CN109444105A - A kind of biological sensor and preparation method thereof detecting DNA glycosylase UDG - Google Patents
A kind of biological sensor and preparation method thereof detecting DNA glycosylase UDG Download PDFInfo
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- CN109444105A CN109444105A CN201811621278.5A CN201811621278A CN109444105A CN 109444105 A CN109444105 A CN 109444105A CN 201811621278 A CN201811621278 A CN 201811621278A CN 109444105 A CN109444105 A CN 109444105A
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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Abstract
The present invention relates to biosensor technology fields, in particular to the biological sensor of feedback rolling circle amplification and restriction endonuclease amplification Fluorometric assay DNA glycosylase UDG is assisted based on polymerase.In order to solve the problems, such as that the above method specificity for detecting UDG in the prior art and sensitivity are all relatively low.The cooperation of phi29 polymerase, endonuclease IV, are realized the fluorescence resonance energy transfer of rolling ring amplification and fluorophor and quencher, homogeneous reaction mixed liquor by a kind of biosensor based on feedback rolling circle amplification detection UDG.Preparation method: the building of annular template and combined probe；Feed back rolling ring amplified signal, fluorescence detection；UDG enzyme is utilized to the specific for hydrolysis of base U, can accurately measure UDG using this special reaction, while the generation of interference can also be avoided；It is recycled and is amplified using endonuclease IV, realize the effect of signal amplification.
Description
Technical field
The present invention relates to biosensor technology fields, in particular to based on feedback rolling circle amplification and endonuclease enzyme signal
The biological sensor of amplification, further relates to preparation method.
Background technique
DNA glycosylase UDG is a kind of important base-excision repair enzymes, responsible for rehabilitation DNA as caused by uracil damage
The integrality of wound and maintenance genome, while the unconventionality expression of UDG is also associated with kinds cancer.It is phonetic to hydrolyze cytimidine generation urine
Pyridine be DNA hydrolysis damage in one of the most common type form, so as to cause during DNA replication dna G:C base-pair be transformed into A:U alkali
Base pair.Initiator and " patrol " of the UDG as base excision repair path, have the high specific to uracil, therefore can
Act on the hydrolysis and division of identification and the N- glycosidic bond on the single-stranded or double-stranded DNA of catalysis.Then, impaired base is released simultaneously
It produces a kind of apyrimidinic site of no purine (i.e. the site AP) and triggers DNA repair process, and pass through additional AP inscribe
Enzyme, archaeal dna polymerase, DNA ligase coordinative role remove AP site, to replace normal cytosine base.As a result,
In DNA damage reparation and connection, enzyme is to have certain correlation with human body diseases for the activity of base-excision repair enzymes.
It includes cancer that abnormal UDG effect can be mutated directly related with a variety of diseases with modificator gene, genotype disease, and human immunity is scarce
It falls into.The active research of base-excision repair enzymes shows critical biological meaning to the physiological reaction for understanding DNA damage reparation.
Therefore, UDG carries out highly sensitive, highly selective detection and its basic function is studied and faced as a kind of potential biomarker
Bed diagnosis is all of great significance.
Classical UDG activity test method mainly has radioactive label method, gel electrophoresis, mass spectrometry.With top
Method short slab is that the consuming time, sensitivity is lower and needs to pre-process sample.
Summary of the invention
In order to solve, the method specificity for detecting UDG in the prior art and sensitivity are all relatively low, detection cycle is long is asked
Topic, the present invention provides a species specificity and high sensitivity, detection are fireballing, based on polymerase assist feedback rolling circle amplification and
Restriction endonuclease amplifies the biosensor of Fluorometric assay DNA glycosylase UDG, while additionally providing preparation method.
