CN109706224A - A kind of method that the Multiple Cycle amplification of exonuclease III auxiliary is used to delicately detect DNA - Google Patents
A kind of method that the Multiple Cycle amplification of exonuclease III auxiliary is used to delicately detect DNA Download PDFInfo
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Abstract
The method that the disclosure is used to delicately detect DNA more particularly to a kind of Multiple Cycle amplification of exonuclease III auxiliary, the technical program utilizes the combination of single-stranded DNA binding protein (SSB) and hairpin probe, the non-specific digestion of exonuclease III (Exo III) is efficiently avoided, to generate nearly zero background signal.The presence of target DNA has caused the multi-cycle amplification of exonuclease III (Exo III) auxiliary, high sensitivity, detection is limited to 3 every liter of femtomoles, and has excellent selectivity, it can identify single base mispairing, there is great potential in terms of medical diagnosis on disease and biomedical research.
Description
Technical field
The disclosure belongs to bioassay technique field, and in particular to a kind of Multiple Cycle expansion of exonuclease III auxiliary
It adds in the method for delicately detecting DNA.
Background technique
Here statement only provides background information related with the disclosure, without necessarily constituting the prior art.
Delicately detection specific dna sequence is in clinical diagnosis, and gene therapy, environmental monitoring and field of food safety are to pass
Important, and developed various DNA biosensors.Based on the amplification of archaeal dna polymerase due to its higher sensitivity
And it is widely used, and micro target DNA can directly be expanded to detectable level by DNA polymerization reaction.Polymerization
Enzyme chain reaction (PCR) is the goldstandard of DNA cloning, but it needs expensive thermal cycler to accurately control reaction temperature,
Its extensive use in non-lab environment is limited, such as instant (POC) test.Isothermal duplication can make DNA in constant temperature
It is carried out amplification reaction under degree, greatly facilitates its practical application, including rolling circle amplification (RCA), exponential amplification (EXPAR), chain is set
It changes amplification (SDA), ring mediated isothermal amplification (LAMP), helicase dependent amplification (HDA) and recombination polymeric enzymatic amplification (RPA)
Deng.Although their sensitivity increases, time-consuming for these amplification based on archaeal dna polymerase, is related to complicated template
And design of primers, and there is intrinsic non-specific amplification.
In existing technical method, it is widely used based on the amplification of archaeal dna polymerase due to its higher sensitivity, and
And micro target DNA can directly be expanded to detectable level by DNA polymerization reaction.Polymerase chain reaction (PCR)
It is the goldstandard of DNA cloning, but it needs expensive thermal cycler to accurately control reaction temperature, limits it in non-experiment
Extensive use in room environmental, such as instant (POC) test.Isothermal duplication can be such that DNA carries out amplification reaction at a constant temperature,
Its practical application, including rolling circle amplification (RCA) are greatly facilitated, exponential amplification reacts (EXPAR), strand displacement amplification (SDA), ring
Mediated isothermality amplification (LAMP), helicase dependent amplification (HDA) and recombination polymeric enzymatic amplification (RPA) etc..Although their spirit
Sensitivity increases, but time-consuming for these amplification based on archaeal dna polymerase, is related to complicated template and design of primers, and have
There is intrinsic non-specific amplification.
The target circulation of the circumscribed III of nuclease (Exo III) auxiliary is due to its higher sensitivity, simple design and easily behaviour
The property made widely is paid close attention to.Nicking endonuclease is a kind of enzyme applied to target circulation, has particular sequence for detecting
The target DNA of (containing nicking sites), and exonuclease III (Exo III) is better than nicking endonuclease, it is a kind of
Enzyme without sequence dependent does not need specific recognition site, is suitable for combining colorimetric, electrochemistry, chemiluminescence and fluorescence method
To develop the multifunctional platform for detecting various target DNA.However, due to only relating to the amplification of single loop signal and higher background
Noise, sensitivity are usually limited.Therefore, we are auxiliary it is necessary to develop a kind of new exonuclease III (Exo III)
The multi-cycle helped expands to detect target DNA.
Summary of the invention
For background technique, the disclosure proposes that a kind of multi-cycle using exonuclease III (Exo III) auxiliary expands
Method delicately to detect DNA.In this technology method, single-stranded DNA binding protein (SSB) can combine closely with probe,
The non-specific digestion of exonuclease III (Exo III) is protected them from, to detect target with zero background signal
DNA.The multi-cycle amplification that target causes substantially increases the sensitivity of detection, and detection is limited to 3 every liter of femtomoles, and dynamic model
It encloses and is often raised to 1 nanomole from 10 femtomoles and is often upgraded to 5 orders of magnitude.In addition, the technical program can be further used for distinguishing list
The target and the target DNA in selective enumeration method complexity blood serum sample of base mispairing.
