CN109251964A - Recycle the method and application of microRNAs detection kit and specific detection circulation microRNAs - Google Patents
Recycle the method and application of microRNAs detection kit and specific detection circulation microRNAs Download PDFInfo
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Abstract
The invention belongs to field of biotechnology, are related to the method and application of a kind of circulation microRNAs detection kit and specific detection circulation microRNAs.The kit includes: the hairpin structure DNA with cohesive end, at least part complementary pairing of part of it and target microRNA；Double-stranded specific nuclease and reaction buffer；Circular DNA probes, a part of complementary pairing of part of it and hairpin structure DNA；One of optional following component: SYBR Gold Fluorescent nucleic acid stain；Archaeal dna polymerase；DNA polymerase reaction buffer；4×dNTPs.The present invention carries out specific detection to circulation miRNAs using the nuclease-mediated target circular response of double-stranded specific and rolling circle amplification reaction, not only detection background can be reduced but also two-way signaling amplification can be played the role of, quickly with easy to operate, detection, the features such as high sensitivity, specificity are good and at low cost.
Description
Technical field
The invention belongs to field of biotechnology, a kind of more particularly, to a kind of circulation microRNAs detection kit
Specific detection recycle microRNAs method and they detection circulation microRNAs in application.
Background technique
Recycling microRNAs (miRNAs) is that one kind is about 19-23nt, endogenous, and non-coding is present in serum, blood plasma
Or the miRNAs of the extracellular free state in the body fluid such as urine, it is by holding noncoding region (3 ' with the 3 ' of its said target mrna molecule
UTR) complementary combine, the expression of controlling gene in translation skill, therefore the miRNAs wide participation a variety of physiology mistakes of organism
Journey, such as ontogeny, cell Proliferation, differentiation and apoptosis, adjusting or even disease of the metabolism with stress reaction occur, including tumour
It is formed.MiRNAs also takes part in the important biological process regulation of tumour cell, plays promotion sensitivity gene and tumor suppressor gene indirectly
Function, has served vital in the occurrence and development of tumour, therefore miRNAs is considered as early diagnosis of tumor, prognosis
With the important potential source biomolecule marker and target for the treatment of.
Recycling miRNAs has short sequence, high homology, low content and the characteristic being easily degraded, therefore in biomedical research
Efficiently with urgent need in terms of early clinical diagnosis, special miRNAs detection method.
The detection method of traditional miRNAs mainly has Northern blot, microarray and real-time fluorescence quantitative PCR
(real-time PCR), these detection methods not only need the instrument and equipment using Large expensive, cumbersome and sensitive
Spend it is low, it is special poor, therefore be difficult to popularize.
Therefore, a kind of method for needing to develop new detection circulation microRNAs.
Summary of the invention
The object of the present invention is to provide a kind of circulation microRNAs detection kits and specific detection to recycle
The method and application of microRNAs.Under conditions of being based on SYBR Gold Fluorescent nucleic acid stain, using double-stranded specific nucleic acid
Target circular response and the rolling circle amplification reaction that enzyme mediates carry out specific detection to circulation miRNAs, can both reduce detection back
Scape can play the role of two-way signaling amplification again, have easy to operate, detection quickly, high sensitivity, specific good and cost
The features such as low.
To achieve the goals above, the first aspect of the present invention provides a kind of circulation microRNAs detection kit, this is followed
Ring microRNAs detection kit includes following component:
(1) a part of hairpin structure DNA, the hairpin structure DNA with cohesive end are with target microRNA's
At least part complementary pairing；Hairpin structure DNA is unfolded in the complementary pairing, forms miRNA- with target microRNA
DNA hybridization body；
(2) double-stranded specific nuclease and double-stranded specific nucleic acid enzyme reaction buffer solution；
(3) circular DNA probes, a part of the circular DNA probes and a part of complementary pairing of hairpin structure DNA；
(4) optional one of following component:
(4-1) SYBR Gold Fluorescent nucleic acid stain；
(4-2) archaeal dna polymerase；
(4-3) DNA polymerase reaction buffer；
(4-4)4×dNTPs。
Said components can be provided separately in kit of the present invention, can also as needed mention constituent part as a mixture
For.
The principle of the present invention is as shown in Figure 1, by taking target microRNA is miR-21 as an example.The detection architecture contains two steps
Reaction, when object miR-21 to be detected there are when, target miR-21 in conjunction with hairpin structure DNA and can spread out shape first
At can will be in hybrid by the miRNA-DNA hybrid that double-stranded specific nuclease identifies and shears, double-stranded specific nuclease
DNA Partial Shear fall after, target miR-21 can be released into the first step reaction circulation.And it is left in hairpin structure DNA
One section of cohesive end it is complementary with cyclic DNA, therefore will do it rolling circle amplification, product under the action of archaeal dna polymerase and dNTP
It is the linear single stranded DNA with a large amount of repetitive sequences (with cyclic DNA complete complementary).Finally SYBR Gold fluorescence nucleic acid is contaminated
Material is added in the amplification system, and the height of target miRNA content is judged by the strong and weak comparison of fluorescence signal.Not to be checked
When surveying object miR-21, hairpin structure DNA will not be opened, therefore subsequent two-step reaction will be unable to carry out, and detect signal
It is extremely weak.