A kind of biological sensor detecting DNA glycosylase UDG, including object UDG, combined probe I, compound spy
Needle II, phi29 DNA polymerase, endonuclease IV, dNTP, 10 × buffer buffer；
The combined probe I by U-DNA and circular template by base pair complementarity in conjunction with formed；
The combined probe II by S-DNA and circular template by base pair complementarity in conjunction with formed；
The circular template by linear padlock-probe C-DNA in conjunction with linking probe L-DNA, in the effect of T4 DNA ligase
Under, the complex of annular template-connection primer is formed, in the presence of having exonuclease I and exonucleaseⅢ, primer is carried out
Digestion, to form annular template；
The base sequence is as follows:
U-DNA sequence is as shown in SEQ No.1；
Linear padlock-probe C-DNA sequence is as shown in SEQ No.2；
Linking probe L-DNA sequence is as shown in SEQ No.3；
S-DNA sequence is as shown in SEQ No.4；
There is a decorating site U between 5 ' the 41st and 42 bit bases of end of the U-DNA, represents uracil base；The 3 ' of U-DNA
Terminal modified Inverted dT, that is, reversed dT is used to inhibit the degradation of exonuclease；
5 ' terminal modified phosphate groups in the C-DNA；
Dabcyl quenching group is modified between 5 ' the 41st and 42 bit bases of end of the S-DNA, then there are also a tetrahydro furans
It mutters decorating site, i.e., without purine without pyrimidine site；FAM fluorophor has been modified between 45th and 46 bit bases；3 ' is terminal modified
Inverted dT, that is, reversed dT be used to inhibit the degradation of exonuclease.
The preparation method of above-mentioned biological sensor, comprising the following steps:
(1) annular template building；
(2) preparation of combined probe I, combined probe II；
(3) homogeneous reaction；
(4) fluorescence detection: it is 486 nm that excitation wavelength, which is arranged, in luminoscope, detects fluorescence intensity at 518 nm, detection range 450
nm-530 nm。
The step (1) specifically comprises the processes of:
C-DNA and L-DNA 6 are added in EP pipe by S1, and 5 min are incubated at 95 DEG C, are slowly cooled to room temperature；Then
It is put into 16 DEG C of water-baths, reaction overnight；
Then S2 adds T4 DNA ligase in reaction system, it is reacted 20 h at 16 DEG C；
T4 DNA ligase in S3 deactivation system；
Exonuclease I and exonucleaseⅲ are added into above-mentioned reaction system by S4, react 2 h at 37 DEG C；Again by reactant
It is 85 DEG C of 10 min of heating water bath, obtains annular template, preservation is spare under the conditions of 4 DEG C.
The preparation process of combined probe I in the step (2) are as follows: buffer aqua sterilisa, annular template, U-DNA, PBS
Liquid is added in EP pipe, in 37 DEG C of 40 min of incubation, is hybridized annular template sufficiently with U-DNA, is prepared into combined probe I.
The preparation process of combined probe II in the step (2) are as follows: by annular template, aqua sterilisa, PBS buffer solution, S-
DNA is prepared into combined probe II in 37 DEG C of 40 min of incubation.
The step (3) specifically comprises the processes of: by aqua sterilisa, 10 × buffer buffer, combined probe I, combined probe II,
UDG, phi29 DNA polymerase, endonuclease IV, dNTP are added in EP pipe, at 37 DEG C, are incubated for 2 h.
10 × buffer buffer are as follows: 50 mM Tris-HCl, 10 mM MgCl2、10 mM (NH4)2SO4、4
mM DTT；pH 7.5.
