RU2525710C1 - METHOD OF DETERMINING NUCLEOTIDE SEQUENCE Pu(5mC)GPy AT PREDETERMINED POSITION OF LONG-DISTANCE DNA - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method comprises obtaining samples of highly purified DNA, fragmentation of the isolated DNA with restriction endonuclease having no recognition site in the amplified region, hydrolysis of fragmented DNA by methyl-dependent site-specific DNA-endonuclease GlaI or its isoschizomer, ligation of the universal oligonucleotide adapter to the hydrolysed DNA followed by amplification in real time using a primer and a probe complementary to the test DNA and the hybrid primer, which 3'end is complementary to at least 3 nucleotides of the 3' terminus of DNA in the test area of hydrolysis GlaI, and the remaining part is complementary to an adapter sequence, and making conclusion on the basis of a fluorescent signal on the presence of the sequence Pu(5mC)GPy.

EFFECT: improved method.

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Description

The invention relates to biotechnology and relates to a method for determining the nucleotide sequence (site) of 5'-Pu (5mC) GPy-3 '/ 3'-PyG (5mC) Pu-5' (hereinafter 5mC - 5-methylcytosine) in a given extended position DNA

DNA methylation is one of the main and most studied mechanisms of epigenetic regulation of gene activity in higher eukaryotes. Methylation of CpG islands in the 5'-regulatory region blocks the transcription of the corresponding genes and normally occurs during inactivation of the X chromosome, as well as imprinting of individual genes [1]. In turn, aberrant methylation of PuCGPy sites (with the formation of the Pu (5mC) GPy sequence) by human DNA methyltransferases DNMT3a and DNMT3b in regulatory regions is associated with the development of certain diseases, in particular, oncopathologies [2, 3]. Hypermethylation of CpG islands in the regulatory regions of tumor suppressor genes, characteristic of tumor tissues, is also retained in malignant cell lines obtained from these tissues [4, 5].

The best known method for determining the epigenetic status of DNA sites is the use of restriction endonucleases blocked by methylation of cytosine bases at recognition sites. Most often, a pair of HpaII and MspI endonucleases is used, which recognize the tetranucleotide sequence of CCGG but are blocked in different ways: MspI does not cleave the sequence (5mC) CGG, and HpaII does not cleave C (5mC) GG [6]. In the case of methylation of the internal CG dinucleotide of this site, it will be cleaved with the MspI endonuclease, and the DNA fragment will not be amplified, while the HpaII site will not cleave and the DNA fragment will be generated during PCR.

The disadvantage of this method is that the CCGG sequence, unlike PuCGPy, is not a de novo aberrant methylation site, so the diagnostic value of this method is lower. Other restriction endonucleases used for analysis (HhaI, BstUI, AciI, etc.) are also not convenient for a complete analysis of the presence of methylation, since their recognition sites include only some of the possible methylation points of eukaryotic DNA and, in addition, do not have isoshizomers differing in sensitivity to the methylated base in the CG dinucleotide (which is desirable for the presence of a positive control in experiments).

An alternative is to use methyl-dependent endonucleases that specifically fragment only methylated DNA to analyze DNA methylation status. Recently, foreign researchers used the McrBC enzyme for this purpose, which recognizes the sequence R (mC) N40-2000R (mC) and cleaves it near one of the two recognizable dinucleotides [7–9]. Currently, this approach is used only in scientific research and in clinical practice has not yet been applied. The use of McrBC endonuclease to analyze the presence of methylated DNA has several disadvantages. In particular, the dependence on two methylated cytosine bases removed at a considerable distance from each other limits the possibility of determining the presence of methylation in an arbitrarily selected region of DNA.

The use of the methyl-dependent site-specific DNA endonuclease GlaI instead of McrBC, the recognition site of which fully corresponds to the reaction product resulting from de novo DNA methylation with cell methylases DNMT3a and DNMT3b [10, 11], allows to eliminate this drawback.