The disclosure specifically uses following technical scheme:
In the first aspect of the disclosure, a kind of Multiple Cycle amplification of exonuclease III auxiliary is provided for sensitive
The method that ground detects DNA, this method comprises:
When there are the single-stranded DNA binding proteins in the case where target DNA, in 1 compound of single-stranded DNA binding protein-probe
It is detached from, target DNA forms 1 duplex of target-probe with the flat end 3'- in conjunction with the 3'- protruding terminus of probe 1, can
It is identified and gradually digested by exonuclease III, therefore, target DNA and triggering primer can be released from duplex, with
Afterwards, the target DNA of release causes the next round circulation digestion of starting probe 1 in conjunction with new probe 1；The triggering primer of release draws
The single-stranded DNA binding protein risen in 2 compound of single-stranded DNA binding protein-probe is detached from, thus the 3'- protruding terminus with probe 2
Complementation, forms 2 duplex of target-probe with the flat end 3'-, then causes digestion of the exonuclease III to probe 2,
To release triggering primer and target analog；The triggering primer of release starts a new round for probe 2 in conjunction with new probe 2
Digestion, the target analog of release start new circular response in conjunction with new probe 1 because it includes the identical sequences of target；
The digestion that target DNA causes probe 1 and probe 2 detects to generate the fluorescence of the fluorophor of enhancing according to fluorescence intensity
DNA。
In the second aspect of the disclosure, a kind of Multiple Cycle amplification of exonuclease III auxiliary is provided for sensitive
Detect the probe combinations of DNA, including probe 1 and probe 2 in ground；
The probe 1 and probe 2 are 3 ' end hair fastener type DNA outstanding, wherein
The probe 1 includes: 3 ' the end outstanding single stranded DNA structures complementary with target DNA；
5 ' ends of the bulbous double-stranded DNA structure being complementarily shaped to by arm 1 and arm 2,1 sequence of arm have fluorophor；And
Cyclic DNA structure 1；
The probe 2 includes: 3 ' end single stranded DNA structures outstanding, and sequence is 2 sequence of arm；
By the bulbous double-stranded DNA that 3 ' end single stranded DNA structure sequences outstanding are complementarily shaped in the target DNA and probe 1
5 ' ends of structure, target DNA have fluorophor；And
Cyclic DNA structure 2.
In terms of the third of the disclosure, a kind of Multiple Cycle amplification of exonuclease III auxiliary is provided for sensitive
Ground detects the kit of DNA, which includes at least following component: exonuclease III, probe 1, probe 2 and single stranded DNA
Binding protein.
At the 4th aspect of the disclosure, the application of the probe combinations or kit in detection DNA is provided.
Compared with the relevant technologies that the present inventor knows, the one of technical solution of the disclosure has following beneficial to effect
Fruit:
(1) at low cost, easy to operate: compared to polymerase chain reaction (PCR), it is anti-to control not need accurate instrument
Temperature is answered, and only relates to a kind of exonuclease III (Exo III), experimental cost is greatly reduced, uses the hair clip of self-quenching
Probe simplifies the design of probe.
(2) high sensitivity, specificity are good: in the technical program, we are assisted due to exonuclease III (Exo III)
Multi-cycle amplification and nearly zero background signal, reached high sensitivity, better than the detection methods of most of reports.This
Outside, the technical program can distinguish the target of single base mismatch, have excellent specificity.
Detailed description of the invention
The Figure of description for constituting disclosure a part is used to provide further understanding of the disclosure, the signal of the disclosure
Property embodiment and its explanation for explaining the disclosure, do not constitute the improper restriction to the disclosure.
The principle explanatory diagram of the multi-cycle amplification of signal detection DNA of Fig. 1: exonuclease III auxiliary.
Fig. 2: when there is no the feelings of (A) and the every liter of target DNA of 10 nanomole that there is (B) single-stranded DNA binding protein (SSB)
To the real-time monitoring of FAM fluorescence under condition, wherein curve 1 indicates that addition single-stranded DNA binding protein (SSB), the expression of curve 2 are not added
Single-stranded DNA binding protein (SSB).The concentration of single-stranded DNA binding protein (SSB) is 200 every liter of nanomoles.
Fig. 3: target DNA+ probe 1+ probe 2+ exonuclease III (curve 1), target DNA+ probe 1+ exonucleaseⅢ
(curve 2), probe 1+ probe 2+ exonuclease III (curve 3), FAM's corresponding to probe 1+ probe 2 (curve 4) is real-time
Fluorescence monitoring.The concentration of its middle probe 1 and probe 2 is 200 every liter of nanomoles, and the concentration of target DNA is 10 every liter of nanomoles.
Fig. 4: the target DNA of (A) various concentration real-time fluorescence corresponding in the range of every liter of 0 to 10 nanomole is bent
Line.(B) there is (■) and when there is no (●) SSB, the logarithm of the fluorescence intensity and target DNA concentration that are obtained at 80 minutes it
Between linear relationship.What error bar indicated is the standard deviation of three repeated experiments.
Fig. 5: target DNA (T), single base mismatch DNA (T1), double alkali yl mismatched dna (T2), random sequence (R) He Wuren
Fluorescence intensity corresponding to the control group (control) of what target DNA.What error bar indicated is the standard deviation of three repeated experiments
Difference.
Fig. 6: the rate of recovery detection in blood serum sample.
Specific embodiment
It is noted that described further below be all exemplary, it is intended to provide further instruction to the disclosure.Unless another
It indicates, all technical and scientific terms used herein has usual with disclosure person of an ordinary skill in the technical field
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root
According to the illustrative embodiments of the disclosure.As used herein, unless the context clearly indicates otherwise, otherwise singular
Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation and/or their combination.
As background technique is introduced, detect that low-abundance DNA is most important, based on the amplification of archaeal dna polymerase due to
Its excellent sensitivity and be widely used, but it also has that time-consuming, complicated template and design of primers and intrinsic non-
The limitations such as specific amplification.The target of exonuclease III (Exo III) auxiliary, which is recycled, is fitted due to it with simplicity and generally
With property, a kind of new method is provided for the detection of DNA, but the non-specific digestion of exonuclease III (Exo III) can produce
Raw high background signal, and the amplification of single cycle signal then will lead to poor sensitivity.Therefore, in order to solve problem above, this hair
The bright multi-cycle amplification for introducing exonuclease III (Exo III) auxiliary has nearly zero for delicately detecting DNA
Background signal.
