claimed invention is a known method for the selection of NK, as claimed in patent No. 2272072 "Method for the isolation of nucleic acids" [4]. A known method for the isolation of nucleic acids involves mixing a sample with a buffer solution containing a chaotropic agent, adsorption of nucleic acids on a glass fiber sorbent, washing it from impurities of a non-nucleotide nature, and eluting the nucleic acids, characterized in that the sorbent is pre-treated with a 0.1-7.0% solution hydrofluoric acid for 1-6 hours

This method is adopted as a prototype. In the claimed modification, a fiberglass sorbent is used as a NK carrier, and buffer solutions containing different ratios of GuSCN, EDTA and Tris-acetate are used for selective isolation of NK of various types. Separation of the sorbent from the liquid phase is carried out under pressure, vacuum or centrifugation, and the elution of the NK is carried out with a buffer solution with bicarbonate and EDTA ions or distilled water.

A common drawback of the above analogue and prototype is the impossibility of their direct use for the isolation of NK from fixed cells and human tissues due to the high content of denaturing substances in the latter. It is also significant that a number of rare and expensive reagents are used to perform these technologies in the laboratory, some of which require special storage and operating conditions. The need for independent preparation of working solutions and materials from them greatly complicates the standardization of methods and, as a result, can negatively affect the qualitative and quantitative characteristics of the final product. All this makes the application of the proposed approaches in a number of laboratories unacceptable.

An object of the invention is to obtain DNA from human lymphocytes that have undergone treatment with ethanol-acetic fixative, while maintaining the qualitative and quantitative characteristics of the sample, acceptable for further polymerase chain reaction.

The term “polymerase chain reaction” is hereinafter used in abbreviated form by PCR.

In the present invention, this technical result is achieved by an additional stage of sample preparation, in which the fixative is replaced by physiological saline, which is a 0.9% NaCl solution, and the subsequent isolation of DNA by a sorbent method. The technology must meet the requirements of safety, simplicity and high reliability, which is achieved using ready-made commercial reagents and materials that have passed certification.

A method is proposed for preparing a suspension of human lymphocytes in an ethanol-acetic fixative for DNA extraction by centrifugation of the suspension, removal of the supernatant, drying of the resulting preparation for 5 minutes at a temperature of 55 ° C, after which physiological saline is added.

The claimed method includes the following sequential stages:

1. A suspension of lymphocytes in an ethanol-vinegar fixative (3: 1) in a volume of 10 ml is placed in 15 ml centrifuge tubes and centrifuged for 10 minutes at 1200 rpm. The supernatant is removed to a volume of 1 ml.

2. The pellet is resuspended, the suspension enriched in lymphocytes is transferred into clean 1.5 ml microtubes. Centrifuge the microtubes for 10 minutes at 10,000 rpm. The supernatant is completely removed.

3. Place microtubes with open caps on a solid-state thermostat preheated to 55 ° C. Dry the drug for 5 minutes.

4. To the dried sediment of lymphocytes add 100 μl of saline, carefully resuspended.

5. To the resulting suspension add 300 μl of a commercial lyse reagent. Thermostat the tube for 5 minutes at 65 ° C.

6. Add 20 μl of the sorbent suspension to the tube. The tube is shaken on a vortex for 10 seconds and then centrifuged for 15 seconds at 5000 rpm.

7. The supernatant is removed, 1.0 ml of alcohol-salt buffer is added to the precipitated sorbent, and intensively mixed.

8. Centrifuge the tube for 15 seconds at 5000 rpm.

9. Repeat washing of the sorbent with an alcohol-salt buffer according to claim 7. Centrifuge the tube for 15 seconds at 10,000 rpm.

10. The supernatant is removed, the precipitate is dried at 65 ° C for 5 minutes.

11. To the dried sorbent add 100 μl of distilled water, TE-buffer or other aqueous eluent.

12. Suspend the contents of the tube on the vortex for 10 seconds, after which the tube is thermostated at 65 ° C for 5 minutes. Resuspend the contents of the tube on a vortex.