5 DNA chain are used altogether in the present invention, sequence is respectively:
U-DNA: CACACGAATTCATCTG TTTTTTTTTTTTACTCTTCCTAGCTUGACTTGCC
GGACTTTAGTCAAGCTATTTTT-Inverted dT
Linear padlock-probe C-DNA (circular DNA):
p-ATTCGTGTGATAGCTTACATGGCAGAGACTGGATAGC
TTACATGGCCAGATGA
Linking probe L-DNA (ligation DNA): CACACGAATTCATCTGT
S-DNA(signal DNA): CACACGAATTCATCTG TTTTTTTTTTTTACTCTTCCTAGC
T(Dabcyl)XACAT(FAM)GGC-Inverted dT
Wherein with the red highlighted decorating site for indicating uracil-DNA nucleosides, the part of italic in U-DNA
It is then the complementary series with annular template, 3 ' terminal modified Inverted dT, that is, reversed dT is used to inhibit the drop of exonuclease
Solution effect.P in C-DNA indicates the modification of a 5 ' phosphate groups, for being held under the action of T4 DNA ligase with 3 '
Hydroxyl form phosphodiester bond, constitute annular template, and underlined region indicates the bond area with L-DNA.In S-DNA tiltedly
Body portion expression can modify FAM fluorophor on two base T respectively and Dabcyl quenches with the binding sequence of circular template
Go out group, and X indicates tetrahydrofuran site (dSpacer) modification, i.e., without purine without pyrimidine site (AP site).3 ' is terminal modified
Inverted dT, that is, reversed dT is used to inhibit the degradation of exonuclease.
The detection of UDG is realized in homogeneous phase solution in the present invention, passes through phi29 DNA polymerase, endonuclease
Dual signal amplification is realized in the cooperation of IV, to realize the highly sensitive detection of UDG, and obtains lower Monitoring lower-cut.
(2) the homogeneous middle reaction occurred mainly has: the preparation of annular template and Complex Probes；Contain the compound of base U
Identification process of the probe to object；The feedback rolling circle amplification of 3 ' -5 ' 5 prime excision enzyme activities based on phi29 archaeal dna polymerase is anti-
It answers.
(1) preparation of annular template and combined probe.Linear padlock-probe C-DNA is in conjunction with linking probe L-DNA, in T4
Under the action of DNA ligase, the complex of annular template-connection primer is formed, when thering is exonuclease I and exonucleaseⅢ to deposit
When, primer is digested, so that it is spare to form annular template.The U-DNA that is modified containing base U can with prepare
Circular template is combined by base pair complementarity and forms combined probe I；And S-DNA can in conjunction with the circular template prepared shape
At combined probe II.
(2) object UDG identifies that the uracil on combined probe generates hydrolysis.In the presence of having object, UDG energy
Enough identification is modified with the combined probe I of uracil and effect is hydrolyzed to glycosidic bond in advance, subsequent generation one without purine without
The AP site of pyrimidine.Then, endonuclease IV identifies AP site and carrying out cutting is broken combined probe, is there is phi29
In the presence of archaeal dna polymerase, 3 ' -5 ' 5 prime excision enzyme activities can hydrolyze the not free base with circular template base pair complementarity,
Form primer-circular template rolling circle amplification reacting precursor.
(3) the feedback rolling circle amplification reaction of 3 ' -5 ' 5 prime excision enzyme activities based on phi29 archaeal dna polymerase.Above-mentioned reaction produces
Raw RCA precursor is able to carry out rolling circle amplification reaction, while the 3 ' ends of combined probe II can be in conjunction with a large amount of RCA products, shape
At double-strand.In the presence of having endonuclease IV, AP site release fluorophor and the primer-with quencher can be cut off
Circular template structure, under 3 ' -5 ' the 5 prime excision enzyme activities effect of phi29 archaeal dna polymerase, feedback forms reaction (2) Bu Zhongxiang
Same primer-circular template RCA precursor.To realize that the feedback rolling of more multiple amplification circulations also expands.
The detection mode of the invention is Fluorometric assay, utilizes luminoscope.Before testing, first by C-DNA and L-DNA shape
Circularize template probe.Then object is added to the homogeneous of combined probe I and combined probe II containing label uracil
Solution, in 37 DEG C of 2 h of incubation, object hydrolyzes the glycosidic bond around uracil, forms abasic site.In phi29 DNA
Polymerase and the lower more multiples of completion of endonuclease IV effect feed back amplification process, to realize the amplification of signal.Then with glimmering
It is 486 nm that excitation wavelength, which is arranged, in light instrument, detects fluorescence intensity at 518 nm, and detection range is 450 nm-530 nm.
