RU2753768C1 - Dna extraction kit - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention relates to medicine and is a set for extracellular DNA extraction consisting of a binding buffer based on 5.5 M guanidine thiocyanate, 5 mM Trilon B, 10 mM Tris-HCl pH 8.0, a washing buffer based on 80% ethanol and an eluting buffer based on 10 mM Tris-HCl and 1 mM Trilon B with pH 8.5, a DNA-affine polypeptide (thermal polylysine with a molecular weight of 10-30 kilodaltons) and suspensions of sorbent: magnetite particles coated with a layer of silica gel, with a total diameter of 1.5 microns.EFFECT: invention makes it possible to increase the yield of extracellular DNA from biological fluids, provides the purity of the released DNA and reduces the time of its isolation.1 cl, 2 dwg, 1 tbl, 1 ex

Description

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

In recent years, a new approach to personalized disease risk assessment has been intensively developed - the so-called "liquid biopsy" - minimally invasive, early diagnosis of tumors, non-oncological pathologies, as well as prenatal diagnostics based on genetic analysis of extracellular, so-called "free circulating extracellular DNA" (cfDNA). The main advantage of the "liquid biopsy" platform being developed is the low invasiveness of taking a biological sample. The key point and complexity of this approach is the need to efficiently isolate ultra-small amounts of extracellular DNA, measured in nanograms per milliliter, from biological fluids containing a large amount of protein, millions of times higher than the amount of cfDNA. The kits widely used for the isolation of genomic DNA from tissues and cells are not suitable for these purposes, since cfDNA is lost during the isolation process.

Existing commercial kits (QIAGEN, Zymo, Applied Biosystems, Invitrogen, Macherey-Nagel) for isolation of cfDNA are based on the preliminary enzymatic removal of excess protein by proteinase K in a buffer containing the detergent sodium dodecyl sulfate, followed by the binding of deproteinized cfDNA with magnetic silica gel layer of silica gel) in the presence of high concentrations of guanidine thiocyanate (GTC), for which 2-3 volumes of GTC solution are added to the initial volume of serum, which significantly increases the initial volume of biofluid and reduces the efficiency of cfDNA isolation, and in addition, it is extremely inconvenient for subsequent manipulations with large volumes liquids requiring a switch from standard 1.5 ml sample tubes and centrifuges to more expensive centrifuges operating in the 15-50 ml range. The use of Proteinase K and additions of 2-3 fold volumes of binding solution forms the basis of all currently available commercial kits for the isolation of extracellular DNA from biological fluids QIAamp Circulating Nucleic Acid kit (Qiagen), MagMAX Cell-Free DNA Isolation Kit (Applied Biosystems) , Quick-cfDNA Serum & Plasma Kit (ZYMO Research), PME Free-Circulating DNA Extraction Kit and PME Free-Circulating DNA (Analyti Jena, AG), Apostle MiniMax ™ High Efficiency Cell-Free DNA Isolation Kit (Beckman Coulter).

The most commonly used and close to the proposed one is the QIAamp Circulating Nucleic Acid kit from Qiagen (QIAamp Circulating Nucleic Acid Handbook, October 2019. QIAGEN), which we have taken as a prototype.

The prototype includes the following components: proteinase K solution, lyophilized RNA for a block of non-specific sorption of the column membrane, 5 buffer solutions (lysis, binding (5.5 M guanidine thiocyanate, 5 mM Trilon B, 10 mM Tris-HCl pH 8.0), the first wash (guanidine chloride), the second wash (80% ethanol) and elution (10 mM Tris-HCl and 1 mM Trilon B with pH 8.5), columns with a DNA-binding membrane, as well as column packings that allow you to filter volumes of liquid 2-10 ml, column and receiver connectors, the actual eluate receiver and a vacuum pump for sucking diluted eluate through the column membrane The kit allows for the isolation of extracellular DNA from blood plasma or urine in high yield.