The technical program utilizes the combination of single-stranded DNA binding protein (SSB) and hairpin probe, efficiently avoids outside nucleic acid
The non-specific digestion of enzyme cutting III (Exo III), to generate nearly zero background signal.The presence of target DNA has caused nucleic acid
The multi-cycle amplification of exonucleaseⅢ (Exo III) auxiliary, high sensitivity, detection is limited to 3 every liter of femtomoles, and has excellent
Selectivity, can identify single base mispairing, have great potential in terms of medical diagnosis on disease and biomedical research.
In first typical embodiment of the disclosure, a kind of Multiple Cycle of exonuclease III auxiliary is provided
Method of the amplification for delicately detecting DNA, in this method, we devise two different hairpin probes, utilize single stranded DNA
Binding protein (SSB) is combined closely with probe, protects them from the non-specific digestion of exonuclease III (Exo III),
To generate nearly zero background signal.It is digested, is finally generated bright by the Multiple Cycle of the target DNA two kinds of hairpin probes caused
The fluorescence signal of the Aminofluorescein (FAM) of aobvious enhancing, and substantially increase the sensitivity and specificity of detection.
It is specific as follows:
When there are the single-stranded DNA binding proteins in the case where target DNA, in 1 compound of single-stranded DNA binding protein-probe
It is detached from, target DNA forms 1 duplex of target-probe with the flat end 3'- in conjunction with the 3'- protruding terminus of probe 1, can
It is identified and gradually digested by exonuclease III, therefore, target DNA and triggering primer can be released from duplex, with
Afterwards, the target DNA of release causes the next round circulation digestion of starting probe 1 in conjunction with new probe 1；The triggering primer of release draws
The single-stranded DNA binding protein risen in 2 compound of single-stranded DNA binding protein-probe is detached from, thus the 3'- protruding terminus with probe 2
Complementation, forms 2 duplex of target-probe with the flat end 3'-, then causes digestion of the exonuclease III to probe 2,
To release triggering primer and target analog；The triggering primer of release starts a new round for probe 2 in conjunction with new probe 2
Digestion, the target analog of release start the anti-of new circulation in conjunction with new probe 1 because it includes the identical sequences of target
It answers；The digestion that target DNA causes probe 1 and probe 2 is examined to generate the fluorescence of the fluorophor of enhancing according to fluorescence intensity
Survey DNA.
In the case where target DNA is not present, since 1 compound of single-stranded DNA binding protein-probe and single stranded DNA combine
2 compound of protein-probe is resistant to the cutting of exonuclease III (Exo III), cannot be by exonuclease III
(Exo III) digestion, and detect low fluorescence signal.
The exonuclease III acts on double-stranded DNA, along the direction 3' → 5' gradually catalytic elimination mononucleotide.
Wherein, 1 compound of single-stranded DNA binding protein-probe is answering of being formed of single-stranded DNA binding protein and probe 1
Close object；2 compound of single-stranded DNA binding protein-probe is the compound that single-stranded DNA binding protein and probe 2 are formed；
The probe 1 and probe 2 are 3 ' end hair fastener type DNA outstanding, wherein
The probe 1 includes: 3 ' the end outstanding single stranded DNA structures complementary with target DNA；
5 ' ends of the bulbous double-stranded DNA structure being complementarily shaped to by arm 1 and arm 2,1 sequence of arm have fluorophor；And
Cyclic DNA structure 1；
The probe 2 includes: 3 ' end single stranded DNA structures outstanding, and sequence is 2 sequence of arm；
By the bulbous double-stranded DNA that 3 ' end single stranded DNA structure sequences outstanding are complementarily shaped in the target DNA and probe 1
5 ' ends of structure, target DNA have fluorophor；And
Cyclic DNA structure 2.
Those skilled in the art can conventional design hair fastener type DNA cyclic DNA structure division, details are not described herein, cyclic annular
DNA structure 1 can be identical or different with 2 structure of DNA structure.
Further, the hydrolysis of exonuclease III, the nucleotide of cyclic DNA structure division can carry out in order to prevent
Phosphorothioateization modification.
The sequence of triggering primer is 5 ' arm 1- cyclic DNA structure 1-3 ' herein, is in a specific embodiment 5 '-
FAM-CAC GCC GAA TCC TAG ACT ATT*T*T*T*-3 ', wherein No. * expression phosphorothioateization modification.
The sequence of target analog herein is 5 '-target DNA- cyclic DNA structure 2-3 ', in a specific implementation
It is 5 '-FAM- in exampleCAG TTC ACG CAT AGA CGG CCT*T*T*T*-3 ', wherein No. * expression phosphorothioateization modification.
In one embodiment of the present disclosure, providing the probe 1 is 3 ' end hair fastener type DNA outstanding, sequence 5 '-
FAM-CAC GCC GAA TCC TAG ACT ATT*T*T*T*ATA GTC TAG GAT TCG GCG TGG GCC GTC TAT
GCG TGA ACT G-3 ', wherein No. * expression phosphorothioateization modification.
The probe 2 is 3 ' end hair fastener type DNA, 5 '-FAM- of sequence outstanding of another kindCAG TTC ACG CAT AGA CGG CCT*T*T*T*GGC CGT CTA TGC GTG AAC TGG GCC TCT AGG ATT CGG CGT G-3 ', wherein *
Number indicate phosphorothioateization modification.
In one of the disclosure or some specific embodiments, the oligonucleotides such as the probe 1 and probe 2 use 10
× trishydroxymethylaminomethane-ethylenediamine tetra-acetic acid (Tris-EDTA) buffer dilution prepares stock solution.
In one of the disclosure or some specific embodiments, (this is slow in annealing buffer for probe 1 and/or probe 2
Trishydroxymethylaminomethane-the hydrochloric acid (Tris-HCl) and 50 mMs every liter of chlorine for the pH 8.0 that fliud flushing is 5 mMs every liter
Change sodium) in 95 DEG C be incubated for 5 minutes, be then slowly cooled to room temperature, be stored in 4 DEG C of uses.
