CN105372303B - A kind of single molecule analysis method of detection methylate DNA - Google Patents

Abstract

The invention relates to a single-molecule analysis method for detecting methylated DNA. In an electrolytic cell containing tetramethylammonium salt, tetraethylammonium salt, tetrapropylammonium salt or tetrabutylammonium salt electrolyte, The pore sensor distinguishes and detects DNA with different levels of methylation, including unmethylated, monomethylated, and polymethylated DNA, through amperometric electrochemical signals, and DNA includes single-stranded, double-stranded, and hairpin structural forms. The method for detecting methylated DNA of the present invention is a single-molecule analysis method, requiring less sample volume, high sensitivity, and single-base resolution; the present invention does not require modification of nanopores and DNA, DNA amplification, and labeling, and is simple to operate Easy to implement and has good applicability.

Description

A single-molecule assay for the detection of methylated DNA

technical field

The invention relates to a single-molecule analysis method for detecting methylated DNA, belonging to the technical field of single-molecule analysis.

Background technique

Nanopore sensors can realize single-molecule detection at the micro/nano scale, and have unique value and significant advantages in the fields of single-molecule analysis and single-molecule chemical reaction research. Traditional nanopore sensors use potassium chloride or sodium chloride as electrolytes. When electrophoresis drives analyte molecules to pass through the nanopore channel, it will cause an instantaneous blocking effect on the ion flow in the channel. According to the amplitude, residence time and frequency of the current blockage, information such as the concentration and chemical structure of the measured sample can be obtained. Nanopore technology has made great progress as a low-cost, high-efficiency DNA detection technology. At present, nanopore sensors are mainly based on hemolysin biological nanopores. α-Hemolysin (α-Hemolysin, αHL) is an exotoxin secreted by Staphylococcus aureus, which can self-assemble into a mushroom-shaped heptamer pore on the phospholipid bilayer. Because the size of its pores only allows single-stranded DNA molecules to pass through, it is mainly used in the detection of single-stranded DNA and RNA, ions, and small molecules.

DNA methylation is an epigenetic modification. Under the action of DNA methyltransferase, a methyl group is added to the fifth carbon atom of cytosine to become 5-methylcytosine (5- ^m C). DNA methylation can regulate the function of the genome without changing the primary structure of DNA molecules, and plays an important role in maintaining normal cell functions, embryonic development, and genetic imprinting. In addition, changes in methylation status, including a decrease in the overall methylation level of the genome and an abnormal increase in the local methylation level of CpG islands, are one of the important factors that cause tumors. Methylated DNA is a very important biomarker of many diseases including cancer. Therefore, the detection of methylated DNA is of great significance for tumor screening and risk assessment, early diagnosis, staging and typing, prognosis judgment and treatment monitoring.

The commonly used method for detecting DNA methylation is to convert the methylated cytosine in the target sequence into a change in the base composition of the DNA sequence. The basic methods are divided into sodium bisulfite method and methylation-sensitive restriction endonuclease Law. The sodium bisulfite method relies on the chemical activity of sodium bisulfite on methylated and unmethylated cytosine to distinguish the two, but the operation is cumbersome, and false positives may be caused by incomplete sodium bisulfite treatment. The methylation-sensitive restriction endonuclease method is a relatively traditional method, which is realized according to the different reactions of methylated cytosine and unmethylated cytosine to specific enzyme digestion treatment. The sensitivity is low, and it may be caused by the enzyme digestion reaction. False positives are caused by the incompleteness of the DNA, and limited by the recognition sites of restriction endonucleases, only the methylation status of some sites can be detected.

Nanopore technology has also been reported for the detection of methylated DNA, but it requires strict control of DNA polymerase, selective modification of methylated DNA or chemical transformation of key sites in the DNA chain. For example, in KCl, the MspA protein nanopore detects the methylation site with the help of phi29 DNA polymerase. This method requires precise single-base level control of single-stranded DNA first passing through the DNA polymerase, and strict implementation of single-stranded DNA. Coupling of molecular DNA polymerases and single protein nanopores; in KCl, α-hemolysin protein nanopores for methylated DNA detection require ferrocene/cucurbit complexes to modify DNA strands and aptamers covalently bond and nanopores, Or the Bisulfite method chemically converts bases and selective ion probes. All the above methods are very complicated, the results are poorly reproducible, and the technical requirements are high.

