WO2019233451A1

WO2019233451A1 - Dna methylation detection probe

Info

Publication number: WO2019233451A1
Application number: PCT/CN2019/090193
Authority: WO
Inventors: 陈琦; 梁昊原; 谷东风
Original assignee: 深圳市圣必智科技开发有限公司
Priority date: 2018-06-08
Filing date: 2019-06-05
Publication date: 2019-12-12
Also published as: CN110577986A

Abstract

Disclosed is a DNA methylation detection probe comprising a sequence for complementary pairing with methylated DNA at the 5' end and the 3' end, in which the last three bases at the 3' end of the detection probe are guanine. The guanine is paired with a methylated cytosine in the middle of the methylated DNA. The sequence at the 3' end of the detection probe is reverse complementary paired with the sequence from the methylated cytosine in the middle of the methylated DNA to the 3' end, and the sequence at the 5' end of the detection probe is reverse complementary paired with the sequence from the adjacent deoxynucleotide of the methylated cytosine in the middle of the methylated DNA to the 5' end. The DNA methylation detection probe can increase the sensitivity of DNA methylation detection.

Description

DNA methylation detection probe

Technical field

The invention relates to the field of molecular biology, in particular to a DNA methylation detection probe.

Background technique

With the completion of the Human Genome Project, one of the next important tasks is to decrypt the genetic system, that is, how human cells use genetic material during growth to determine when and where a particular gene is expressed.

DNA methylation has become an important part of the epigenetic system because it can affect the genetic state of gene expression. Mammalian DNA methylation mostly occurs on cytosine in CpG dinucleotides, that is, a methyl group is added to the 5th carbon position on the pyrimidine ring of cytosine.

In normal cells, methylation occurs mainly in repeated genomic regions, including genetic elements and satellite deoxynucleotides. However, CpG islands associated with gene promoters and exons are usually unmethylated. But mutational DNA methylation in these regions can cause transcriptional silencing of cancer suppressor genes, and this mutational DNA methylation can be used as a marker for various diseases, including cancer. Methylation of specific regions on CpG islands may be associated with specific types of cancer. Therefore, accurate quantification of DNA methylation at any given location in the human genome is important for the early treatment of human cancer.

Traditionally, there are various detection methods for analyzing DNA methylation in a single CpG position or a short sequence. Methylation-specific PCR (MSP) opens the door to the use of polymerase chain reaction (PCR) for methylation analysis, but because MSP analysis results are observed by gel electrophoresis, MSP can only provide experiments Qualitative analysis rather than quantitative analysis. Fluorescent label real-time monitoring PCR and methylation-specific quantum dot fluorescence resonance energy transfer (MSq-FRET) and other methods require precision temperature-controlled PCR instruments, and generally require the use of fluorescent dye molecularly labeled probes, which greatly aggravates Experiment costs. COBRA combined with sulfite provides another option for quantitative and sensitive detection of DNA methylation, but the prerequisite for COBRA is that methylation-sensitive restriction in the analysis sample Digestion enzyme digestion site. Based on surface enhanced Raman spectroscopy and single nucleotide amplification methods, applications are limited due to lower sensitivity.

technical problem

The main purpose of the present invention is to provide a DNA methylation detection probe, which aims to improve the sensitivity of DNA methylation detection.

Technical solutions

To achieve the above object, the present invention proposes a DNA methylation detection probe, which includes sequences for complementary pairing with methylated DNA at the 5 ′ end and the 3 ′ end, wherein the last three bases at the 3 ′ end of the detection probe The bases are all guanine, the guanine is paired with the methylated cytosine in the middle of the methylated DNA, and the sequence at the 3 'end of the detection probe matches the methylated cytosine to 3' from the middle of the methylated DNA. The sequence at the end of the probe is reversely complementary paired, and the sequence at the 5 ′ end of the detection probe is reversely complementary paired with the sequence from the deoxynucleotide to the 5 ′ end of the methylated cytosine in the middle of the methylated DNA.

Preferably, the DNA sequence of the detection probe is SEQ ID NO: 1.

Preferably, the 3 'end includes 13 bases.