Closest to the claimed prototype method is a method for determining the nucleotide sequence of Pu (5mC) GPy in a predetermined position of extended DNA, including preliminary hydrolysis of the studied DNA with methyl-dependent site-specific DNA endonuclease GlaI and subsequent amplification in real time with PCR helpers [12] . The use of helpers, which are complex oligonucleotide structures, 3 'half of which is complementary to the studied DNA to the site of the proposed hydrolysis, and the 5' end encodes the sequence for the primer, allows you to accurately mark the site of GlaI hydrolysis, thus, the presence of the PCR signal clearly indicates the presence of the Pu sequence 5mC) GPy.

The disadvantages of this method are the duration, high cost and limited functionality. So, each round of amplification contains two stages of annealing and elongation: on the first, the helper is hybridized and then a DNA sequence complementary to the primer is completed from it, and then, after denaturation, the primer is hybridized and the amplicon continues to work. Since the helper and primer have similar structures, there is strong competition between them, to reduce which it is necessary to carefully select the primer / helper ratio and use single nucleotide substitutions for inosine in the helper structure. In addition, such a system is characterized by low PCR efficiency, a long reaction time (up to 85 rounds of PCR are necessary to obtain data), a high probability of premature decomposition of the components of the reaction mixture, and the need to synthesize a unique helper for each test sequence.

The objective of the invention is to simplify the known method and expand its functionality.

The problem is achieved by the proposed method, which consists in obtaining samples of highly purified DNA, preliminary fragmentation of the extracted DNA with a restriction endonuclease that does not have a recognition site in the amplified region, in particular TaqI; by hydrolysis of fragmented DNA by a methyl-dependent site-specific DNA endonuclease GlaI or its isoshizomer, ligation of a universal oligonucleotide adapter to a hydrolyzed DNA, followed by real-time amplification using a primer and probe complementary to the studied DNA and a hybrid primer, 5 'end of which is at least 3 3 'of the DNA end at the studied site of GlaI hydrolysis, and the remainder is complementary to the adapter sequence, and drawing up a conclusion on the appearance of fluorine stsentnogo signal in the case of sequence 5'-Pu (5mC) GPy-3 '/ 3'-PyG (5mC) Pu-5'.

Highly purified DNA is isolated as described previously in [13]. Preliminary DNA fragmentation is carried out in a reaction mixture containing DNA and TaqI restriction endonuclease (or its isoshizomer) for 2 hours at 65 ° C in buffer: 33 mM Tris-acetate, pH 7.9; 10 mM magnesium acetate, 66 mM potassium acetate, 1 mM dithiothreitol. Then the fragmented DNA is cleaved by adding 1-2 ea. GlaI methyl-dependent site-specific DNA endonuclease (or its isoschisomer), in the reaction buffer (20 mM Tris-SO ₄ , pH 8.0, 4 mM MgCl ₂ , 1 mM β-mercaptoethanol, 100 ng / μl BSA) for 1 hour at 30 ° C followed by inactivation at 65 ° C for 20 minutes. Further, ATP is added to the reaction mixture to a final concentration of 0.5-1.0 mM, PEG to 1-2%, β-mercaptoethanol to 6.8-8.0 mM, universal adapter (5'-CTCCCGCCTGCTCTTTCATCG-3 '/ 3'-ACGAGAAAGTAGC-5 ') to a final concentration of 500-1000 nM and 500-1000 ea highly active T4 DNA ligase. The adapter ligation reaction is carried out for 15 minutes at 25 ° C, then the ligase is thermally inactivated at 65 ° C for 20 minutes. To carry out a PCR reaction, the mixture is added until the final concentrations are reached: 50 mM Tris-SO ₄ , pH 9.0, 30 mM KCl, 10 mM ammonium sulfate, 3 mM MgCl ₂ , 0.2 mM, a mixture of deoxyribonucleoside triphosphates, 0.01% Tween- 20, a mixture of primers and probe of 0.4-0.8 μm each and 0.04-0.1 EA / μl of Hot Start DNA polymerase. The appearance of a fluorescent signal during PCR unambiguously indicates the presence of a Pu (5mC) GPy sequence at a given position of extended DNA.