Contents of the invention

The purpose of the present invention is to provide a simple, cheap, high accuracy, high sensitivity single molecule analysis method that can distinguish and detect DNA with different methylation levels without DNA pretreatment and chemical modification.

The realization process of the present invention is as follows:

A single-molecule analysis method for detecting methylated DNA, characterized in that: in an electrolytic cell containing tetramethylammonium salt, tetraethylammonium salt, tetrapropylammonium salt or tetrabutylammonium salt electrolyte, nano The pore sensor distinguishes and detects DNA with different levels of methylation, including unmethylated, monomethylated, and polymethylated DNA, through amperometric electrochemical signals, and DNA includes single-stranded, double-stranded, and hairpin structural forms. The electrolyte is a buffer solution dissolved in tetramethylammonium salt, tetraethylammonium salt, tetrapropylammonium salt or tetrabutylammonium salt.

The above-mentioned nanopore sensor is a protein nanometer single channel or a solid nanopore sensor. The protein nano single channel is to insert the membrane protein into the lipid bilayer to form the protein nano single channel, and the membrane protein is α-hemolysin protein, MspA or Phi29. The protein nano single channel distinguishes and detects DNA with different methylation levels, including unmethylated, monomethylated and polymethylated DNA, through the difference of DNA residence time distribution and event frequency. DNA includes single-stranded, double-stranded and Issuing structure form.

The above-mentioned solid nanopore sensor is a nanopore formed by inorganic materials, high molecular polymers, carbon nanotubes and graphene.

The above-mentioned single-molecule analysis methods for detecting methylated DNA include:

(1) In the tetramethylammonium chloride electrolyte, the α-hemolysin protein inserts into the lipid bilayer to form a protein nano single channel;

(2) Unmethylated, monomethylated and polymethylated DNAs are added alone or mixed in cis (protein nano single-channel stem) side of the electrolytic cell, presenting different current signal characteristics, with hairpin DNA methylation As the number of sites increases, the proportion of long retention events of the current signal decreases, the DNA retention time decreases, and the event frequency increases.

Specifically, single-molecule assays for the detection of hairpin methylated DNA include:

(1) In the tetramethylammonium chloride electrolyte, the wild-type α-hemolysin protein inserts into the lipid bilayer to form a protein nano single channel;

(2) The hairpin structure DNA containing 0/1/2 methylated cytosines in the stem region was added in cis to the electrolytic cell, and three kinds of DNA current signals were recorded. As the number of hairpin DNA methylation sites increased, the proportion of long retention events > 30 ms decreased, the retention time decreased, and the event frequency increased.

Single-molecule assays for the detection of single-stranded methylated DNA include:

(1) In the tetramethylammonium chloride electrolyte, the α-hemolysin protein inserts into the lipid bilayer to form a protein nano single channel;

(2) Add the hairpin probe and complementary target single-stranded DNA containing 0/1/2 methylated cytosines in cis to the electrolytic cell, and record the current signals of the three DNA mixtures, with the methylation of the single-stranded DNA As the number of sites increased, the proportion of long-dwelling events >300 ms decreased, and the frequency of events increased.

Compared with the traditional potassium chloride electrolyte, different degrees of methylated DNA can be clearly distinguished in the tetramethylammonium chloride electrolyte, and the change trend of the current signal is uniform with the increase of the number of methylation sites. As the number of DNA methylation sites in the hairpin structure increases, the residence time of DNA in the nanopore becomes shorter, the proportion of long retention events decreases, and the frequency of DNA passing through the nanopore increases. Use tetramethylammonium chloride instead of traditional potassium chloride or sodium chloride or lithium chloride electrolyte, can distinguish and detect DNA with different degrees of methylation, without nanopores and DNA modification, without DNA amplification, without labeling , with single-base resolution.