Preferably, the 5 'end includes 21 bases.

Preferably, the DNA methylation detection probe further includes a specific sequence in a middle portion for initiating a hyperbranched rolling circle amplification reaction, and the specific sequence includes a forward primer and a reverse primer.

Preferably, the length of the forward primer is 25.

Preferably, the forward primer is SEQ ID NO: 3.

Preferably, the length of the reverse primers is 23.

Preferably, the reverse primer is SEQ ID NO: 4.

Beneficial effect

In this technical solution, the last three bases at the 3 ′ end of the DNA methylation detection probe are guanine, which can detect the DNA methylation on cytosine in the CpG dinucleotide with a higher probability. Therefore, Compared with the prior art, it has the characteristics of high sensitivity.

Embodiments of the invention

In order to further explain the technical means and effects adopted by the present invention to achieve the above-mentioned objectives, specific implementations of the present invention will be described in detail below with reference to preferred embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

A DNA methylation detection probe according to an embodiment includes a sequence for complementary pairing with methylated DNA at the 5 ′ end and a 3 ′ end, and a specific sequence for initiating a hyper-branch rolling circle amplification reaction in the middle portion, wherein The last three bases of the 3 'end of the detection probe are guanine, which is paired with the methylated cytosine in the middle of the methylated DNA, and the sequence of the 3' end of the detection probe and the methylated cell from the middle of the methylated DNA The sequence from the pyrimidine to the 3 'end is reversely complementary paired, and the sequence at the 5' end of the detection probe is reversely complementary paired with the sequence of methylated DNA from the middle methylated cytosine adjacent to the deoxynucleotide to the other 5 'end.

The last base at the 3 'end of the DNA methylation detection probe corresponds to the methylated cytosine in the middle of the methylated DNA, and can specifically pass through three hydrogen bonds with the methylated cytosine in the middle of the methylated DNA. Formation and complementary pairing.

In an alternative embodiment, if the DNA to be tested is methylated DNA, it can be completely complementary paired with the detection probe, and after complementary pairing, the 3 'end and the 5' end of the detection probe are close to each other. Under the action of ligase, the 3 'end and 5' end of the detection probe are connected to form a circular DNA, which will not be digested by the digestive juice.

Among them, unmethylated DNA is treated with bisulfite, and the amino group at position 4 on the cytosine ring is replaced by a carbonyl group, that is, cytosine has been converted to uracil, but uracil cannot be paired with guanine. The DNA cannot be completely complementary to the detection probe, and no circular DNA is formed, so all DNA is digested by exonuclease III and I.

Circular DNA can be used to quantitatively analyze the degree of methylation of the DNA to be tested through subsequent HRCA reactions and signal detection. Because HRCA has ultra-high amplification capabilities (109-fold signal amplification), it guarantees ultra-high sensitivity requirements for detection. And the detection probe can be fused with many methylated DNAs, and has good compatibility. In addition, using this probe does not require the use of expensive fluorescently labeled probes or PCR amplification during the detection process, and the cost of detection is greatly reduced.

In an alternative embodiment, the methylation DNA detection process of H157 (human non-small cell lung cancer cell line) and H209 (human small cell lung cancer cell line) cell lines is as follows:

1.DNA extraction and digestion

1.1 Cultivate two cell lines, H157 and H209, respectively.

The p16 promoter region in a normal H157 cell line is highly methylated, whereas these regions in a normal H209 cell line are unmethylated.

Culture conditions: The two cell lines were respectively placed in a DMEM culture solution containing 10% bovine serum albumin, and cultured in a humidified 37 ° C incubator containing 5% carbon dioxide.

1.2 Extracting DNA

The genomic DNA of the two cell lines was extracted with a DNA extraction kit, and the absorption value of the DNA extraction solution at 260 nm was detected by a spectrophotometer to convert the DNA concentration.

1.3 The extracted DNA was treated with two restriction digestive enzymes, Pst I and BstE II, and genomic DNA was digested into shorter base fragments to obtain the test DNA.

In other embodiments, the step of extracting DNA from the cell line can be omitted, and methylated DNA and unmethylated DNA can be obtained by direct purchase.