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

1. Preliminary DNA fragmentation is carried out with a TaqI restriction endonuclease or another that does not have a recognition site in the studied region, which allows for a more complete cleavage of the original DNA;

2. The universal oligonucleotide adapter 5'-CTCCCGCCTGCTCTTTCATCG-3 '/ 3'-CGATGAAAGAGCA-5' is ligated to the hydrolyzed DNA, which makes it possible to simplify the method and expand its functionality due to the fact that all sequences are marked during the ligation of the hydrolyzed DNA with the universal adapter Pu (5mC) GPy, which allows you to analyze any Pu (5mC) GPy sequence on extended DNA, including several sequences simultaneously (multiplex);

3. Amplification is carried out using a primer and a probe complementary to the studied DNA and a hybrid primer, the 3 'end of which is complementary to at least three nucleotides of the 3' end of the DNA from the given site of hydrolysis of the methyl dependent site-specific DNA endonuclease, and the remainder is complementary to the adapter sequence, which improves the specificity of hybridization to the studied DNA region.

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

Example 1. The sequence determination of Pu (5mC) GPy in plasmid pHspAI2.

Plasmid pHspAI2 carries the M.HspAI methylase gene, which recognizes the 5'-GCGC-3 '/ 3'-CGCG-5' sequence and methylates internal cytosine to form the 5'-G (5mC) GC-3 '/ 3' sequence -CG (5mC) G-5 '. This sequence is a special case of the recognition site of methyl-dependent GlaI DNA endonuclease.

The preliminary fragmentation of plasmid pHspAI2 DNA was performed in 50 μl of the reaction mixture, including 5 μg of DNA and 20 units of GsaI restriction endonuclease for 2 hours at 65 ° C in buffer: 33 mM Tris-acetate, pH 7.9; 10 mM magnesium acetate, 66 mM potassium acetate, 1 mM dithiothreitol. As the control DNA, the unmethylated plasmid pBR322 was used, which was subjected to preliminary hydrolysis in the same manner with HindIII DNA endonuclease for 2 hours at 37 ° C in buffer: 10 mM Tris chloride, pH 8.5; 10 mm magnesium chloride, 100 mm sodium chloride, 1 mm dithiothreitol. Then, DNA hydrolysates were purified by phenol-chloroform extraction and precipitated with 96% ethanol according to the standard procedure [13], after which the DNA precipitates were dried at room temperature and dissolved in 40 μl of TE buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA ) The concentration of fragmented DNA was measured using a spectrophotometer and adjusted to a concentration of 1,500 copies / μl with a TE buffer solution containing 5 ng / μl of phage λ DNA. 6 different tubes were taken in the experiment (Table 1), 15 μl of the corresponding DNA was added to each. Test tube 1 - experiment, 2-4 - used as control of the corresponding stages, 5 - negative control. Then the fragmented DNA was digested by adding 1 e.a. GlaI methyl-dependent site-specific DNA endonuclease, in reaction buffer (20 mM Tris-SO ₄ , pH 8.0, 4 mM MgCl ₂ , 1 mM β-mercaptoethanol, 100 ng / μl BSA) for 1 hour at 30 ° C followed by inactivation at 65 ° C for 20 minutes. Further, ATP was added to the reaction mixture to a concentration of 0.5 mM, PEG to 1%, β-mercaptoethanol to 6.8 mM, a universal adapter to 500 nM and 1000 ea. highly active T4 DNA ligase. Ligated for 15 minutes at 25 ° C, then thermally inactivated at 65 ° C for 20 minutes. To conduct a PCR reaction, the mixture was added until the final concentrations were reached: 50 mM Tris-SO ₄ , pH 9.0, 30 mM KCl, 10 mM ammonium sulfate, 3 mM MgCl ₂ , 0.2 mM deoxyribonucleoside triphosphate mixture, 0.01% Tween-20 , a mixture of primers and probe of 0.4 μm each and 0.04 EA / μl of Hot Start DNA polymerase. The structure of the primers and the probe is as follows: direct - 5'-GACACATGCAGCTCCCGGAGA-3 ', hybrid - 5'-CCTGCTCTTTCATCG GCCCT -3' (the part of the primer complementary to the studied DNA is underlined), probe - 5'-FAM-TCTGCTCCCGTG AT3 '. The resulting mixture was divided into 3 parts and amplified on amplifiers DT-322 (DNA technology) or CFX-96 (Bio-Rad) according to the program: 95 ° C for 3 min, then 5 cycles without detection: 95 ° C 10 sec, 61 ° C 30 sec, 72 ° C 10 sec; then 45 cycles with detection (FAM channel) at the annealing stage: 95 ° C for 10 sec, 61 ° C for 30 sec, 72 ° C for 10 sec.