Innovations and positive effects of the present invention:

(1) The method for detecting methylated DNA of the present invention is a single-molecule analysis method, which is different from existing detection methods, requiring less sample volume, high sensitivity, and single-base resolution;

(2) The present invention does not require modification of nanopores and DNA, DNA amplification, and labeling, and is simple and easy to operate;

(3) The present invention can directly detect hairpin DNA with different levels of methylation, including unmethylated, monomethylated and polymethylated DNA, and can simultaneously detect different methylated DNA using only one DNA probe The level of single-stranded DNA has good applicability.

Description of drawings

Figure 1 is the detection result of methylated DNA with hairpin structure in tetramethylammonium chloride electrolyte (voltage is +40 mV);

Figure 2 is the detection result of methylated DNA with hairpin structure in potassium chloride electrolyte (voltage is +120 mV);

Figure 3 is the detection result of the mixed sample of single-stranded methylated DNA and probe (voltage is +40 mV).

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials and reagents used can be obtained from commercial sources unless otherwise specified. The instrument involved in the embodiment is a single-molecule electron detection system (mainly including Axon 200B amplifier, digital-to-analog converter and function generator). It should be understood that these examples are only for illustrating the invention and are not intended to limit the scope of the invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes and modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

The protein nanochannel-based single-molecule detection technology involved in the present invention has been widely used in the detection of metal ions, small organic molecules, proteins, enzymes and other substances and the third-generation DNA sequencing technology. However, in nanopore single-molecule detection, only one DNA probe is used to detect DNA with different degrees of methylation (including unmethylated, monomethylated and polymethylated DNA). Hairpin DNA and single-stranded DNA are representative DNA. In tetramethylammonium chloride electrolyte system, a single-molecule analysis method was designed to detect DNA with different degrees of methylation by using α-hemolysin nanopore.

The DNA sequence (tetramethylammonium chloride) used in the present invention is more likely to interact with A-T base pairs, and poly(dG) itself easily forms a hybrid structure. Therefore, the hairpin DNA adopts poly(dC) as the tail, and the double helix region for C-G base pairs)

0 methylated cytosine hairpin DNA:

5'-CGCGCGCCCCCGCGCGCGCCCCCCCCCCCCCCCCCCCCC-3'

1 methylated cytosine hairpin DNA (underlined part is methylated cytosine):

5'-CGCG ^m C GCCCCCGCGCGCGCCCCCCCCCCCCCCCCCCCCC-3'

2 methylated cytosine hairpin DNA (underlined part is methylated cytosine):

5'-CGCG ^m C GCCCCCG ^m C GCGCGCCCCCCCCCCCCCCCCCCCCC-3'

The designed hairpin probe (the shaded part is the complementary pairing region with the target DNA. The stability of C-G base pairs is higher than that of A-T base pairs. If the double helix region is all C-G base pairs, it is easy to cause DNA to stay too long and block Nanopore, therefore, target DNA and probe pairing region both A-T and C-G base pairs exist):

5'-GGCCGCCCCCGGCCTGCGAGTCCCCCCCCCCCCCCC-3'

0 methylated cytosines target ssDNA

5'-ACTCGCA-3'

1 methylated cytosine target ssDNA (underlined part is methylated cytosine):

5'-ACTCG ^m C A-3'

2 methylated cytosine target single-stranded DNA (underlined part is methylated cytosine):

5'-ACT ^m C G ^m C A-3'.

Example 1

Specifically, the detection of methylated hairpin DNA by the present invention comprises the following steps:

(1) Install the pre-punched Telfon membrane in the center of the electrolytic cell, divide the electrolytic cell into two parts, and drop a mixture of pentane and hexadecane containing 1% hexadecane around the hole with a capillary tube. Add 4 M tetramethylammonium chloride, 10 mMTris-HCl, pH 8.5 buffer solution into the tank, add a small amount of phospholipid dropwise above the liquid surface, add buffer solution to make the liquid surface cross the small hole, and form a stable phospholipid bilayer. The electrolytic cell is cis-added with wild-type α-hemolysin, which is inserted into the phospholipid bilayer to form a nano single channel.

(2) Add the hairpin DNA containing 0/1/2 methylated cytosines in the stem region in cis to the electrolytic cell, stir evenly, and detect the DNA current signal under positive voltage (+40 mV~+120 mV). The tail of the hairpin DNA first enters the cavity of the α-hemolysin nanopore, and under the drive of voltage, the double helix region unwinds and passes through the nanopore.