In one of the disclosure or some specific embodiments, 1 compound of single-stranded DNA binding protein-probe and/or
The preparation method of 2 compound of single-stranded DNA binding protein-probe, comprising the following steps: probe 1 and/or probe 2 and single stranded DNA knot
Hop protein (SSB) is in (the buffer are as follows: 50 mMs every liter of potassium acetate, 20 mMs every liter of pH of 1 × NEB buffer 4
7.9 trishydroxymethylaminomethane-acetic acid, 10 mMs every liter of magnesium acetate and 1 mM every liter of dithiothreitol (DTT)), in
37 DEG C of incubations.
In one of the disclosure or some specific embodiments, a kind of the multiple of exonuclease III auxiliary is provided
Method of the cyclic amplification for delicately detecting DNA, the reaction system of this method include:
The amplification of signal of exonuclease III (Exo III)-auxiliary carries out in 20 microlitres of reaction system, including target
Marking DNA, 1 compound of single-stranded DNA binding protein-probe and 2 compound of single-stranded DNA binding protein-probe, (totally 200 nanomoles are every
Rise, molar ratio 1:1), the exonuclease III (Exo III) of 5 units (5U), 1 × NEB buffer 4 (buffer are as follows:
50 mMs every liter of potassium acetate, trishydroxymethylaminomethane-acetic acid of 20 mMs every liter of pH 7.9,10 mMs every liter
Magnesium acetate and 1 mM every liter of dithiothreitol (DTT)), in 37 DEG C be incubated for.
In Bio-Rad CFX ConnectTMReal-time fluorescence measurement is carried out in real-time system, and glimmering with the monitoring of 30 seconds intervals
Luminous intensity.
In one of the disclosure or some specific embodiments, this method is specifically included:
1) standard curve is made:
By the target DNA of various concentration, 1 compound of single-stranded DNA binding protein-probe and single-stranded DNA binding protein-probe
2 compounds, 1 × NEB buffer 4 are uniformly mixed, and are incubated for；After incubation, its fluorescence intensity is detected under 518nm wavelength, is made
Make standard curve；
2) sample to be tested is taken, fluorescence detection is carried out according to the method in step 1), is obtained according to standard curve to be detected
The concentration of DNA in sample.
Further, the sample to be tested is blood serum sample, water sample (can monitor the content for drinking Inactivation of Enteric Viruses In Water or determine water
Different plant species present in raw environment) etc. samples.
In a specific embodiment of the disclosure, the standard curve is F=1886.6+732.2log10C, phase relation
Number (R2) it is 0.979, wherein F is the obtained fluorescence intensity when being incubated for 80 minutes, and C is the concentration of target DNA.
In the second aspect of the disclosure, a kind of Multiple Cycle amplification of exonuclease III auxiliary is provided for sensitive
Detect the probe combinations of DNA, including probe 1 and probe 2 in ground；
The probe 1 and probe 2 are 3 ' end hair fastener type DNA outstanding, wherein
The probe 1 includes: 3 ' the end outstanding single stranded DNA structures complementary with target DNA；
5 ' ends of the bulbous double-stranded DNA structure being complementarily shaped to by arm 1 and arm 2,1 sequence of arm have fluorophor；And
Cyclic DNA structure 1；
The probe 2 includes: 3 ' end single stranded DNA structures outstanding, and sequence is 2 sequence of arm；
By the bulbous double-stranded DNA that 3 ' end single stranded DNA structure sequences outstanding are complementarily shaped in the target DNA and probe 1
5 ' ends of structure, target DNA have fluorophor；And
Cyclic DNA structure 2.
In terms of the third of the disclosure, a kind of Multiple Cycle amplification of exonuclease III auxiliary is provided for sensitive
Ground detects the kit of DNA, which includes at least following component: exonuclease III, probe 1, probe 2 and single stranded DNA
Binding protein.
Further, further includes: annealing buffer, the composition of the annealing buffer are as follows: 5 mMs every liter of pH's 8.0
Trishydroxymethylaminomethane-hydrochloric acid (Tris-HCl) and 50 mMs every liter of sodium chloride；
1 × NEB buffer 4, the composition of the buffer are as follows: 50 mMs every liter of potassium acetate, 20 mMs every liter
Trishydroxymethylaminomethane-acetic acid, 10 mMs every liter of magnesium acetate and 1 mM every liter of the dithiothreitol (DTT) of pH7.9；
10 × trishydroxymethylaminomethane-ethylenediamine tetra-acetic acid (Tris-EDTA) buffer is dilute.
In the 4th typical embodiment of the disclosure, the probe combinations or kit are provided in detection DNA
Application.
In order to enable those skilled in the art can clearly understand the technical solution of the disclosure, below with reference to tool
The technical solution of the disclosure is described in detail in the embodiment of body.
In following embodiment, the sequence of target DNA is 5 '-CAG TTC ACG CAT AGA CGG CC-3 ', such as SEQ IN
Shown in NO.1, the sequence of probe 1 is 5 '-FAM-CAC GCC GAA TCC TAG ACT ATT*T*T*T*ATA GTC TAG GAT TCG GCG TGG GCC GTC TAT GCG TGA ACT G-3 ', as shown in SEQ IN NO.2, the sequence of probe 2 is
5’-FAM-CAG TTC ACG CAT AGA CGG CCT*T*T*T*GGC CGT CTA TGC GTG AAC TGG GCC TCT
AGG ATT CGG CGT G-3 ', as shown in SEQ IN NO.3, the sequence of the target (T1) of single base mismatch is 5 '-CAG TTC
ACT CAT AGA CGGCC-3 ', as shown in SEQ IN NO.4, the sequence of the target (T2) of double alkali yl mispairing is 5 '-CTG TTC
ACA CAT AGA CGG CC-3 ', as shown in SEQ IN NO.5.Random sequence (T3) is 5 '-TAG GAT AAC ATG GGT
TGG CC-3 ', as shown in SEQ IN NO.6.No. * expression phosphorothioateization modification in sequence.