Table 1 presents the results of a qualitative analysis of the presence of the Pu (5mC) GPy sequence in extended bacterial DNA.

Table 1. Test tube No. one 2 3 four 5 Plasmid DNA pHspAI2 pHspAI2 pHspAI2 pHspAI2 pBR322 Adding GlaI + - + - + Adding ligase + + - - + The presence of the sequence Pu (5mC) GPy (3 repetitions) + - - - - + - - - - + - - - -

Figure 1 shows the accumulation curve of a modified DNA product over time. It can be seen that the PCR reaction occurs only when the adapter is ligated to the studied DNA after preliminary hydrolysis of GlaI, thereby creating conditions for the correct annealing of the hybrid primer (Table 1, tube 1). If at least one of the stages is skipped, then product production is impossible (Table 1, tubes 2-4), as well as the presence of nonspecific DNA does not give false positive results (Table 1, tube 5).

Example 2. Sequencing of Pu (5mC) GPy in the first exon of the CST6 tumor suppressor gene.

To analyze the presence of the Pu (5mC) GPy sequence, DNA was selected from Raji cells (Burkitt's lymphoma), in which the studied region of the CST6 gene was hypermethylated as an unmethylated negative control of mouse A / He line DNA.

Preliminary fragmentation and purification of DNA was carried out analogously to example 1, but as a restriction endonuclease that does not have a recognition site in the study area, TaqI was used in a buffer: 33 mM Tris-acetate, pH 7.9; 10 mM magnesium acetate, 66 mM potassium acetate, 1 mM dithiothreitol. For analysis, DNA was used at a concentration of 1500 copies / μl without additional addition of phage λ DNA. Subsequent splitting and subsidizing of the obtained fragments was also carried out under the conditions described in example 1, but with increased concentrations of reagents during ligation. Final concentrations were: ATP 1 mM, PEG 2%, β-mercaptoethanol 8 mM, universal adapter 1000 nM and 1000 ea were used. highly active T4 DNA ligase in the reaction. PCR was carried out as described in example 1, except that DNA primers were used: direct - 5'-GC ATGGTCGGAGAACTCC-3 ', hybrid - 5'-CTGCTCTTTCATCG GCCGC- 3' (underlined part of the primer complementary to the DNA being studied) and probe - 5'-FAM-CTTCTGC ACCTGCGGGTCGT-BHQ1-3 '.

Table 2 presents the results of the analysis of the presence of the Pu (5mC) GPy sequence in the first exon of the CST6 tumor suppressor gene.

Table 2. Test tube No. one 2 3 four 5 6 7 8 DNA Raji Mouse Adding GlaI + - - + + - - Adding ligase + - + - + - + - The presence of the sequence Pu (5mC) GPy (3 repetitions) + - - - - - - - + - - - - - - - + - - - - - - -

Figure 2 shows the accumulation curve of a product with modified Raji DNA over time. Analogously to example 1, the PCR reaction occurs only when the adapter is ligated to the studied DNA after preliminary hydrolysis of GlaI, thereby creating conditions for the correct annealing of the hybrid primer (Table 2, tube 1). In the case of human genomic DNA, skipping one or more stages also interferes with product development (Table 2, tubes 2-4). Unmethylated DNA eukaryotic DNA does not give false-positive results regardless of the missed stages (Table 2, tubes 5-8).

Example 3. Sequence Determination of Pu (5mC) GPy in the first exon of the RARB cancer suppressor gene.