(3) Analyze and statistic the current signal, as shown in Figure 1, Figure 1-a, b, and c from left to right are the schematic diagram of the DNA detection principle, the single-channel current signal diagram, and the statistical diagram of the residence time distribution; Figure 1-a is Hairpin DNA without methylated cytosine; Figure 1-b is hairpin DNA with 1 methylated cytosine; Figure 1-c is hairpin DNA with 2 methylated cytosines; Figure 1-d is hairpin DNA with The event frequency of the three hairpin DNAs; Fig. 1-e is the variation trend graph of the residence time of the three hairpin DNAs with voltage.

There are significant differences in the retention time of the three kinds of DNA. With the increase of the number of methylation sites, the proportion of long retention events (>30ms) decreases, the retention time decreases, and the event frequency increases. This shows that the increase in the number of methylation sites makes it difficult for DNA to form a hairpin structure, and the stability of DNA with a hairpin structure decreases. Therefore, the proportion of long retention events, retention time, and event frequency can be used to distinguish hairpin DNA with three different degrees of methylation.

The relationship between the residence time of the hairpin DNA and the voltage: under the voltage of +40 mV~+120 mV, as the voltage increases, the residence time of the hairpin DNA decreases, as shown in Figure 1-e, which proves that the hairpin DNA passes through the nanopore. Moreover, at +40 mV~+120 mV voltage, the retention time and event frequency of the three methylated DNAs are significantly different. Therefore, three hairpin DNAs with different methylation levels can be distinguished at any of the voltages. , among which the residence time is the longest under the condition of +40 mV, and the discrimination is the highest.

Example 2

Potassium chloride is a traditional nanopore single-molecule detection electrolyte, similar to Example 1, under the condition that other experimental conditions are kept constant, the detection results of methylated hairpin DNA in potassium chloride electrolyte are shown in Figure 2 Figure 2-a, b, c are schematic diagrams of the DNA detection principle (left), and single-channel current signal diagrams (right). Figure 2-a is the hairpin DNA without methylated cytosine; Figure 2-b is the hairpin DNA with 1 methylated cytosine; Figure 2-c is the hairpin DNA with 2 methylated cytosines; Figure 2-d is a statistical diagram of the residence time distribution of three kinds of hairpin DNA.

First, in the KCl electrolyte, the difference in the retention time of the three hairpin DNAs is only ~10 ms, which is much smaller than the ~2600 ms in the tetramethylammonium chloride electrolyte; moreover, the retention time of the hairpin DNA with different numbers of methylation sites The size relationship of hairpin DNA with 1 ^mC > hairpin DNA with 0 ^mC > hairpin DNA with 2 ^mC , the three DNAs did not show a uniform rule. Therefore, the purpose of distinguishing and detecting methylated DNA cannot be achieved in potassium chloride electrolyte. Tetramethylammonium chloride can selectively interact with DNA methylated cytosine to change the stability of the DNA double helix structure, thereby affecting the electrical signal of DNA passing through the nanopore, such as retention time and event frequency, which is an advantageous Electrolyte for single molecule detection of methylated DNA.

Example 3

Detection of methylated single-stranded DNA includes the following steps:

(1) Hairpin probe DNA and target DNA were mixed at a concentration ratio of 1:1, heated at 95°C for 5 min, and cooled naturally.

(2) Install the pre-punched Telfon membrane in the center of the electrolytic cell, divide the electrolytic cell into two parts, and drop a mixture of pentane and hexadecane containing 1% hexadecane around the hole with a capillary tube. Add 4 M tetramethylammonium chloride, 10 mMTris-HCl, pH 8.5 buffer solution into the tank, add a small amount of phospholipid dropwise above the liquid surface, add buffer solution to make the liquid surface submerge the small holes, and form a stable phospholipid bilayer. The electrolytic cell is cis-added with wild-type α-hemolysin, which is inserted into the phospholipid bilayer to form a nano single channel.

(3) Add the mixture of hairpin probe DNA and target DNA containing 0/1/2 methylated cytosine in cis to the electrolytic cell, stir evenly, and detect the DNA current signal under positive voltage. The tail of the hairpin DNA first enters the cavity of the α-hemolysin nanopore. Driven by the voltage, the double helix region formed by the target DNA and the probe DNA is untied, and the probe DNA passes through the nanopore.