13. Centrifuge the tube for 1 minute at 12,000 rpm. The supernatant containing DNA is transferred to a clean tube.

The essence of the proposed method and the results obtained are illustrated in FIG. 1, FIG. 2 and FIG. 3. In FIG. 1 shows the results of measuring the concentration of DNA isolated from a number of fixed lymphocyte samples of the claimed method, FIG. 2 - the results of measuring the ratio 260/280 in the obtained DNA preparations isolated by the claimed method, in FIG. 3 is an electrophoregram of PCR products carried out using DNA isolated by the claimed method.

In FIG. 1 presents the results of spectrophotometric determination of DNA concentration, confirming the implementation of the claimed method. DNA was isolated from 43 samples of lymphocytes in an ethanol-acetic fixative. Measurements were carried out on a NanoDrop 2000 instrument using equipment developer software (Thermo Fisher Scientific Inc.) by determining the absorption of a 260-nm wavelength of UV light. DNA concentration ranged from 18.9 ng / μl to 213.2 ng / μl with a median of 58.7 ng / μl. Thus, all samples contain DNA in an amount exceeding the minimum necessary for conducting a PNR (about 10 ng / μl) [5]. Transferring the starting material to physiological saline allowed us to successfully extract DNA from fixed lymphocytes. The absence of DNA preparations to be discarded in this experiment shows that the sign of “replacing the ethanol-acetic fixative with physiological saline” is significant, is in a causal relationship with the technical result, and allows to achieve the expected quantitative characteristics of the final product.

The degree of purification of DNA preparations from impurities, primarily of protein nature, is presented in FIG. 2. When evaluating this indicator for each drug, the ratio of the degree of absorption of UV light at 260 nm (maximum absorption for double-stranded DNA) to the degree of absorption of UV light at 280 nm (maximum absorption for proteins) was calculated. The value of the indicator 260/280 varied in the range of 1.47-2.53 with a median value of 1.96. Most of the samples were characterized by a good and excellent degree of DNA purification from protein impurities (1.7–2.0) [5].

Therefore, the sign “replacement of ethanol-acetic fixative with physiological saline” is essential and is in a causal relationship with the technical result and allows to achieve the expected quality characteristics of the final product.

Moreover, the defining hallmarks of the method compared with the prototype and confirming the advantage of the claimed invention are:

1. The possibility of using to obtain the DNA of human lymphocytes treated with ethanol-vinegar fixative.

2. The concentration of DNA in the obtained samples and the level of impurities in them allow further PCR.

3. To implement the method, ready-made commercial reagents and materials are used to ensure the simplicity and reliability of the method.

In FIG. 3 shows an electrophoregram of PCR products carried out using DNA isolated by the claimed method, which is explained in the example below, confirming the practical application of the claimed method.

Example 1 (studies were carried out by the inventors in the central research laboratory of GBOU VPO KemGMA).

To establish the genetic causes of male infertility in a group of patients, it was necessary to conduct a PCR analysis of microdeletions in the AZF region of the Y chromosome. The only available DNA source was peripheral blood lymphocytes prepared for cytogenetic studies and stored for a long time in suspension in ethanol-acetic fixative at a temperature of + 4 ° С.

A suspension of lymphocytes in an ethanol-acetic fixative in a volume of 10 ml was centrifuged in 15 ml tubes for 10 minutes at 1200 rpm. The supernatant was removed to a volume of 1 ml, and the pellet was resuspended and transferred to 1.5 ml microtubes. The microtubes were centrifuged for 10 minutes at 10,000 rpm. The supernatant was completely removed, the precipitate was dried for 5 minutes at 55 ° C. 100 μl of physiological saline (0.9% NaCl) was added to the dried lymphocyte pellet, carefully resuspended.

To the resulting suspension was added 300 μl of a commercial lysing reagent from the Universal Sample Preparation (UP) kit manufactured by Genlab Research and Production Company LLC, the tube was thoroughly mixed and heated for 5 minutes at 65 ° C. 20 μl of a sorbent suspension from the UP kit were added to the tube. The tube was shaken on a vortex, and then centrifuged for 15 seconds at 5000 rpm. The supernatant was removed.