Embodiment 1
Fluorescence detection: all oligonucleotides are with 10 × trishydroxymethylaminomethane-ethylenediamine tetra-acetic acid (Tris-EDTA)
Buffer dilution prepares stock solution.The hairpin probe (including probe 1 and probe 2, molar ratio 1:1) of 1 every liter of micromole
In annealing buffer, (trishydroxymethylaminomethane-hydrochloric acid (Tris-HCl) of 5 mMs every liter of pH 8.0,50 mMs every
Rise sodium chloride) in 95 DEG C be incubated for 5 minutes, be then slowly cooled to room temperature, be stored in 4 DEG C of uses.In order to prepare single stranded DNA
Binding protein (SSB)-probe complex, two different hairpin probes and single-stranded DNA binding protein (SSB) are buffered in 1 × NEB
Liquid 4 (50 mMs every liter of potassium acetate, trishydroxymethylaminomethane-acetic acid of 20 mMs every liter of pH 7.9,10 mMs
Every liter of magnesium acetate, 1 mM every liter of dithiothreitol (DTT)), it is incubated in 37 DEG C.Exonuclease III (Exo III)-auxiliary
Amplification of signal carried out in 20 microlitres of reaction system, the DNA including various concentration, two different single stranded DNA combination eggs
White-probe complex (totally 200 every liter of nanomole, molar ratio 1:1), the exonuclease III (Exo III) of 5 units, 1
× NEB buffer 4 (50 mMs every liter of potassium acetate, trishydroxymethylaminomethane-vinegar of 20 mMs every liter of pH 7.9
Acid, 10 mMs every liter of magnesium acetate, 1 mM every liter of dithiothreitol (DTT)), it is incubated in 37 DEG C.In Bio-Rad CFX
ConnectTMReal-time fluorescence measurement is carried out in real-time system, and fluorescence intensity was monitored with 30 seconds intervals.
Embodiment 2
Experimental principle (such as Fig. 1): the technical program devises two kinds of hairpin probe (1 Hes of probe with 3 '-protruding terminus
Probe 2), two kinds of probes are modified fluorophor Aminofluorescein (FAM) in its 5 '-end, the fluorescence of Aminofluorescein (FAM)
It can effectively be quenched by the continuous guanine base on its opposite by Photo-induced electron transfer (PIET).When there is no targets
In the case where marking DNA, since the 3'- protruding terminus of probe is resistant to the cutting of exonuclease III (Exo III), no
It can be digested by exonuclease III (Exo III), and detect low-down fluorescence signal.When there are target DNA's (Fig. 1)
In the case of, (Fig. 1) forms 1 duplex of target-probe with the flat end 3'- in conjunction with the 3'- protruding terminus of probe 1, can
It is identified and gradually digested by exonuclease III (Exo III).Therefore, target DNA and triggering primer can be discharged from duplex
Out.Then, the target DNA of release can cause in conjunction with new probe 1 starting probe 1 next round circulation digestion (Fig. 1,
Recycle I).In addition, the triggering primer of release is complementary with the 3'- protruding terminus of probe 2 (Fig. 1), the target with the flat end 3'- is formed
2 duplex of mark-probe then causes the digestion of exonuclease III (Exo III) to probe 2, to release triggering primer
(Fig. 1) and target analog (Fig. 1).The triggering primer of release can start the new round digestion of probe 2 in conjunction with new probe 2
(Fig. 1 recycles II), and the target analog discharged can be opened because it includes the identical sequences of target in conjunction with new probe 1
The reaction of dynamic new circulation I and II (Fig. 1 recycles III).Since multiple circulations of exonuclease III (Exo III) auxiliary disappear
Change, a target can cause the digestion of enough probe 1 and probe 2, generate the glimmering of the Aminofluorescein (FAM) of enhancing
Light, and only relate to a kind of enzyme (i.e. Exo III).
In order to eliminate the highback as caused by exonuclease III (Exo III) non-specific digestion single stranded DNA (ssDNA)
Scape signal, we introduce single-stranded DNA binding protein (SSB).Single-stranded DNA binding protein (SSB) be capable of high-affinity with non-sequence
The mode of column dependence is combined closely with single stranded DNA (ssDNA).Studies have shown that specific protein and single stranded DNA (ssDNA)
Combination can protect DNA from the digestion of exonuclease III (Exo III).In order to assess single-stranded DNA binding protein
(SSB) and whether the combination of single stranded DNA (ssDNA) can effectively inhibit the non-specificity of exonuclease III (Exo III) to disappear
Change, single-stranded DNA binding protein (SSB) is incubated for probe 1 and probe 2 with identical molar ratio to prepare egg in conjunction with single stranded DNA
White-probe complex.As shown in Figure 2 A, high background letter is observed in the case where no addition single-stranded DNA binding protein (SSB)
Number (Fig. 2A, curve 2).On the contrary, detected in the case where adding single-stranded DNA binding protein (SSB) zero background signal (Fig. 2A,
Curve 1), show that single-stranded DNA binding protein (SSB) can effectively inhibit exonuclease III (Exo III) to 1 He of probe
The non-specific digestion of probe 2.In the presence of target DNA, there is the fluorescence letter obtained when single-stranded DNA binding protein (SSB) participation
Obtained fluorescence signal (Fig. 2 B) when number being participated in much higher than no single-stranded DNA binding protein (SSB).Therefore, single stranded DNA is introduced to combine
Albumen (SSB) can generate nearly zero background signal and increase reaction efficiency.