According to the prototype kit protocol, 1120 ng of synthetic RNA is added to each blood plasma sample to block nonspecific DNA sorption by the column membrane. The RNA introduced into the sample is isolated along with the desired extracellular DNA. Any impurity in the purified DNA preparation is extremely undesirable, since it can inhibit the activity of thermopolymerase, thus reducing the efficiency of PCR analysis.

The use in the prototype kit of enzymatic degradation of protein using proteinase K and a special lysis buffer to remove excess plasma protein leads to additional dilution of the sample and lengthening of the DNA extraction procedure.

Also, a significant limitation of the prototype kit is that the processed maximum sample volume according to the Qiagen protocol does not exceed 5 ml for plasma and 4 ml of urine. This especially limits the isolation of the amount of cfDNA required for subsequent analysis from urine, where it is often required to isolate from a volume of 30–40 ml.

The prototype kit requires the use of filter columns, column packings for increasing the volume of filtered liquid, a filtering receiver of the eluate, a vacuum pump, a thermostat and a centrifuge, that is, it is resource-intensive and labor-intensive. For this reason, the number of samples simultaneously processed using the prototype is limited by the number of receivers of the columns of the filtering device and amounts to 24 samples, which is insufficient for routine analysis of a large number of samples.

In connection with the foregoing, the procedure for isolating DNA with a prototype kit is quite laborious and time-consuming, it takes 2-3 hours, according to the prototype developer's company, and the isolated DNA also has an admixture of synthetic RNA and an admixture of protein.

The technical result of the present invention is to increase the yield of extracellular DNA from biological fluids, the purity of the extracted DNA, and also to reduce the time of its isolation.

This result is achieved by the fact that in a known kit for DNA isolation, consisting of a binding buffer based on 5.5 M guanidine thiocyanate, 5 mM Trilon B, 10 mM Tris-HCl pH 8.0, a wash buffer based on 80% ethanol and an elution buffer based on 10 mM Tris-HCl and 1 mM Trilon B with pH 8.5, according to the invention, the kit additionally contains a DNA-affinity polypeptide (thermal polylysine with a molecular weight of 10-30 kilodaltons) and a suspension of a sorbent - magnetite particles coated with a layer of silica gel, with a total diameter of 1.5 microns.

To date, the protocols of known kits for DNA isolation use the stage of enzymatic degradation of the protein (hydrolysis by proteinase K), followed by dilution of the original sample with lysis and binding solutions. This leads to a dilution of the sample and a decrease in the yield of extracellular DNA, as well as to the complication and lengthening of the isolation procedure.

In this regard, we tried to apply the method of DNA extraction based on affinity binding of DNA with a thermal polypeptide with polylysine and precipitation of the DNA-polylysine complex by centrifugation.

The use in the kit of a solution of DNA-affinity peptide (thermal polylysine with a molecular weight of 10-30 kilodaltons), which is 1/10 of the initial sample volume, provides DNA binding in a biological fluid. That allows you to concentrate DNA and separate it from excess proteins in biological fluids, followed by precipitation of the DNA-peptide complex by centrifugation.

Thus, instead of the enzymatic treatment of the sample and its substantial dilution with lysis and then binding buffers, we obtained a cfDNA-polylysine complex concentrated in the form of a precipitate and purified from the protein of the biological fluid in one quick procedure.

Suspension of a sorbent - magnetite particles covered with a layer of silica gel, with a total diameter of 1.5 microns, does not exhibit nonspecific DNA sorption. This makes it possible to exclude the use of synthetic RNA and which, ultimately, provides a higher purity of the desired DNA preparation. And when the binding buffer and the suspension of the sorbent are added to the precipitate of the DNA-polylysine complex, its dissociation occurs, the DNA binds to the sorbent, and the thermal polylysine is released into the aqueous phase.

All this allows a significant time of DNA isolation and to increase its yield and purity.