The present invention is based on the specific recognitions of the nucleic acid probe of label uracil and object, with chain extension function
Phi29 DNA polymerase realizes the extension of chain, generates largely extended chain complementary with combined probe II, phi29 DNA polymerase
Polymerization and its 3 ' -5 ' 5 prime excision enzyme activities and endonuclease IV cooperation rolling circle amplification amplification and fluorescent base
Group and the fluorescence resonance energy transfer of quencher construct aptamer biosensors.The sensor has detection speed fast, inspection
The advantages that limit is low, and specificity is high is surveyed, the shortcomings and deficiencies of the existing detection method of UDG can be made up, is realized fast and accurate to its
Quantitative detection.
Beneficial effects of the present invention:
1. specific recognition
The nucleic acid probe of uracil and the specific recognition of object are marked, the hydrolysis of UDG and uracil, phi29 are utilized
The 5 prime excision enzyme activity of the 3 ' of archaeal dna polymerase -5 ' realizes that the booster action of feedback amplification and endonuclease IV are realized to target
The high specific of object detects；
2. ultrasensitiveness detects
The cleavage site (AP site) of endonuclease IV is utilized, realizes that positional dissection, release fluorophor generate certain strong
The fluorescence signal of degree；Polymerization and 3 ' -5 ' 5 prime excision enzyme activities using phi29 archaeal dna polymerase are put in realization rolling circle amplification
It realizes that feedback is amplified again and endonuclease amplifies while big effect, realizes fluorescence signal amplification, improve detection
Sensitivity, lowest detection are limited to 4.7 × 10-5U/mL is realized and is detected to the ultrasensitiveness of object UDG；
3. detection is rapid
The reaction condition of the sensor is mild, and reaction speed is fast；Due to using fluorescence method, detection method is easy to operate, detects
Period is short；The main process of testing principle is to improve reaction speed in homogeneous middle realization, reduce the complicated journey of operation
Degree, realizes the quick of object, simply, sensitive to detect；
4. reproducible
Preparation method is simple, performance stablize, fluorescence detection it is reproducible, be suitable for UDG relevant to disease detection and biology
The practical application of sensor industrialization；The process costs for making the biosensor are low, the inexpensive requirement suitable for industrialization.
Detailed description of the invention
Fig. 1 is the schematic diagram of the test；
Fig. 2 is the testing result figure of embodiment 2；
Fig. 3 is the testing result figure of embodiment 3；
Fig. 4 is the testing result figure of embodiment 4；
Fig. 5 is the testing result figure of embodiment 5.
Fig. 6 is the standard curve that UDG enzyme is detected in embodiment 6.
Specific embodiment
Invention is further explained combined with specific embodiments below.
The preparation of the annular template of embodiment 1 and combined probe
It prepares and contains 50 mM Tris-HCl, 10 mM MgCl2, the T4 DNA ligase reaction of 10 mM DTT and 1 mM ATP is slow
Fliud flushing.It prepares and contains 10 mM Na2HPO4, 10 mM NaH2PO4, 140 mM NaCl, 1 mM KCl, 1 mM MgCl2, 1
mM CaCl2, the PBS buffer solution of pH=7.4.
(1) by 42 μ L aqua sterilisas, 6 μ L linear dies (10 μM), 6 μ L linking probes (10 μM) and 6 μ L 10 ×
T4 DNA ligase buffer mixes, 95 DEG C of 5 min of denaturation, is then slowly cooled to room temperature and completes hybridization, then in reactant
3 μ L T4 DNA ligases (60 U/ μ L) is added in system, it is reacted 20 hours at 16 DEG C；Later, reaction system is at 65 DEG C
Temperature condition is lauched bath 15 minutes, the T4 DNA ligase in deactivation system.
(2) exonucleaseⅲ of I (20 U/ μ L) and 3 μ L of 3 μ L exonuclease are added in Xiang Shangshu reaction system
2 h are reacted at 37 DEG C of (100 U/ μ L)；Again by 85 DEG C of 10 min of heating water bath of reaction system, annular template is obtained, is protected under the conditions of 4 DEG C
It hides spare.
