CN105372303B - A kind of single molecule analysis method of detection methylate DNA - Google Patents
A kind of single molecule analysis method of detection methylate DNA Download PDFInfo
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Abstract
The present invention relates to a kind of single molecule analysis methods of detection methylate DNA, it is in the electrolytic cell containing tetramethyl ammonium, tetraethyl ammonium salt, tetrapropyl ammonium salt or tetrabutylammonium salt electrolyte, the DNA that nanopore sensor passes through current electrochemical signal distinguishing methylation level different with detection, including not methylating, monomethylation and more methylate DNAs, DNA includes single-stranded, double-strand and hairpin structure form.The method that the present invention detects methylate DNA is single molecule analysis method, needs sample size few, high sensitivity, has single base resolution ratio；The present invention is not necessarily to, to nano-pore and DNA modification, be not necessarily to DNA cloning, and without label, operation is simple, has good application.
Description
Technical field
The present invention relates to a kind of single molecule analysis methods of detection methylate DNA, belong to single molecule analysis technical field.
Background technology
The Single Molecule Detection on micro-/ nano scale may be implemented in nanopore sensor, in single molecule analysis, unimolecule chemistry
The fields such as repercussion study have unique value and notable superiority.Traditional nanopore sensor uses potassium chloride or sodium chloride
For electrolyte.When electrophoresis driving analyte molecule passes through nanopore-channel, momentary blockage effect, root are caused to duct ion stream
According to the amplitude of current blockade, residence time and frequency, the information such as concentration and the chemical constitution of institute's sample can be obtained.Nano-pore skill
Art has been achieved for huge advance as low cost, efficient DNA detection techniques.At present nanopore sensor mainly with
Based on hemolysin biological nano hole.Alpha hemolysin（α-Hemolysin, α HL）It is outside the one kind secreted by staphylococcus aureus
Toxin can be self-assembled into the haptamer hole of a mushroom-shaped on phospholipid bilayer.Since the size in its duct only allows list
Ssdna molecule passes through, and is mainly used in the detection of single stranded DNA and RNA, ion, small molecule.
DNA methylation is a kind of epigenetic modification, under the action of dnmt rna, the 5th carbon original of cytimidine
A methyl group is added on son, becomes 5-methylcytosine（5-mC）.DNA methylation can not change DNA molecular level-one
The function that genome is adjusted in the case of structure, rises emphatically in maintaining normal cell function, embryo development procedure, genetic imprinting
It acts on.In addition, the variation of methylation state, includes reduction and the islands the CpG part methyl of genome entirety methylation level
Change it is horizontal it is abnormal increase, be one of an important factor for causing tumour.Methylate DNA is many diseases including cancer
One very important Biological indicators.Therefore, the detection of methylate DNA to the screening and risk assessment of tumour, early diagnosis, point
Phase parting, Index for diagnosis and Treatment monitoring all have great importance.
The method of common detection DNA methylation is to convert the methylated cytosine in aim sequence to DNA sequence dna alkali
The variation of base composition, Basic practice are divided into the restriction enzyme enzyme process of sodium bisulfite method and methyl-sensitive.Sodium hydrogensulfite
Method be rely on sodium hydrogensulfite to methylate it is different with the chemism of non-methylated cytosine both distinguish, but operate numerous
It is trivial, not exclusively it may lead to false positive because of sodium hydrogensulfite processing.The restriction enzyme enzyme process of methyl-sensitive is to compare biography
The method of system, the differential responses that are handled specific digestion according to methylated cytosine and non-methylated cytosine realize, spirit
Sensitivity is low, may because endonuclease reaction it is incomplete due to generate false positive, and the limitation of conditionality endonuclease recognized site, only
The methylation state of moiety site can be detected.
Nano-pore technology also has been reported that for methylate DNA detection, but need to use the stringent control of archaeal dna polymerase, methylates
The chemical conversion of the selective modification or DNA chain critical sites of DNA.For example, in KCl, MspA protein nanos hole by
Phi29 archaeal dna polymerases realize that the detection of methylation sites, this method need accurately single base level control single stranded DNA head
Archaeal dna polymerase is first passed through, and needs the coupling of stringent realization single-molecule DNA polymerase and single protein nano hole；In KCl
In, alpha hemolysin protein nano hole needs ferrocene/cucurbit compound modifying DNA chains for methylate DNA detection
With aptamers covalent bond and nano-pore or Bisulfite forensic chemistries conversion base and selective ion probe.Above-mentioned all sides
Method is extremely complex, as a result poor reproducibility, and technology requires high.
Invention content
The object of the present invention is to provide a kind of simple, cheap, accuracy height, high sensitivities, without DNA pre-treatments and chemistry
Modification, can distinguish and detect the single molecule analysis method of different methylation level DNA.