The precipitated sorbent was washed with 1 ml of alcohol-salt buffer, the tube was centrifuged for 15 seconds at 5000 rpm, the supernatant was removed. The washing of the sorbent with 1 ml of alcohol-salt buffer was repeated. The tube was centrifuged for 15 seconds at 10,000 rpm, the supernatant was removed, and the pellet was dried at 65 ° C for 5 minutes.

DNA was transferred into the aqueous phase using 100 μl of the eluent from the UP kit at 65 ° C for 5 minutes, the tube was centrifuged for 1 minute at 12000 rpm. The supernatant containing DNA was transferred to a clean tube and used for PCR.

Multiplex PCR was performed to simultaneously diagnose AZF deletions in the AZFa, AZFb, AZFc Y chromosome subregions. At the same time, a commercial set of “AZF-deletions” produced by the Scientific and Production Company Litekh LLC was used. As targets for PCR, STS markers are used, which are localized in the subregions AZFa (sY84 and sY86), AZFb (sY127 and sY134), AZFc (sY254 and sY255) (Fig. 3). The targets of internal control are the sites of the ZFX / Y and SRY loci located in the short arm of Y chromosome. For each test sample, 2 tests are performed simultaneously (system A and system B), which increases the reliability of the analysis.

The DNA of the fertile man included in the reagent kit (K +) was used as a positive control sample. As can be seen, the DNA samples of all patients (No. 1-6) were characterized by high PCR efficiency for all markers, not inferior in quality to the positive control sample. This indicates, on the one hand, the absence of DNA degradation during the isolation procedure itself and its high concentration in the final solution. In addition, there are no PCR inhibitors in the sample or their content is not sufficient for a significant deterioration in the quality of PCR.

Based on the results of genotyping, it was possible to draw a conclusion about the presence of all studied STS markers in patients, which indicates the absence of AZF microdeletions in them. Since in this experiment a batch of biological samples from several people was used, we can talk about the high reproducibility of the result of applying the claimed invention. The claims "replacement of ethanol-acetic fixative with physiological solution, while replacing the fixative by centrifuging the cell suspension, removing the supernatant, drying the resulting preparation for 5 min at 55 ° C, adding physiological saline and resuspending the cell mass" allows you to get the DNA preparation, complying with the requirements of PCR. Therefore, the claimed technical result is achieved.

Used Books

1. Kornienko I.V. Methods of researching human DNA. DNA isolation, its qualitative and quantitative assessment in the aspect of the forensic medical study of material evidence of biological origin / I.V. Kornienko, S.G. Kharlamov. - Rostov-on-Don: publishing house of the Southern Federal University, 2012. - 216 p.

2. Klaus J. Lymphocytes: Methods / J. Klaus. - M .: Mir, 1990 .-- 394 p.

3. Boom R. Rapid and simple method for purification of nucleic acids / R. Boom, C.J.A. Sol, M.M.M. Salimans et al. // J. Clin. Microbiol. - 1990. - V. 28, No. 3. - R. 495-503.

4. A method of producing nucleic acids: US Pat. No. 2272072 (application No. 2004126133, IPC class C12N 15/10) / P.L. Laktionov S.M. Tamkovich, E.Yu. Rykova et al .; publ. 03/20/2006.

5. Rethmeyer J.A. Comparison of biological specimens and DNA collection methods for PCR amplification and microarray analysis / A.J. Rethmeyer, X. Tan, A. Manzardo et al. // Clin Chem Lab Med. - 2013 .-- 51 (5): doi: 10.1515 / cclm-2012-0429.

Claims (1)

A method of preparing a suspension of human lymphocytes in an ethanol-acetic fixative for DNA extraction, including centrifuging the suspension, removing the supernatant, drying the resulting preparation for 5 minutes at 55 ° C, after which physiological saline is added.

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