2. Sodium bisulfite treats the test DNA to convert unmethylated cytosine in the test DNA to uracil.

The processing conditions are as follows: 1 μg of the test DNA is added to a 20 μL volume of 0.35 mol / L sodium hydroxide solution, and after reacting at 37 ° C for 20 minutes, a certain volume of sodium bisulfite solution and hydroquinone are added. To the above solution so that their final concentrations are 3.2mol / L and 0.5mmol / L, and react at 50 ° C for 16-18 hours, and then pass the solution through a desalting column to recover DNA; add a certain volume of sodium hydroxide to the recovered DNA The solution was made to have a final concentration of 0.3 mol / L in a 50 μL solution, and reacted at 37 ° C. for 15 minutes, and then the solution was completely neutralized with ammonia acetate, finally precipitated in ethanol, and dried to obtain a DNA powder. The obtained DNA powder was dissolved in ultrapure water and stored at -20 ° C until use.

3. Connection reaction

3.1 Designing lock probes based on the sequence of the DNA to be tested

In this embodiment, the sequences of the DNA to be tested are as follows: The methylated DNA sequence is:

GAG GGT GGG GmCG GAC mCGmC GTG mCGC TmCGGmCG GCT G (SEQ ID NO: 2), wherein mC represents methylated cytosine;

The unmethylated DNA sequence is: GAG GGT GGG GCG GAC CGC GTG CGC TCGGCG GCT G (SEQ ID NO: 5).

Designed lock probe sequence:

AC GCG ATC CGC CCC ACC CTC ATT AGG TTACTG CGA TTA GCA CAA GCA CCA AGA GCA ACT ACA CGA ATT CCA ACCGCC GAA CG (SEQ ID NO: 1).

In this example, in order to enhance the specificity of detection, a special design was made on the sequence of the lock probe: the total length of the lock probe is 83 bases, and the 5 'end of the complementary pairing with the methylated DNA has 21 bases, 13 bases at the 3 'end, and 49 bases in the middle loop. This asymmetric sequence structure with complementary pairing at both ends will facilitate the binding of the target gene to the lock probe; The last base at the 3 'end of the padlock probe is set to guanine, which specifically complements the methylated cytosine of methylated DNA through the formation of three hydrogen bonds to complement the unmethylated DNA. With the treatment of sodium bisulfite, the amino group at position 4 on the cytosine ring was replaced by a carbonyl group, that is, cytosine has been converted to uracil and cannot be complementary paired with guanine.

In other embodiments, the lock probe may adopt other reasonable design schemes, for example, the length of the sequence for complementary pairing at the 5 'end is not limited to 21 bp, and the length of the sequence for complementary pairing at the 3' end is not limited to 13 bp. The design of the lock probe only needs to meet the experimental requirements. That is, make sure that the sequences at the 5 'and 3' ends of the lock probe are reversely complementary paired with the sequences at the 5 'and 3' of the methylated DNA of interest, respectively.

3.2 Conditions for the ligation reaction

Mix 2L of test DNA of different concentration with 2L of 1mol / L lock probe to prepare 20L of mixed solution. The mixed solution contains: 20mmol / L Tris-HCl (pH7.6) buffer, 25mmol / L potassium acetate , 10mmol / L magnesium acetate, 1mmol / L nicotinamide adenosine dinucleotide and 0.1% Triton X-100. The reaction was performed at 95 ° C for 5 minutes, and then 12 units of Taq DNA ligase were added, and the reaction was performed at 65 ° C for 60 minutes.

After the test DNA is treated with bisulfite, unmethylated cytosine is converted into uracil, but methylated cytosine does not change. During the ligation reaction, the methylated DNA can interact with the lock probe. The two ends are completely complementary and paired, so that the 3 'end and the 5' end of the lock probe are close to each other. Under the action of the ligase, the 3 'end and the 5' end of the lock probe are connected to form a circular lock probe. needle. Due to the significant difference in sequence between the unmethylated DNA and the lock probe, the unmethylated DNA cannot be completely complementary to each other. Therefore, the 3 'and 5' ends cannot be ligated by ligase. It is distinguished from this that, because of the specificity of the circular connection of the lock probe, this method of using the circular connection of the lock probe for methylation detection has high specificity.