The present invention realizes that process is as follows：
A kind of single molecule analysis method of detection methylate DNA, it is characterised in that：Containing tetramethyl ammonium, tetraethyl ammonium
In the electrolytic cell of salt, tetrapropyl ammonium salt or tetrabutylammonium salt electrolyte, nanopore sensor passes through current electrochemical signal distinguishing
The DNA of methylation levels different with detection, including do not methylate, monomethylation and more methylate DNAs, DNA include single-stranded, double-strand
With hairpin structure form.The electrolyte is dissolved with tetramethyl ammonium, tetraethyl ammonium salt, tetrapropyl ammonium salt or 4-butyl ammonium
Buffer solution.
Above-mentioned nanopore sensor is protein nano single channel or solid nano hole sensor.Protein nano single channel
Be by memebrane protein be inserted into lipid bilayer formed protein nano single channel, the memebrane protein be alpha hemolysin protein, MspA or
Phi29.Protein nano single channel is distributed by the DNA residence times to be distinguished and detection is different methylates with the difference of event frequency
Horizontal DNA, including do not methylate, monomethylation and more methylate DNAs, DNA include single-stranded, double-strand and hairpin structure form.
Above-mentioned solid nano hole sensor is by inorganic material, high molecular polymer, carbon nanotube and the dilute formation of graphite
Nano-pore.
The single molecule analysis method of above-mentioned detection methylate DNA includes：
（1）In tetramethyl ammonium chloride electrolyte, alpha hemolysin protein is inserted into lipid bilayer and forms protein nano single channel；
（2）Electrolytic cell is cis-（Protein nano single channel stem）Side individually or mixing be added do not methylate, monomethylation
With more methylate DNAs, different current signal features is presented, as hairpin dna methylation sites number increases, the length of current signal
Delay event ratio reduces, and the DNA residence times reduce, and event frequency increases.
Specifically, the single molecule analysis method of detection hairpin structure methylate DNA includes：
（1）In tetramethyl ammonium chloride electrolyte, wild type alpha hemolysin protein is inserted into lipid bilayer and forms protein nano
Single channel；
（2）The cis- hairpin structure DNA for being separately added into pedicle region and containing 0/1/2 methylated cytosine of electrolytic cell, note
Record three kinds of DNA current signals.As hairpin dna methylation sites number increases , >The length delay event ratio of 30ms reduces, and is detained
Time reduces, and event frequency increases.
The single molecule analysis method for detecting single-stranded methylate DNA includes：
（1）In tetramethyl ammonium chloride electrolyte, alpha hemolysin protein is inserted into lipid bilayer, forms protein nano single-pass
Road；
（2）In the cis- target containing 0/1/2 methylated cytosine that hair fastener probe and complementary pairing is added of electrolytic cell
Single stranded DNA records three kinds of DNA mixture current signals, as single stranded DNA methylation sites number increases , >The long of 300 ms is detained
Event ratio reduces, and event frequency increases.
Relative to traditional potassium chloride electrolyte, different degrees of first can be obviously distinguished in tetramethyl ammonium chloride electrolyte
Base DNA, and increase with methylation sites number, the variation tendency of current signal is unified.With hairpin structure DNA methylation position
Points increase, and DNA residence times in nano-pore shorten, and long delay event ratio reduces, and nano-pore frequency is passed through to increase.Make
Traditional potassium chloride or sodium chloride or chlorination lithium electrolyte are replaced with tetramethyl ammonium chloride, can distinguish and detect difference and methylate journey
The DNA of degree is not necessarily to nano-pore and DNA modification, is not necessarily to DNA cloning, without label, has single base resolution ratio.
The innovative point and good effect of the present invention：
（1）The method that the present invention detects methylate DNA is single molecule analysis method, different from existing detection method, is needed
Want sample size few, high sensitivity has single base resolution ratio；
（2）The present invention is not necessarily to, to nano-pore and DNA modification, be not necessarily to DNA cloning, and without label, operation is simple；
（3）The present invention can directly detect the hairpin structure DNA of different methylation levels, including not methylate, monomethyl
Change and more methylate DNAs, also, the single stranded DNAs of different methylations can be detected simultaneously using only a DNA probe, tool
There is good application.
Description of the drawings
Fig. 1 is the testing result of hairpin structure methylate DNA in tetramethyl ammonium chloride electrolyte（Voltage is+40 mV）;
Fig. 2 is the testing result of hairpin structure methylate DNA in potassium chloride electrolyte（Voltage is+120 mV）;
Fig. 3 is the testing result of single-stranded methylate DNA and probe mixing sample（Voltage is+40 mV）.