DNA isolation kit contains:

The kit consists of 4 solutions and a suspension of the sorbent, each of which is in a separate test tube and is added separately to the reaction mixture in accordance with the DNA extraction procedure below, as well as instructions for DNA extraction. The test tubes with the ingredients of the kit are in a cardboard box with cells for their vertical position.

The set has the following composition.

1. DNA-affinity polypeptide (thermal polylysine with a molecular weight of 10-30 kilodaltons) - 50 ml.

2. Binding buffer (5.5 M guanidine thiocyanate, 5 mM Trilon B, 10 mM Tris-HCl pH 8.0) - 50 ml.

3. Wash buffer (80% ethanol) - 50 ml.

4. Elution buffer (10 mM Tris-HCl and 1 mM Trilon B with pH 8.5) -1 ml.

5. Sorbent (10% suspension of magnetite, covered with a layer of silica gel with a particle size of 1.5-2 microns in a solution of 0.5 M sodium chloride) - 2 ml.

Examples of using the set

Example 1

To illustrate the production of genomic DNA, we give example No. 1 - 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 the sorbent suspension was added, stirred again and left for 10 min at room temperature, after which 0.5 ml of wash buffer, mixed and centrifuged at 5000 rpm for 1 min. The supernatant was carefully collected and discarded, and 30 μL of elution buffer was added to the sediment, 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. Was selected containing DNA supernatant and measured the concentration of DNA using a fluorometer. The yield was usually 5-20 ng of DNA from 1 ml of blood plasma, which corresponds to the maximum value. The technical result of the present invention is to simplify and accelerate the production of extracellular DNA from biological fluids (blood plasma, urine, saliva) by binding, aggregating and precipitating DNA. This result is achieved by the fact that in the known method for obtaining DNA, including the addition of 0.1 part of a solution of thermal polylysine (10 mg / ml) to 1 part of a biological fluid, thoroughly stirring the mixture for 10 min and precipitation of the DNA-thermal polylysine complex by centrifugation at 14 000 g for 5 minutes.

Figure 1 shows the comparative efficiency of extracellular DNA isolation from blood plasma using the Qiagen prototype kit - QIAamp Circulating Nucleic Acid kit (Fig.1A) and our kit (Fig.1B), assessed by the fluorescence method (Qubit dsDNA HS Assay Kit and Qubit 4 fluorometer) and quantitative PCR (ABI 7500 Fast Real Time PCR). The fluorescence method showed that both kits release the same amount of DNA within the measurement error, while the functional test - quantitative PCR showed that our kit releases 34.6% more DNA, which may be due to the presence of polymerase inhibitors in the DNA preparation obtained with using the Qiagen kit, or with differences in the composition of the DNA population obtained with the two kits. To verify this, we checked the spectra of the DNA extracted with these kits. Since the extracellular DNA of biofluids is highly degraded and its size is about 150 base pairs or less, the efficiency of isolation of short fragments less than 100 base pairs (bp) is very important. The efficiency of isolation of the kits was assessed using a mixture of low molecular weight DNA fragments of different lengths of known size (Ultra Low Range DNA ladder, Thermo Fisher) as a reference DNA, added to the urine before isolation with our kit and the Qiagen kit relative to the original mixture of DNA fragments.

After isolation from urine, DNA was separated by electrophoresis in 10% polyacrylamide gel, stained with SYBR Green fluorescent dye, photographed in UV light, and the peak areas were digitized (ImageQuant 7.0 software, Cytiva).

Table 1
Efficiency of isolation of DNA fragments of different lengths DNA fragment length Efficiency
Our set (A) Efficiency
Qiagen set (B) 100 bp 100% 100% 75 bp 98.7% 69.9% 50 bp 55.9% 34.9% 35 bp 35.3% 0.4%

The results are summarized in Table 1, they indicate that both kits with equal efficiency isolate DNA fragments of 100 base pairs, DNA fragments of 75 bp. are distinguished by our kit by 30% more than Qiagen, the difference is even more noticeable when considering the results of isolation of a fragment of 50 bp, and DNA fragments of 35 bp in size. the Qiagen kit barely extracts, whereas our kit extracts 35.3% of these fragments.