1. the experimental verification of principle
In the technical program, the detection performance of enhancing can be generated in order to verify the amplification of multi-cycle signal, we supervise in real time
Survey the fluorescence signal under different experimental conditions.As shown in figure 3, exonuclease III is added there is no in the case where target DNA
Apparent fluorescence signal (Fig. 3, curve 3) cannot be generated, similar with the signal for only passing through probe 1 and the acquisition of probe 2 (Fig. 2, curve
2), show in no target DNA, probe cannot be cut, and can achieve zero background signal.When target DNA, 1 He of probe
In the presence of exonuclease III (Exo III), it is able to detect that apparent fluorescence signal (Fig. 3, curve 2), shows target DNA
The digestion of probe 1 has been caused (Fig. 1 recycles I).Then, after adding probe 2 (Fig. 3, curve 1), with only addition probe 1 (Fig. 3, song
Line 2) when the fluorescence signal that obtains compare, fluorescence signal greatly enhances, and shows to be assisted by exonuclease III (Exo III)
Multi-cycle amplification produce stronger fluorescence signal (Fig. 1, recycle I, II and III).
2. sensitivity technique
In order to assess the sensitivity of the technical program detection DNA, we probe into various concentration DNA under optimum experimental condition
Corresponding real-time fluorescent signals.As shown in Figure 4 A, fluorescence intensity is received as target DNA concentration from 10 femtomoles is often raised to 10 and rubs
You every liter increase and gradually increase.In logarithmic scale, the concentration of fluorescence intensity and target DNA often rise to 1 in 10 femtomoles
Linear correlation (Fig. 4 B) in the concentration range of every liter of nanomole.Regression equation is F=1886.6+732.2log10C, phase relation
Number (R2) it is 0.979, wherein F is the obtained fluorescence intensity at 80 minutes, and C is the concentration of target DNA.This method flies 10
The Larger Dynamic range with 5 orders of magnitude in the range of 1 every liter of nanomole mole is often risen to, according to 3 σ/K method, detection limit
(LOD) 3 every liter of femtomoles are calculated as, wherein σ is the standard deviation (SD) of control group, and K is the slope of linear regression curves.Phase
Instead, in the case where no single-stranded DNA binding protein (SSB), detection is limited to 23.67 every liter of picomoles (Fig. 4 B), detection limit
Than 4 orders of magnitude of detection limit for height obtained in the presence of single-stranded DNA binding protein (SSB).It is worth noting that, this technology method ratio
The DNA analysis for exonuclease III (Exo III) auxiliary reported before is much sensitive, with exonuclease III (Exo
III) fluorescence detection (36 every liter of picomole) assisted is compared, and sensitivity improves 4 orders of magnitude, with exonuclease III
The electrochemical analysis (0.1 every liter of picomole) of (Exo III) auxiliary is compared, and 2 orders of magnitude is improved, with exonuclease III
The colorimetric method of (Exo III) auxiliary is compared, and 1 order of magnitude is improved.The sensitivity of raising is attributed to following: (1) single stranded DNA knot
Hop protein (SSB) is effectively combined with probe, produces nearly zero background signal (Fig. 2)；(2) exonuclease III (Exo
III) multi-cycle assisted expands (Fig. 3).In addition, the sensitivity of the technical program and it is related to two cyclic amplifications, and with
The sensitivity phase of (20 every liter of femtomoles and 4.83 every liter of femtomoles) that DNAzyme is obtained as the method for amplification output signal
When.
3. specific detection
In order to assess the specificity of the technical program, we use the target (T1) of single base mismatch, the target of double alkali yl mispairing
(T2) and random sequence (T3) are marked as negative control and carries out specificity experiments.As shown in figure 5, the target DNA of base exact matching
(T) fluorescence intensity corresponding to the target (T1) of the fluorescence intensity ratio single base mismatch corresponding to is 1.2 times high, and compares double alkali yl
Fluorescence intensity corresponding to the target (T2) of mispairing is 3.8 times high.In addition, random sequence (R) produces insignificant fluorescence signal,
It is similar with the fluorescence signal of no any target DNA compareed.These results indicate that the technical program has excellent spy
The opposite sex can distinguish the target of exact matching and mispairing, have very big application potential to the analysis of single nucleotide polymorphism.
4. the rate of recovery detects: total volume is that 20 microlitres of reaction mixture includes 5% blood containing various concentration DNA
Clearly, 1 × NEB buffer 4 (50 mMs every liter of potassium acetate, trishydroxymethylaminomethane-of 20 mMs every liter of pH 7.9
Acetic acid, 10 mMs every liter of magnesium acetate, 1 mM every liter of dithiothreitol (DTT)), 200 every liter of nanomoles it is two different
Single-stranded DNA binding protein (SSB)-probe complex, the exonuclease III (Exo III) of 5 units, is incubated at 37 DEG C, with
Real-time fluorescence measurement is carried out afterwards.
Rate of recovery analysis: for the ability of the target DNA tested in the technical program detection of complex biological sample, we will
In the blood serum sample of the target DNA incorporation 5% and 10% of various concentration and calculate the rate of recovery.As shown in fig. 6, quantitative recovery rate meter
Calculating is 92.73% to 104.3%, and relative standard deviation (RSD) is 1.61% to 5.22%, with exonuclease III (Exo
III it is suitable that the target recycling amplification coupling liposome auxiliary) assisted expands the obtained rate of recovery.Should the result shows that, this skill
Art method can be used for the target DNA in accurate quantification complex biological sample.