Specific implementation mode
Experimental method used in following embodiment is conventional method unless otherwise specified, used material, reagent
Deng being commercially available unless otherwise specified.Instrument involved by embodiment is unimolecule electronic detection system（It is main
To include Axon 200B amplifiers, D-A converter and function generator）.It should be understood that these embodiments are merely to illustrate hair
It is bright rather than limit the scope of the invention.In addition, it should also be understood that, after having read present disclosure, those skilled in the art
Can make various changes and modification to the present invention, such equivalent forms equally fall within the application attached claims limited
Range.
Molecule detection according to the present invention based on protein nano channel has been widely used in metal ion,
The detection of the substances such as organic molecule, protein, enzyme and the DNA sequencing technology of the third generation.But in nanometer pore single-molecule detection,
The DNA (including not methylating, monomethylation and more methylate DNAs) of different methylations is detected using only a DNA probe
It has not been reported, the embodiment of the present invention is using hairpin structure DNA and single stranded DNA as representative DNA, in tetramethyl ammonium chloride electrolyte
In system, a kind of single molecule analysis method detecting different methylation DNA is devised using alpha hemolysin nano-pore.
DNA sequence dna used in the present invention（Tetramethyl ammonium chloride is more likely to act on A-T base-pairs, and poly (dG) is certainly
Body is easy to form hybrid structure again, and therefore, it is C-G base-pairs that hairpin dna is used as tail, double helix region using poly (dC)）
0 methylated cytosine hairpin dna：
5’-CGCGCGCCCCCGCGCGCGCCCCCCCCCCCCCCCCCCCC-3’
1 methylated cytosine hairpin dna（Underscore part is methylated cytosine）：
5’-CGCG mCGCCCCCGCGCGCGCCCCCCCCCCCCCCCCCCCC-3’
2 methylated cytosine hairpin dnas（Underscore part is methylated cytosine）：
5’-CGCG mCGCCCCCG mCGCGCGCCCCCCCCCCCCCCCCCCCC-3’
The hair fastener probe of design（Dash area be and target dna complementary pairing region.The stability of C-G base-pairs is higher than
A-T base-pairs, if all C-G base-pairs in double helix region, it is too long to block nanometer to be easy to cause the DNA residence times
Hole, therefore, target dna exist with Probe pairings region A-T and C-G base-pairs）：
5’-GGCCGCCCCCGGCCTGCGAGTCCCCCCCCCCCCCCC-3’
0 methylated cytosine target single stranded DNA
5’-ACTCGCA-3’
1 methylated cytosine target single stranded DNA (underscore part is methylated cytosine)：
5’-ACTCG mCA-3’
2 methylated cytosine target single stranded DNAs (underscore part is methylated cytosine)：
5’-ACT mCG mCA-3’。
Embodiment 1
Specifically, present invention detection methylates, hairpin dna includes the following steps：
（1）By the Telfon films for accomplishing fluently aperture in advance mounted in electrolytic cell center, electrolytic cell is divided into two parts, uses capillary
Pentane and hexadecane mixture containing 1% hexadecane are added dropwise around hole, 4 M tetramethyl ammonium chlorides, 10 mM are added in two slots
A small amount of phosphatide is added dropwise in the buffer solution of Tris-HCl, pH 8.5, ullage, and addition buffer solution makes liquid level get over small holes, is formed steady
Fixed phospholipid bilayer.The cis- addition wild type alpha hemolysin of electrolytic cell is inserted into phospholipid bilayer and forms nanometer single channel.
（2）The cis- hairpin dna that pedicle region is added and contains 0/1/2 methylated cytosine of electrolytic cell, stirs evenly, just
Voltage（+40 mV~+120 mV）Lower detection DNA current signals.The tail portion of hairpin dna is introduced into alpha hemolysin nano-pore cavity,
Under voltage driving, double helix region unlocks and passes through nano-pore.
（3）Analysis statistics current signal, as shown in Figure 1, Fig. 1-a, b, c are followed successively by the signal of DNA testing principles from left to right
Figure, single channel current signal graph, residence time distribution statistics figure；Fig. 1-a are the hairpin dna without containing methylated cytosine；Figure
1-b is the hairpin dna containing 1 methylated cytosine；Fig. 1-c are the hairpin dna containing 2 methylated cytosines；Fig. 1-d are
The event frequency of three kinds of hairpin dnas；Fig. 1-e are trend chart of three kinds of hairpin dna residence times with voltage.
The residence time of three kinds of DNA, there are notable differences, with increasing for methylation sites number, long delay event（>30
ms）Ratio reduces, and the residence time is reduced, and event frequency increases.This explanation, methylation sites number increases so that DNA is not easy to be formed
Hairpin structure, and the stability of hairpin structure DNA reduces.Therefore, long delay event ratio, residence time, event frequency are utilized
Rate can distinguish the hairpin dna of three kinds of different methylations.