3.3 Verification of ligation reaction products

In order to prove the feasibility of the method, in this embodiment, electrophoresis experiments are used for verification. Here, the electrophoresis experiments may be a standard silver stained denatured 10% polyacrylamide gel electrophoresis connected with the reaction product. Since the linear lock probe runs faster than the circular lock probe, electrophoresis proves the existence of the ligation reaction product, and the method is feasible.

4.Digestive response

4.1 Conditions for digestive reactions

10L of the ligation reaction solution was mixed with 10L of digestive juice, reacted at 37 ° C for 2 hours, and finally inactivated at 95 ° C for 10 minutes. 10 L of digestive juice contained 1 mmol / L of DTT, 6.7 mmol / L of magnesium chloride, 67 mmol / L of glycine-potassium hydroxide pH 9.5, 10 units of exonuclease I and exonuclease III.

Exonuclease I and exonuclease III can digest non-circular DNA, but cannot digest circular DNA, thus obtaining pure circular lock probes. In this embodiment, the non-circular DNA refers to methylated DNA, unmethylated DNA, and a lock probe that has not undergone a ligation reaction.

4.2 Verification of digestion reaction products

In this example, electrophoresis experiments were used to verify that all non-circular DNA was digested cleanly, except for the circular lock probe. The presence of the loop-locked probe further proves the feasibility of the ligation reaction. At the same time, in order to reduce the occurrence of non-ligation-specific amplification in the subsequent amplification reaction and further improve the specificity, the digestion of exonase is very important.

5. Hyperbranch rolling circle amplification reaction

5.1 Design two primers based on a lock probe

The sequence of primer 1 (forward primer) is: 3’CTT GTG CTA ATC GCA GTA ACC TAA T 5 ’(SEQ ID NO: 3);

The sequence of primer 2 (reverse primer) is: 3'ACC AAG AGC AAC TAC ACG AAT TC 5 '(SEQ ID NO: 4).

5.2 Conditions for Hyperbranch Rolling Circle Amplification

10L of the digestion reaction product was mixed with 20L of the amplification solution, and reacted at 63 ° C for 1 hour. The amplification solution contained: 0.05 μmol / L of primer 1 and primer 2, 400 μmol / L of deoxynucleoside triphosphate mixed solution and 8 units of Bst DNA polymerase.

As the concentration of the two primers decreases from 1 μmol / L to 0.05 μmol / L, the difference between the fluorescence intensity of methylated genes and unmethylated genes gradually increases. Therefore, in this example, 0.05 μmol / L is selected as The optimal concentration of the two primers, because higher concentrations of primers will cause dimerization and non-specific amplification of the primers themselves. In addition, as the substrate concentration of dNTPs increased from 2 μmol / L to 400 μmol / L, the difference in fluorescence intensity between methylated genes and unmethylated genes gradually increased. Therefore, in this embodiment, 400 μmol / L is used as the dNTPs substrate. Optimal concentration of substances.

5.3 Verification of Hyperbranched Rolling Circle Amplification Reaction Products

In this example, electrophoresis experiments are used to verify that the agarose gel electrophoresis map of SYBR Green I as the dye of the hyperbranched rolling circle amplification reaction product shows that the methylated genes can pass the cyclized lock probe Furthermore, a large number of DNA products were obtained by amplification, but the blank experiments and unmethylated DNA could not be amplified, so no products were obtained.

6, detection

6.1 Testing conditions

30 μL of the solution after the super-branch rolling circle amplification reaction was mixed with 1 μL of SYBR Green I (20 times), and deionized water was added to 100 μL. After incubating at room temperature for 10 minutes, the solution was detected with a fluorescence photometer. The fluorescence detection conditions were: the excitation light wavelength was 488 nanometers, the spectrum collection range was 500-650 nanometers, and the emission wave intensity was measured at 520 nanometers.