(3) by 24 μ L aqua sterilisas, 4 μ L annular templates (1 μM), 4 μ L U-DNA(1 μM), 8 μ L PBS buffer solution add
Enter into EP pipe, in 37 DEG C of 40 min of incubation, hybridizes annular template sufficiently with U-DNA, be prepared into combined probe I；With identical
4 μ L annular templates (1 μM), 4 μ L S-DNA(1 μM) are prepared into combined probe II in 37 DEG C of 40 min of incubation by method.
2 fluorescence intensity of embodiment with combined probe I concentration variation
A kind of preparation method of biological sensor of the present invention, comprising the following steps:
(1) be respectively 50 nM, 100 nM, 500 nM, 1 μM, 5 μM by 2 μ L combined probe I(concentration), 2 μ L dNTP(1
MM), 2 μ L phi29 archaeal dna polymerases (1 U/ μ L), 2 μ L endonuclease IV(1 U/ μ L) in 2 μ L buffer (50 mM
Tris-HCl, 10 mM MgCl2, 10 mM (NH4)2SO4, 4 mM DTT, pH 7.5) in mixing after be separately added into 2 μ L
UDG enzyme solution (1 U/mL), 37 DEG C of 60 min of isothermal reaction after mixing；
(2) 2 II(1 μM of μ L combined probes are added into the solution of step (1)), 37 DEG C of 60 min of isothermal reaction after mixing；
(3) step (2) acquired solution is diluted with water to 100 μ L, will then carries out fluorescence detection；Excitation wavelength is set as 486
Nm, launch wavelength are 518 nm, 450 nm-530 nm of detection range, read fluorescence signal variation.
Testing result is shown in Fig. 2, it can be seen from the figure that fluorescence intensity constantly enhances with the increase of combined probe I amount,
After combined probe I amount reaches 1 μM, fluorescence intensity is basically unchanged.
The preparation method for the solution used in the above process:
Ultrapure water is both needed to carry out high-temperature sterilization processing.Specific method is that ultrapure water is individually positioned in conical flask, then uses tin
Foil paper and newspaper are sealed.In high-pressure sterilizing pot 120 DEG C at a temperature of sterilize 20 min.10 × buffer
(buffer) it is to be purchased with polymerase, can be used directly.
3 fluorescence intensity of embodiment with combined probe II concentration variation
A kind of preparation method of biological sensor of the present invention, comprising the following steps:
(1) by 2 I(1 μM of μ L combined probes), 2 μ L dNTP(1 mM), 2 μ L phi29 archaeal dna polymerases (1 U/ μ L), 2 μ
L endonuclease IV(1 U/ μ L) in 2 μ L buffers (50 mM Tris-HCl, 10 mM MgCl2, 10 mM (NH4)2SO4, 4 mM DTT, pH 7.5) in mixing after be separately added into 2 μ L UDG enzyme solutions (1 U/mL), 37 DEG C of isothermal reactions after mixing
60 min；
(2) it is respectively 50 nM, 100 nM, 500 nM, 1 μM that 2 μ L combined probe II(concentration are added into the solution of step (1)
, 5 μM), 37 DEG C of 60 min of isothermal reaction after mixing；
(3) step (2) acquired solution is diluted with water to 100 μ L, will then carries out fluorescence detection；Excitation wavelength is set as 486
Nm, launch wavelength are 518 nm, 450 nm-530 nm of detection range, read fluorescence signal variation.
Testing result is shown in Fig. 3, it can be seen from the figure that fluorescence intensity constantly enhances with the increase of combined probe II amount,
After combined probe II amount reaches 1 μM, fluorescence intensity is basically unchanged.
The preparation method for the solution used in the above process:
Ultrapure water is both needed to carry out high-temperature sterilization processing.Specific method is that ultrapure water is individually positioned in conical flask, then uses tin
Foil paper and newspaper are sealed.In high-pressure sterilizing pot 120 DEG C at a temperature of sterilize 20 min.10 × buffer
(buffer) it is to be purchased with polymerase, can be used directly.