The residence time of hairpin dna and the relationship of voltage：Under+40 mV voltages of mV ~+120, as voltage increases, hair fastener
The DNA residence times reduce, such as Fig. 1-e, it was demonstrated that hairpin dna has passed through nano-pore.Also, under+40 mV voltages of mV ~+120,
There are notable difference in the residence time of three kinds of methylate DNAs, event frequency, therefore, can distinguish three under any voltage wherein
The hairpin dna of the different methylations of kind, wherein with residence time longest under the conditions of+40 mV, discrimination highest.
Embodiment 2
Potassium chloride is that traditional nanometer pore single-molecule detects electrolyte, similar to Example 1, is ensureing other experiment conditions
In the case of constant, testing result of the hairpin dna in potassium chloride electrolyte that methylate is as shown in Fig. 2, Fig. 2-a, b, c DNA
Testing principle schematic diagram（It is left）, single channel current signal graph（It is right）.Fig. 2-a are the hairpin dna without containing methylated cytosine；Figure
2-b is the hairpin dna containing 1 methylated cytosine；Fig. 2-c are the hairpin dna containing 2 methylated cytosines；Fig. 2-d are
The residence time distribution statistics figure of three kinds of hairpin dnas.
First, in KCl electrolyte, the residence time difference of three kinds of hairpin dnas is only ~ 10 ms, is much smaller than tetramethyl chlorination
In ammonium electrolyte ~ 2600 ms；Also, the magnitude relationship of the residence time of different methylation sites number hairpin dnas is containing 1mThe Fa KaDNA > of C;Containing 0mThe Fa KaDNA > of C;Containing 2mThe hairpin dna of C, three kinds of DNA do not show unified rule.Cause
This, can not realize differentiation and detect the purpose of methylate DNA in potassium chloride electrolyte.Tetramethyl ammonium chloride can selectively with
DNA methylation cytimidine acts on, and changes the stability of DNA double helical structure, to influence the telecommunications across nano-pore of DNA
Number, it is a kind of superior methylate DNA Single Molecule Detection electrolyte such as residence time, event frequency.
Embodiment 3
The single stranded DNA that methylates is detected to include the following steps：
（1）Hair fastener DNA probe is with target dna with concentration ratio 1:1 mixing, 95 DEG C of 5 min of heating, slow natural cooling.
（2）By the Telfon films for accomplishing fluently aperture in advance mounted in electrolytic cell center, electrolytic cell is divided into two parts, uses capillary
Pentane and hexadecane mixture containing 1% hexadecane are added dropwise around hole, 4 M tetramethyl ammonium chlorides, 10 mM are added in two slots
A small amount of phosphatide is added dropwise in the buffer solution of Tris-HCl, pH 8.5, ullage, and addition buffer solution makes liquid level not have small holes, is formed steady
Fixed phospholipid bilayer.The cis- addition wild type alpha hemolysin of electrolytic cell is inserted into phospholipid bilayer and forms nanometer single channel.
（3）The cis- mixing that hair fastener DNA probe and the target dna containing 0/1/2 methylated cytosine is added of electrolytic cell
Object stirs evenly, and DNA current signals are detected under positive voltage.The tail portion of hairpin dna is introduced into alpha hemolysin nano-pore cavity,
Under voltage driving, the double helix region that target dna is formed with DNA probe is unlocked, and DNA probe passes through nano-pore.
（4）Analysis statistics current signal, if Fig. 3 shows, Fig. 3-a, b, c be followed successively by from left to right DNA testing principles schematic diagram,
Single channel DNA current signals figure, residence time distribution statistics figure；Fig. 3-a do not contain the single stranded DNA and probe of methylated cytosine
Mixing sample；Fig. 3-b are the mixing sample of single stranded DNA and probe containing 1 methylated cytosine；Fig. 3-c are to contain 2
The single stranded DNA of methylated cytosine and the mixing sample of probe；Fig. 3-d are the event of three kinds of single stranded DNAs and probe mixing sample
Frequency.It is long delay event that Magen David, which marks,（>300 ms）Signal.
The residence time of three kinds of DNA, there are notable differences, with increasing for methylation sites number, long delay event（>300
ms）Ratio reduces, and event frequency increases.This explanation, methylation sites number increases so that target dna is not easy to form double spiral shells with probe
Revolve structure.Therefore, the hairpin dna of three kinds of different methylations can be distinguished using long delay event ratio, event frequency.
The single stranded DNA that the present invention detects different methylations is sent out different methylations based on tetramethyl ammonium chloride
The good discrimination of card structure DNA.After the pairing of target single stranded DNA and hairpin structure probes complementary, configuration similar to hairpin structure DNA,
So as to achieve the purpose that distinguish the single stranded DNA that methylates.
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.
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| CN112280875B (en) * | 2019-07-22 | 2023-07-18 | 四川大学华西医院 | Method, device and system for rapidly detecting bacterial drug resistance by utilizing nanopores |
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