6.2 Test results

The high sensitivity and accurate quantification of DNA methylation is very important for the early treatment of cancer. In order to prove the high sensitivity of the method, the present embodiment studies the results of fluorescence detection of methylated genes at different concentrations. As the methylated gene concentration increases, its fluorescence detection value also increases, and the concentration has an exponential relationship with the signal intensity value, that is, the logarithmic value of the concentration has a linear relationship with the signal intensity value, and the linear relationship covers 4 orders of magnitude. From 1fmol / L to 10 pmol/L. The linear relationship is: If = 34.54 + 102.98log10C, where If is the fluorescence signal intensity value, and C is the concentration of methylated genes (female per liter). Using this equation to analyze the fluorescence intensity of the blank value plus 3 times the deviation value, the detection limit is 0.8 mol per liter. This detection limit is 8 orders of magnitude higher than the colorimetric method using gold nanometers, and 3 orders of magnitude higher than the Raman enhanced spectrum obtained using single base amplification.

In addition, in this embodiment, an artificial mixed solution obtained by mixing methylated genes and unmethylated genes at a certain ratio is also tested for the degree of methylation. As the degree of methylation increases, the resulting fluorescence intensity value also increases. The degree of methylation detected almost coincides with the degree of methylation actually added. In addition, this method can successfully detect 0.01% methylation groups in mixed samples, which has significantly higher resolution than the following methods: MALDI-MS mass spectrometry (5%), quantum dot-based fluorescence energy transfer (1%), cations Conjugated polyelectrolyte method (1%), methylation-specific PCR (0.1%), can even be compared with MS-qFRET (0.01%).

In this embodiment, in order to further verify the reliability of the above detection method, an actual sample is tested. This method was used to investigate the methylation of six CpG islands in the p16 promoter region segment of non-small cell lung cancer cell line H157 and small cell lung cancer cell line H209. When using this analysis method to detect actual samples of cell lines, this example treated the genomic DNA with restriction digestive enzymes before the experiment in order to prevent secondary formation such as supercoil or hypercyclization of DNA in subsequent experiments. structure. With the increase of the amount of genomic DNA, the fluorescence intensity obtained by detecting H157 increased, while H209 remained unchanged, and the detection limit of H157 was 2ng. This shows that the detection method in this patent can detect DNA methylation in lung cancer cell lines with higher sensitivity.

Industrial applicability

The above are only preferred embodiments of the present invention, and thus do not limit the patent scope of the present invention. Any equivalent structure or equivalent function transformation made by using the content of the description of the present invention, or directly or indirectly used in other related technical fields, the same The principle is included in the scope of patent protection of the present invention.

Claims

A DNA methylation detection probe, characterized in that it comprises sequences for complementary pairing with methylated DNA at the 5 'end and the 3' end, wherein the last three bases at the 3 'end of the detection probe are birds Purine, which is paired with methylated cytosine in the middle of the methylated DNA, and the sequence of the 3 'end of the detection probe is reversed from the sequence of the methylated cytosine to the 3' end of the methylated DNA Complementary pairing. The sequence at the 5 'end of the detection probe is reverse complementary paired with the sequence of methylated DNA from the deoxynucleotide to the 5' end of the methylated cytosine in the middle.
The DNA methylation detection probe according to claim 1, wherein the DNA sequence of the detection probe is SEQ ID NO: 1.
The DNA methylation detection probe according to claim 1, wherein the 3 'end includes 13 bases.
The DNA methylation detection probe according to claim 1, wherein the 5 'end includes 21 bases.
The DNA methylation detection probe according to claim 1, wherein the DNA methylation detection probe further comprises a specific sequence in the middle portion for initiating a hyper-branch rolling circle amplification reaction, and Specific sequences include forward and reverse primers.
The DNA methylation detection probe according to claim 5, wherein the length of the forward primer is 25.
The DNA methylation detection probe according to claim 5, wherein the forward primer is SEQ ID NO: 3.
The DNA methylation detection probe according to claim 5, wherein the length of the reverse primer is 23.
The DNA methylation detection probe according to claim 5, wherein the reverse primer is SEQ ID NO: 4.