4 fluorescence intensity of embodiment with endonuclease IV concentration variation
A kind of preparation method of biological sensor of the present invention, comprising the following steps:
(1) by 2 I(1 μM of μ L combined probes), 2 μ L dNTP(1 mM), 2 μ L phi29 archaeal dna polymerases (1 U/ μ L), 2 μ
L endonuclease IV(concentration is respectively 0.05 U/ μ L, 0.1 U/ μ L, 0.5 U/ μ L, 1 U/ μ L, 5 U/ μ L) it is buffered in 2 μ L
Liquid (50 mM Tris-HCl, 10 mM MgCl2, 10 mM (NH4)2SO4, 4 mM DTT, pH 7.5) in mixing after respectively
2 μ L UDG enzyme solutions (1 U/mL), 37 DEG C of 60 min of isothermal reaction after mixing is added；
(2) 2 II(1 μM of μ L combined probes are added into the solution of step (1)), 37 DEG C of 60 min of isothermal reaction after mixing；
(3) step (2) acquired solution is diluted with water to 100 μ L, will then carries out fluorescence detection；Excitation wavelength is set as 486
Nm, launch wavelength are 518 nm, 450 nm-530 nm of detection range, read fluorescence signal variation.
Testing result is shown in Fig. 4, it can be seen from the figure that with the increase of endonuclease IV amount, fluorescence intensity constantly increases
By force, after endonuclease IV amount reaches 1 U/ μ L, fluorescence intensity is basically unchanged.
The preparation method for the solution used in the above process:
Ultrapure water is both needed to carry out high-temperature sterilization processing.Specific method is that ultrapure water is individually positioned in conical flask, then uses tin
Foil paper and newspaper are sealed.In high-pressure sterilizing pot 120 DEG C at a temperature of sterilize 20 min.10 × buffer
(buffer) it is to be purchased with polymerase, can be used directly.
5 fluorescence intensity of embodiment with archaeal dna polymerase concentration variation
A kind of preparation method of biological sensor of the present invention, comprising the following steps:
(1) by 2 I(1 μM of μ L combined probes), 2 μ L dNTP(1 mM), (concentration is respectively 2 μ L phi29 archaeal dna polymerases
0.05 U/ μ L, 0.1 U/ μ L, 0.5 U/ μ L, 1 U/ μ L, 5 U/ μ L), 2 μ L endonuclease IV(1 U/ μ L) 2 μ L buffer
Liquid (50 mM Tris-HCl, 10 mM MgCl2, 10 mM (NH4)2SO4, 4 mM DTT, pH 7.5) in mixing after respectively
2 μ L UDG enzyme solutions (1 U/mL), 37 DEG C of 60 min of isothermal reaction after mixing is added；
(2) 2 II(1 μM of μ L combined probes are added into the solution of step (1)), 37 DEG C of 60 min of isothermal reaction after mixing；
(3) step (2) acquired solution is diluted with water to 100 μ L, will then carries out fluorescence detection；Excitation wavelength is set as 486
Nm, launch wavelength are 518 nm, 450 nm-530 nm of detection range, read fluorescence signal variation.
Testing result is shown in Fig. 5, it can be seen from the figure that with the increase of phi29 archaeal dna polymerase amount, fluorescence intensity is continuous
Enhancing, after phi29 archaeal dna polymerase amount reaches 1 U/ μ L, fluorescence intensity is basically unchanged.
The preparation method for the solution used in the above process:
Ultrapure water is both needed to carry out high-temperature sterilization processing.Specific method is that ultrapure water is individually positioned in conical flask, then uses tin
Foil paper and newspaper are sealed.In high-pressure sterilizing pot 120 DEG C at a temperature of sterilize 20 min.10 × buffer
(buffer) it is to be purchased with polymerase, can be used directly.