An important criterion for the purity of the cfDNA obtained using the kit is the amount of protein and RNA admixture. Figure 2 shows the results of quantitative protein measurement using the highly sensitive Qubit Protein Assay Kit fluorescence method and the Qubit 4 fluorometer (Invitrogen). The protein content in the cfDNA preparation from blood plasma isolated by the Qiagen kit (Fig. 2A) was 4.9 μg, while in the cfDNA preparation from blood plasma isolated by our method, the protein impurity was 2 times less and amounted to 2.4 μg ( Fig. 2 B).

The content of impurity RNA in cfDNA preparations was measured by the highly sensitive and specific fluorescence method Qubit RNA HS Assay Kit (Invitrogen). In the cfDNA preparations isolated by our kit, no RNA impurities were detected, while all cfDNA preparations isolated by the Qiagen kit contained an RNA impurity of about 770 ng, which is due to the fact that, according to the company's protocol, synthetic RNA is added to each blood plasma sample in the amount of 1120 ng (QIAamp Circulating Nucleic Acid Handbook, October 2019. QIAGEN). Thus, in terms of purity, our kit allows for a more highly purified cfDNA compared to the kit from QIAGEN.

The procedure for DNA isolation with the QIAGEN kit takes 2-3 hours, while ours ends within 1 hour, while it does not include the stage of enzymatic degradation of the protein in the sample using proteinase K, does not require the addition of synthetic RNA to block nonspecific sorption of the column membrane. The QIAGEN kit requires the use of filter columns, column packings for increasing the volume of filtered liquid, a filter eluate receiver, a vacuum pump, an oven and a centrifuge, while our kit's instruments are limited to a centrifuge and an oven. For this reason, the maximum sample volume processed by the QIAGEN kit does not exceed 5 ml of plasma and 4 ml of urine, while the maximum sample volume processed by our kit is 45 ml, that is, 9-11 times more and does not depend on the nature of the biological fluid. The number of samples processed simultaneously using the QIAGEN kit is limited by the number of column receivers of the filtering device and amounts to 24 samples, while the format of our kit - sorbent in suspension - allows the simultaneous extraction of 36 or more samples.

As can be seen from the above examples, the proposed kit provides a high yield of extracellular DNA corresponding to the prototype, while the DNA extraction time is 2-3 times shorter than that of the prototype, it is not associated with a two-fold dilution of the sample as in the prototype, which makes it possible to isolate cfDNA from volumes up to 45 ml, does not require the use of filter columns, nozzles and a pump, as well as a high degree of DNA purity, which allows it to be used in medical practice for diagnostic purposes.

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

1. The use of a DNA-precipitating agent (thermal polylysine) instead of enzymatic degradation of the protein in the sample provides for obtaining more pure DNA preparations in comparison with the prototype.

2. The use of a DNA-affinity polypeptide (thermal polylysine) provides higher quality extracellular DNA preparations, enriched with DNA fragments with a lower molecular weight, which are lost during isolation by the prototype.

The kit was developed in the genetic engineering laboratory of the Federal State Budgetary Institution “Russian Scientific Center for Radiology and Surgical Technologies named after Academician A.M. Granov "of the Ministry of Health of the Russian Federation.

Claims (1)

Extracellular DNA isolation kit, consisting of binding buffer based on 5.5 M guanidine thiocyanate, 5 mM Trilon B, 10 mM Tris-HCl pH 8.0, wash buffer based on 80% ethanol and elution buffer based on 10 mM Tris-HCl and 1 mM Trilon B with a pH of 8.5, characterized in that the additional kit for DNA isolation contains a DNA-affinity polypeptide, namely thermal polylysine with a molecular weight of 10-30 kilodaltons, and a suspension of a sorbent - magnetite particles coated with a layer of silica gel, total with a diameter of 1.5 microns.

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