To analyze the presence of the Pu (5mC) GPy sequence, DNA was selected from Raji cells (Burkitt’s lymphoma), in which the studied region of the RARB gene was hypermethylated, and mouse DNA of the A / He line and plasmid pHspAI2 were chosen as a negative control for non-specific DNA.

Preliminary fragmentation and purification of DNA was carried out analogously to example 1, but as a restriction endonuclease that does not have a recognition site in the study area, TaqI was used in a buffer: 33 mM Tris-acetate, pH 7.9; 10 mM magnesium acetate, 66 mM potassium acetate, 1 mM dithiothreitol. For analysis, DNA was used at a concentration of 1500 copies / μl. Subsequent cleavage and ligation of the obtained fragments was also carried out under the conditions described in example 1, but with differences: in the case of human and mouse DNA, phage λ DNA was not added, and 2 ea were taken into the reaction. restriction endonuclease GlaI.

PCR was carried out as described in example 1, except that the following DNA primers were used: direct - 5'-TTC AGAGGC AGGAGGGTCTATTC-3 ', hybrid - 5'-CCTGCTCTTTCATCG GTTCTCG- 3' (underlined part of the primer complementary to the DNA being studied ) and the probe - 5'-FAM-TCCCAGTCCTCAAACAGCTCGCATGG-BHQ1-3 '. The concentrations of primers and probe were also increased to 0.8 μM each and Hot Start DNA polymerase to 0.1 ea / μl

Table 3 presents the results of the analysis of the presence of the Pu (5mC) GPy sequence in the first exon of the RARB cancer suppressor gene.

Table 3. Test tube No. one 2 3 four 5 6 7 8 9 10 eleven 12 DNA Raji Mouse pHspAI2 Adding GlaI + + - - + + - - + + - - Adding ligase + - + - + - + - + - + - The presence of the sequence Pu (5mC) GPy (3 repetitions) + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - -

Figure 3 shows the accumulation curve of a product with modified Raji DNA over time. Analogously to example 1, the PCR reaction occurs only when the adapter is ligated to the studied DNA after preliminary hydrolysis of GlaI, thereby creating conditions for the correct annealing of the hybrid primer (Table 3, tube 1). In the case of human genomic DNA, skipping one or more stages also prevents the production of the product (Table 3, tubes 2-4). Nonspecific eukaryotic and bacterial DNA does not give false positive results, regardless of the missed stages (Table 3, tubes 5-12).

Example 4. The use of the GlaI isoschizomer to determine the sequence of Pu (5mC) GPy in the first exon of the RARB cancer suppressor gene.

To analyze the presence of the Pu (5mC) GPy sequence, DNA was selected from Raji cells (Burkitt's lymphoma), in which the studied region of the RARB gene was hypermethylated, and mouse DNA of the A / He line was selected as a negative control for non-specific DNA.

Preliminary fragmentation and purification of DNA was carried out analogously to example 1, but as a restriction endonuclease that does not have a recognition site in the study area, TaqI was used in a buffer: 33 mM Tris-acetate, pH 7.9; 10 mM magnesium acetate, 66 mM potassium acetate, 1 mM dithiothreitol. For analysis, DNA was used at a concentration of 1500 copies / μl without additional addition of phage λ DNA. Subsequent cleavage and ligation of the obtained fragments was also carried out under the conditions described in example 1, but for specific hydrolysis of methylated DNA, the GlaI isoschisomer methyl-dependent MoxI DNA endonuclease was used. PCR was carried out as described in example 1, with the primers and probe from example 3.

Table 4 presents the results of the analysis of the presence of the Pu (5mC) GPy sequence in the first exon of the RARB tumor suppressor gene.