Detection of the embodiment 6 to UDG enzyme
A kind of preparation method of biological sensor of the present invention, comprising the following steps:
(1) by 2 I(1 μM of μ L combined probes), 2 μ L dNTP(1 mM), 2 μ L phi29 archaeal dna polymerases (1 U/ μ L), 2 μ
L endonuclease IV(1 U/ μ L) in 2 μ L buffers (50 mM Tris-HCl, 10 mM MgCl2, 10 mM (NH4)2SO4, 4 mM DTT, pH 7.5) in be separately added into 2 μ L UDG enzyme solutions (concentration be from 5 × 10-5 U/mL to 1 U/ after mixing
ML), 37 DEG C of 60 min of isothermal reaction after mixing；
(2) 2 II(1 μM of μ L combined probes are added into the solution of step (1)), 37 DEG C of 60 min of isothermal reaction after mixing；
(3) step (2) acquired solution is diluted with water to 100 μ L, will then carries out fluorescence detection；Excitation wavelength is set as 486
Nm, launch wavelength are 518 nm, 450 nm-530 nm of detection range, read fluorescence signal variation.
According to the fluorescence intensity of serial UDG enzyme solution, make standard curve, as shown in fig. 6, calculating regression equation is F=986.8+
195.6×LgCUDG (U/mL), related coefficient 0.9925 is limited to 4.7 × 10 by can be calculated lowest detection-5U/mL.Root
According to the fluorescence intensity 836 of prepare liquid, calculate wherein UDG enzyme concentration be 0.169 U/mL.
The preparation method for the solution used in the above process:
Ultrapure water is both needed to carry out high-temperature sterilization processing.Specific method is that ultrapure water is individually positioned in conical flask, then uses tin
Foil paper and newspaper are sealed.In high-pressure sterilizing pot 120 DEG C at a temperature of sterilize 20 min.10 × buffer
(buffer) it is to be purchased with polymerase, can be used directly.
The research of 7 experimental repeatability of embodiment
A kind of preparation method of biological sensor of the present invention, comprising the following steps:
(1) by 2 I(1 μM of μ L combined probes), 2 μ L dNTP(1 mM), 2 μ L phi29 archaeal dna polymerases (1 U/ μ L), 2 μ
L endonuclease IV(1 U/ μ L) in 2 μ L buffers (50 mM Tris-HCl, 10 mM MgCl2, 10 mM (NH4)2SO4, 4 mM DTT, pH 7.5) in be separately added into 2 μ L UDG enzyme solutions (concentration be respectively 1 U/mL, 0.1 U/ after mixing
ML, 0.01 U/mL, 0.001 U/mL, 0.0001 U/mL), 37 DEG C of 60 min of isothermal reaction after mixing；
(2) 2 II(1 μM of μ L combined probes are added into the solution of step (1)), 37 DEG C of 60 min of isothermal reaction after mixing；
(3) above-mentioned reaction system is repeated 5 times respectively.
(4) step (2) acquired solution is diluted with water to 100 μ L, will then carries out fluorescence detection；Excitation wavelength setting
For 486 nm, launch wavelength is 518 nm, 450 nm-530 nm of detection range, reads fluorescence signal variation.
Testing result is as shown in table 1, as can be seen from the table, carries out acquired by 3 identical experiments for same concentration
Fluorescence signal intensity value is almost the same, has good repeatability.
Table 1 is to the biological sensor repetitive research
The preparation method for the solution used in the above process:
Ultrapure water is both needed to carry out high-temperature sterilization processing.Specific method is that ultrapure water is individually positioned in conical flask, then uses tin
Foil paper and newspaper are sealed.In high-pressure sterilizing pot 120 DEG C at a temperature of sterilize 20 min.10 × buffer
(buffer) it is to be purchased with polymerase, can be used directly.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by the limit of embodiment
System, other any changes made without departing from the spirit and principles of the present invention, modification, combination, substitution, simplification should be
Equivalence replacement mode, is included within the scope of the present invention.