(4) Analysis and statistics of the current signal, as shown in Figure 3, Figure 3-a, b, and c from left to right are the schematic diagram of the DNA detection principle, the single-channel DNA current signal diagram, and the statistical diagram of the residence time distribution; Figure 3-a is not The mixed sample of single-stranded DNA and probe containing methylated cytosine; Figure 3-b is the mixed sample of single-stranded DNA and probe containing one methylated cytosine; Figure 3-c is the mixed sample containing two methylated cytosine Mixed samples of single-stranded DNA with methylated cytosine and probe; Figure 3-d shows the event frequency of three mixed samples of single-stranded DNA and probe. The six-pointed star marks the long-dwelling event (>300 ms) signal.

There are significant differences in the retention time of the three kinds of DNA. As the number of methylation sites increases, the proportion of long retention events (>300ms) decreases and the event frequency increases. This shows that the increase in the number of methylation sites makes it difficult for the target DNA to form a double helix structure with the probe. Therefore, the ratio of long retention events and event frequency can be used to distinguish three types of hairpin DNA with different methylation levels.

The detection of single-stranded DNA with different methylation degrees in the present invention is based on the good distinction of hairpin structure DNA with different methylation degrees by tetramethylammonium chloride. After the target single-stranded DNA is complementary to the hairpin structure probe, the configuration is similar to the hairpin structure DNA, so that the purpose of distinguishing methylated single-stranded DNA can be achieved.

Claims (6)

1. a kind of single molecule analysis method of detection methylate DNA, it is characterised in that：Containing tetramethyl ammonium, tetraethyl ammonium salt, In the electrolytic cell of tetrapropyl ammonium salt or tetrabutylammonium salt electrolyte, nanopore sensor passes through current electrochemical signal distinguishing and inspection Survey the DNA of different methylation levels, including do not methylate, monomethylation and more methylate DNAs, DNA include single-stranded, double-strand and hair Card structure form；

The electrolyte is the buffer solution dissolved with tetramethyl ammonium, tetraethyl ammonium salt, tetrapropyl ammonium salt or 4-butyl ammonium；Institute The nanopore sensor stated is protein nano single channel or solid nano hole sensor.

2. the single molecule analysis method of detection methylate DNA according to claim 1, it is characterised in that：Memebrane protein is inserted Enter lipid bilayer and form protein nano single channel, the memebrane protein is alpha hemolysin protein, MspA or Phi29.

3. the single molecule analysis method of detection methylate DNA according to claim 2, it is characterised in that：Protein nano Single channel is distributed by the DNA residence times and the DNA of different methylation levels is distinguished and detected to the difference of event frequency, including not It methylates, monomethylation and more methylate DNAs, DNA include single-stranded, double-strand and hairpin structure form.

4. the single molecule analysis method of detection methylate DNA according to claim 1, it is characterised in that：The solid Nanopore sensor is the nano-pore by inorganic material, high molecular polymer, carbon nanotube and the dilute formation of graphite.

5. the single molecule analysis method of detection methylate DNA according to claim 1, it is characterised in that：

（1）In tetramethyl ammonium chloride electrolyte, alpha hemolysin protein is inserted into lipid bilayer and forms protein nano single channel；

（2）Protein nano single channel stem, that is, cis- side of electrolytic cell individually or mixing be added do not methylate, monomethylation and Different current signal features is presented in more methylate DNAs, and as hairpin dna methylation sites number increases, the length of current signal is stagnant The reduction of event ratio, DNA residence times is stayed to reduce, event frequency increases.

6. the single molecule analysis method of detection methylate DNA according to claim 1, it is characterised in that：

（1）In tetramethyl ammonium chloride electrolyte, alpha hemolysin protein is inserted into lipid bilayer, forms protein nano single channel；

（2）It is single-stranded in the cis- target containing 0/1/2 methylated cytosine that hair fastener probe and complementary pairing is added of electrolytic cell DNA records three kinds of DNA mixture current signals, as single stranded DNA methylation sites number increases , >The length delay event of 300 ms Ratio reduces, and event frequency increases.