Table 4. Test tube No. one 2 3 four 5 6 7 8 DNA Raji Mouse Adding GlaI + - - + + - - Adding ligase + - + - + - + -

The presence of the sequence Pu (5mC) GPy (3 repetitions) + - - - - - - - + - - - - - - - + - - - - - -

Figure 4 shows the accumulation curve of a product with modified Raji DNA over time. Analogously to example 1, the PCR reaction occurs only when the adapter is ligated to the studied DNA after preliminary hydrolysis of MoxI, thereby creating conditions for the correct annealing of the hybrid primer (Table 4, tube 1). In the case of human genomic DNA, skipping one or more stages also prevents the production of the product (Table 4, tubes 2-4). Nonspecific murine DNA does not give false-positive results, regardless of the missed stages (Table 4, tubes 5-8).

Example 5. Determination of the minimum number of detectable Pu (5mC) GPy sequences in Raji genomic DNA.

Preliminary fragmentation and purification of DNA was carried out analogously to example 1, but as a restriction endonuclease that does not have a recognition site in the study area, TaqI was used in a buffer: 33 mM Tris-acetate, pH 7.9; 10 mM magnesium acetate, 66 mM potassium acetate, 1 mM dithiothreitol. For analysis, DNA was used in concentrations of 1500, 750, 300, 100, 50, 25, 10, 5, 2.5; 1.6; 0.8; 0.4; and 0.2 copies / μl (Table 5, tubes 1-13). To create a nucleotide load, phage X DNA samples were added to a concentration of 10 ng / μl. Subsequent cleavage and ligation of the obtained fragments was carried out under the conditions described in example 1. PCR was carried out as described in example 1, with the primers and probe from example 3.

Table 5 presents the results of the analysis of the presence of the sequence Pu (5mC) GPy in the extended human genomic DNA at various concentrations.

Table 5. Test tube No. one 2 3 four 5 6 7 8 9 10 eleven 12 13 fourteen The concentration of the original Raji DNA (copies / μl) 1500 750 300 one hundred fifty 25 10 5 2,5 1,6 0.8 0.4 0.2 0 The number of sequences of Pu (5mC) GPy per 1 well of PCR 7500 3750 1500 500 250 125 fifty 25 12.5 8 four 2 one 0 Pu (5mC) GPy sequence detection (3 repeats) + + + + + + + + + + + + - - + + + + + + + + - - + - + - + + + + + + + + + - - - -

From table 5 it is seen that the proposed method has a high specificity and allows you to identify up to single amounts of Pu (5mC) GPy sequences in the study drug.

Using the proposed method will allow more fully and sensitively determine the sequence of Pu (5mC) GPy in DNA samples, as well as reduce analysis time, reduce the cost of the method by using a universal adapter for all Pu (5mC) GPy sequences in extended DNA, and expand its functionality beyond analysis of any Pu (5mC) GPy sequence, including several sequences simultaneously (multiplex).

Information sources

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Claims (4)

1. A method for determining the nucleotide sequence of Pu (5mC) GPy at a given position of extended DNA, including the isolation of highly purified DNA, hydrolysis of fragmented DNA with methyl-dependent site-specific DNA endonuclease GlaI, followed by real-time amplification and the conclusion on the presence of the sequence Pu (5mC) GPy by the appearance of a fluorescent signal, characterized in that the extracted DNA is pre-fragmented with a restriction endonuclease DNA that does not have a recognition site in the amplified region, and is hydrolyzed the universal DNA oligonucleotide adapter 5'-CTCCCGCCTGCTCTTTCATCG-3 '/ 3'-CGATGAAAGAGCA-5' is ligated into the DNA, followed by real-time amplification using a primer and probe complementary to the studied DNA and hybrid primer, 3 'of which is complementary to at least 3 nucleotides 'of the end of DNA from a given site of hydrolysis with a methyl-dependent site-specific DNA endonuclease, and the remainder is complementary to the adapter sequence. 2. The method according to claim 1, characterized in that the fragmentation of the isolated DNA is carried out by restriction endonuclease TaqI, GsaI, NaeIII, or any other that does not have a recognition site in the amplified region. 3. The method according to claim 1, characterized in that the hydrolysis of the fragmented DNA is carried out by a methyl-dependent site-specific DNA endonuclease GlaI or its isoshizomer. 4. The method according to claim 1, characterized in that the adapter is used in a concentration of 500-1000 nm.

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