Sequence table
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aagctatttt t 71
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cacacgaatt catctgtttt ttttttttac tcttcctagc tacatggc 48
Claims (7)
1. a kind of biological sensor for detecting DNA glycosylase UDG, which is characterized in that including object UDG, compound spy
Needle I, combined probe II, phi29 DNA polymerase, endonuclease IV, dNTP, 10 × buffer buffer；
The combined probe I by U-DNA and circular template by base pair complementarity in conjunction with formed；
The combined probe II by S-DNA and circular template by base pair complementarity in conjunction with formed；
The circular template by linear padlock-probe C-DNA in conjunction with linking probe L-DNA, in the effect of T4 DNA ligase
Under, the complex of annular template-connection primer is formed, in the presence of having exonuclease I and exonucleaseⅢ, primer is carried out
Digestion, to form annular template；
The base sequence is as follows:
U-DNA sequence is as shown in SEQ No.1；
Linear padlock-probe C-DNA sequence is as shown in SEQ No.2；
Linking probe L-DNA sequence is as shown in SEQ No.3；
S-DNA sequence is as shown in SEQ No.4；
There is a decorating site U between 5 ' the 41st and 42 bit bases of end of the U-DNA, represents uracil base；The 3 ' of U-DNA
Terminal modified Inverted dT, that is, reversed dT is used to inhibit the degradation of exonuclease；
5 ' terminal modified phosphate groups in the C-DNA；
Dabcyl quenching group is modified between 5 ' the 41st and 42 bit bases of end of the S-DNA, then there are also a tetrahydro furans
It mutters decorating site, i.e., without purine without pyrimidine site；FAM fluorophor has been modified between 45th and 46 bit bases；3 ' is terminal modified
Inverted dT, that is, reversed dT be used to inhibit the degradation of exonuclease.
2. the preparation method of biological sensor described in claim 1, which comprises the following steps:
(1) annular template building；
(2) preparation of combined probe I, combined probe II；
(3) homogeneous reaction；
(4) fluorescence detection: it is 486 nm that excitation wavelength, which is arranged, in luminoscope, detects fluorescence intensity at 518 nm, detection range 450
nm-530 nm。
3. preparation method according to claim 2, which is characterized in that the step (1) specifically comprises the processes of:
C-DNA and L-DNA 6 are added in EP pipe by S1, and 5 min are incubated at 95 DEG C, are slowly cooled to room temperature；Then
It is put into 16 DEG C of water-baths, reaction overnight；
Then S2 adds T4 DNA ligase in reaction system, it is reacted 20 h at 16 DEG C；
T4 DNA ligase in S3 deactivation system；
Exonuclease I and exonucleaseⅲ are added into above-mentioned reaction system by S4, react 2 h at 37 DEG C；Again by reactant
It is 85 DEG C of 10 min of heating water bath, obtains annular template, preservation is spare under the conditions of 4 DEG C.
4. preparation method according to claim 2, which is characterized in that the preparation work of combined probe I in the step (2)
Skill are as follows: aqua sterilisa, annular template, U-DNA, PBS buffer solution are added in EP pipe, in 37 DEG C of 40 min of incubation, make circular die
Plate sufficiently hybridizes with U-DNA, is prepared into combined probe I.
5. preparation method according to claim 2, which is characterized in that the preparation of combined probe II in the step (2)
Technique are as follows: annular template, PBS buffer solution, aqua sterilisa, S-DNA are prepared into combined probe II in 37 DEG C of 40 min of incubation.
6. preparation method according to claim 2, which is characterized in that the step (3) specifically comprises the processes of: by aqua sterilisa,
10 × buffer buffer, combined probe I, combined probe II, UDG, phi29 DNA polymerase, endonuclease IV, dNTP
It is added in EP pipe, at 37 DEG C, is incubated for 2 h.
7. the preparation method according to claim 2 or 6, which is characterized in that 10 × buffer buffer are as follows: 50
mM Tris-HCl、10 mM MgCl2、10 mM (NH4)2SO4,4 mM DTT；pH 7.5.
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