Above-described embodiment is the preferable embodiment of the disclosure, but embodiment of the present disclosure is not by above-described embodiment
It limits, made changes, modifications, substitutions, combinations, simplifications under other any spiritual essence and principles without departing from the disclosure,
It should be equivalent substitute mode, be included within the protection scope of the disclosure.
SEQUENCE LISTING
<110>Shandong Normal University
<120>method of a kind of Multiple Cycle amplification of exonuclease III auxiliary for delicately detecting DNA
<130> 2018
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213>artificial sequence
<400> 1
cagttcacgc atagacggcc 20
<210> 2
<211> 64
<212> DNA
<213>artificial sequence
<400> 2
cacgccgaat cctagactat ttttatagtc taggattcgg cgtgggccgt ctatgcgtga 60
actg 64
<210> 3
<211> 64
<212> DNA
<213>artificial sequence
<400> 3
cagttcacgc atagacggcc ttttggccgt ctatgcgtga actgggcctc taggattcgg 60
cgtg 64
<210> 4
<211> 20
<212> DNA
<213>artificial sequence
<400> 4
cagttcactc atagacggcc 20
<210> 5
<211> 20
<212> DNA
<213>artificial sequence
<400> 5
ctgttcacac atagacggcc 20
<210> 6
<211> 20
<212> DNA
<213>artificial sequence
<400> 6
taggataaca tgggttggcc 20
Claims (10)
1. a kind of method of Multiple Cycle amplification of exonuclease III auxiliary for delicately detecting DNA, characterized in that should
Method includes:
When there are in the case where target DNA, the single-stranded DNA binding protein in 1 compound of single-stranded DNA binding protein-probe is detached from,
Target DNA forms 1 duplex of target-probe with the flat end 3'- in conjunction with the 3'- protruding terminus of probe 1, can be by nucleic acid
ExonucleaseⅢ identifies and gradually digests that therefore, target DNA and triggering primer can be released from duplex, then, release
Target DNA cause in conjunction with new probe 1 starting probe 1 next round circulation digestion；The triggering primer of release causes single-stranded
Single-stranded DNA binding protein in 2 compound of DNA binding protein-probe is detached from, so that the 3'- protruding terminus with probe 2 is complementary,
2 duplex of target-probe with the flat end 3'- is formed, then causes digestion of the exonuclease III to probe 2, to release
Release triggering primer and target analog；The triggering primer of release starts the new round digestion of probe 2 in conjunction with new probe 2,
The target analog of release starts the reaction of new circulation in conjunction with new probe 1 because it includes the identical sequences of target；Target
DNA causes the digestion of probe 1 and probe 2, to generate the fluorescence of the fluorophor of enhancing, detects DNA according to fluorescence intensity.
2. the method as described in claim 1, characterized in that wherein, 1 compound of single-stranded DNA binding protein-probe is single
The compound that chain DNA binding protein and probe 1 are formed；2 compound of single-stranded DNA binding protein-probe is that single stranded DNA combines
The compound that albumen and probe 2 are formed；
The probe 1 and probe 2 are 3 ' end hair fastener type DNA outstanding, wherein
The probe 1 includes: 3 ' the end outstanding single stranded DNA structures complementary with target DNA；
5 ' ends of the bulbous double-stranded DNA structure being complementarily shaped to by arm 1 and arm 2,1 sequence of arm have fluorophor；And
Cyclic DNA structure 1；
The probe 2 includes: 3 ' end single stranded DNA structures outstanding, and sequence is 2 sequence of arm；
The bulbous double-stranded DNA structure for holding single stranded DNA structure sequences outstanding to be complementarily shaped to by the target DNA and probe 13 ',
5 ' the ends of target DNA have fluorophor；And
Cyclic DNA structure 2；
The triggering primer is 5 ' arm 1- cyclic DNA structure 1-3 '；
The target analog is 5 '-target DNA- cyclic DNA structure 2-3 '.
3. the method as described in claim 1, characterized in that 1 compound of single-stranded DNA binding protein-probe and/or single stranded DNA
The preparation method of 2 compound of binding protein-probe, comprising the following steps:
Probe 1 and/or probe 2 and single-stranded DNA binding protein are incubated in 1 × NEB buffer 4.
4. the method as described in claim 1, characterized in that the reaction system of this method includes: exonuclease III- auxiliary
Amplification of signal carried out in 20 microlitres of reaction system, including target DNA, 1 compound of single-stranded DNA binding protein-probe and
2 compound of single-stranded DNA binding protein-probe, the exonuclease III of 5 units, 1 × NEB buffer 4 are incubated for.
5. the method as described in claim 1, characterized in that this method specifically includes:
1) standard curve is made:
The target DNA of various concentration, 1 compound of single-stranded DNA binding protein-probe and single-stranded DNA binding protein-probe 2 are answered
It is uniformly mixed to close object, 1 × NEB buffer 4, is incubated for；After incubation, its fluorescence intensity, production mark are detected under 518nm wavelength
Directrix curve；
2) sample to be tested is taken, fluorescence detection is carried out according to the method in step 1), sample to be tested is obtained according to standard curve
The concentration of middle DNA.
6. method as claimed in claim 5, characterized in that the standard curve is F=1886.6+732.2log10C is related
Coefficient is 0.979, and wherein F is the obtained fluorescence intensity when being incubated for 80 minutes, and C is the concentration of target DNA；
Further, the sample to be tested is blood serum sample or water sample.
7. a kind of Multiple Cycle amplification of exonuclease III auxiliary is for delicately detecting the probe combinations of DNA, feature
It is, including probe 1 and probe 2；
The probe 1 and probe 2 are 3 ' end hair fastener type DNA outstanding, wherein
The probe 1 includes: 3 ' the end outstanding single stranded DNA structures complementary with target DNA；
5 ' ends of the bulbous double-stranded DNA structure being complementarily shaped to by arm 1 and arm 2,1 sequence of arm have fluorophor；And
Cyclic DNA structure 1；
The probe 2 includes: 3 ' end single stranded DNA structures outstanding, and sequence is 2 sequence of arm；
The bulbous double-stranded DNA structure for holding single stranded DNA structure sequences outstanding to be complementarily shaped to by the target DNA and probe 13 ',
5 ' the ends of target DNA have fluorophor；And
Cyclic DNA structure 2；
Further, the probe 1 is 3 ' end hair fastener type DNA, 5 '-FAM-CAC GCC GAA TCC TAG of sequence outstanding
ACT ATT*T*T*T*ATA GTC TAG GAT TCG GCG TGG GCC GTC TAT GCG TGA ACT G-3 ', wherein *
Number indicate phosphorothioateization modification；
The probe 2 is 3 ' end hair fastener type DNA, 5 '-FAM-CAG TTC ACG CAT AGA CGG of sequence outstanding of another kind
CCT*T*T*T*GGC CGT CTA TGC GTG AAC TGG GCC TCT AGG ATT CGG CGT G-3 ', wherein * table
Show that phosphorothioateization is modified.
8. a kind of Multiple Cycle amplification of exonuclease III auxiliary is for delicately detecting the kit of DNA, characterized in that
The kit includes at least following component: exonuclease III, probe 1, probe 2 and single-stranded DNA binding protein；
The probe 1 is 3 ' end hair fastener type DNA, 5 '-FAM-CAC GCC GAA TCC TAG ACT ATT* of sequence outstanding
T*T*T*ATA GTC TAG GAT TCG GCG TGG GCC GTC TAT GCG TGA ACT G-3 ', wherein No. * expression phosphorus
Sulfonylization modification；
The probe 2 is 3 ' end hair fastener type DNA, 5 '-FAM-CAG TTC ACG CAT AGA CGG of sequence outstanding of another kind
CCT*T*T*T*GGC CGT CTA TGC GTG AAC TGG GCC TCT AGG ATT CGG CGT G-3 ', wherein * table
Show that phosphorothioateization is modified.
9. kit as claimed in claim 8, characterized in that the kit further include: annealing buffer, the annealing buffer
Composition are as follows: the trishydroxymethylaminomethane-hydrochloric acid and 50 mMs every liter of sodium chloride of 5 mMs every liter of pH 8.0；
1 × NEB buffer 4, the composition of the buffer are as follows: 50 mMs every liter of potassium acetate, 20 mMs every liter of pH 7.9
Trishydroxymethylaminomethane-acetic acid, 10 mMs every liter of magnesium acetate and 1 mM every liter of dithiothreitol (DTT)；
10 × trishydroxymethylaminomethane-ethylenediamine tetra-acetic acid buffer is dilute.
10. the application of probe combinations as claimed in claim 7 or kit according to any one of claims 8 in detection DNA.
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Cited By (5)
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| CN110964817A (en) * | 2019-11-20 | 2020-04-07 | 江西师范大学 | Functional hairpin probe and composition based on exonuclease III and method for improving sensitivity of detecting Pax-5a gene |
| CN112114154A (en) * | 2020-09-27 | 2020-12-22 | 江苏省原子医学研究所 | Kit for detecting nuclear factor-kappa B and application thereof |
| CN112251493A (en) * | 2020-10-22 | 2021-01-22 | 江南大学 | Substance detection method based on fluorescence resonance energy transfer and exonuclease-assisted cyclic amplification strategy |
| CN113637730A (en) * | 2021-09-09 | 2021-11-12 | 华中农业大学 | Visual nucleic acid detection method combining isothermal amplification technology with exonuclease mediation |
| WO2023087706A1 (en) * | 2021-11-16 | 2023-05-25 | 圣湘生物科技股份有限公司 | Primer/probe design method, detection composition and kit for mirna detection |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110964817A (en) * | 2019-11-20 | 2020-04-07 | 江西师范大学 | Functional hairpin probe and composition based on exonuclease III and method for improving sensitivity of detecting Pax-5a gene |
| CN110964817B (en) * | 2019-11-20 | 2022-09-23 | 江西师范大学 | Functional hairpin probe and composition based on exonuclease III and method for improving sensitivity of detecting Pax-5a gene |
| CN112114154A (en) * | 2020-09-27 | 2020-12-22 | 江苏省原子医学研究所 | Kit for detecting nuclear factor-kappa B and application thereof |
| CN112114154B (en) * | 2020-09-27 | 2023-04-25 | 江苏省原子医学研究所 | Kit for detecting nuclear factor-kappa B and application thereof |
| CN112251493A (en) * | 2020-10-22 | 2021-01-22 | 江南大学 | Substance detection method based on fluorescence resonance energy transfer and exonuclease-assisted cyclic amplification strategy |
| CN113637730A (en) * | 2021-09-09 | 2021-11-12 | 华中农业大学 | Visual nucleic acid detection method combining isothermal amplification technology with exonuclease mediation |
| CN113637730B (en) * | 2021-09-09 | 2024-02-09 | 华中农业大学 | Isothermal amplification technology combined exonuclease mediated visual nucleic acid detection method |
| WO2023087706A1 (en) * | 2021-11-16 | 2023-05-25 | 圣湘生物科技股份有限公司 | Primer/probe design method, detection composition and kit for mirna detection |
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