Double-stranded specific nuclease (Duplex-specific nuclease, DSN) is a commercialized heat resistant type core
Sour enzyme, it can DNA in degradation selectivity double-stranded DNA (10-12bp) and DNA RNA hybrid (15bp), but to single-stranded
DNA, single stranded RNA and double-stranded RNA are without degradation.And the enzyme can distinguish the DNA hybridization of exact matching and Incomplete matching
Body has high specificity.
Rolling circle amplification (rolling circle amplification, RCA) is to use for reference ring-shaped DNA molecule rolling in nature
A kind of detection method for signal amplification that the amplification mechanism of ring type grows up.RCA is constant temperature enzymatic reaction, at a constant temperature
Using single stranded circle DNA/RNA as template, by the effect of specific DNA/RNA polymerase, short single stranded DNA/RNA primer is expanded
Increase single stranded DNA/RNA of growth, the product of RCA contains hundreds and thousands of tandem repetitive sequences complementary with circular template, can be used for
The detection of denier biomarker.
SYBR Gold Dye is a kind of low toxicity, and stable commercialization Fluorescent nucleic acid stain, autofluorescence is faint, but with
After nucleic acid combines (single stranded DNA, single stranded RNA, double-stranded DNA, double-stranded RNA), fluorescence signal can be remarkably reinforced (1000 times) therefore can
Background is detected to significantly reduce.
According to the present invention, the effect can be achieved in the hairpin structure DNA of various structures based on the above principles.Preferably,
A part and at least part complementary pairing of target microRNA close to long cohesive end of the hairpin structure DNA；More
Preferably, a part and target microRNA complete complementary that the long cohesive end of the hairpin structure DNA starts are matched；Into one
Preferably, a part of the long cohesive end of the hairpin structure DNA mutually recruits step at least part of target microRNA
Pair base number be 15-24.
Correspondingly, a part of the circular DNA probes and the short cohesive end of hairpin structure DNA is a part of complementary
Pairing.
The concept of heretofore described " long cohesive end " and " short cohesive end " can be it will be appreciated by those skilled in the art that
Respectively refer to one end outstanding and one end not outstanding in cohesive end.
A kind of specific embodiment according to the present invention, the target microRNA are miR-21, have such as SEQ ID NO:1
Shown sequence；The hairpin structure DNA has the sequence as shown in SEQ ID NO:2；The circular DNA probes have such as SEQ ID
Sequence shown in NO:3.
In accordance with the present invention it is preferred that the hairpin structure DNA is provided in the form of the buffer of hairpin structure DNA, it is dense
Degree is 1nM-100nM；The buffer such as Tris-HCl buffer (100mM NaCl, 5mM MgCl2, pH7.4).
Preferably, the circular DNA probes are provided in the form of the rolling circle amplification reaction solution of circular DNA probes, concentration
For 1nM-100nM.The rolling circle amplification reaction solution can be the conventional various reaction solutions suitable for rolling circle amplification reaction.
The second aspect of the present invention provides the method for specific detection circulation microRNAs a kind of, and this method includes following
Step:
1) determinand is mixed with hairpin structure DNA, double-stranded specific nuclease is added, carry out enzyme digestion reaction；
2) circular DNA probes are added into enzyme digestion reaction product, carry out rolling circle amplification；
3) rolling circle amplification product is detected；
Wherein,
The hairpin structure DNA has cohesive end, and at least part of part of it and target microRNA mutually recruits
It is right；Hairpin structure DNA is unfolded in the complementary pairing, forms miRNA-DNA hybrid with target microRNA；
A part of the circular DNA probes and a part of complementary pairing of hairpin structure DNA.
In accordance with the present invention it is preferred that detecting rolling circle amplification product using SYBR Gold Fluorescent nucleic acid stain.
According to the present invention, the effect can be achieved in the hairpin structure DNA of various structures based on the above principles.Preferably,
A part and at least part complementary pairing of target microRNA close to long cohesive end of the hairpin structure DNA；More
Preferably, a part and target microRNA complete complementary that the long cohesive end of the hairpin structure DNA starts are matched；Into one
Preferably, a part of the long cohesive end of the hairpin structure DNA mutually recruits step at least part of target microRNA
Pair base number be 15-24.
Correspondingly, a part of the circular DNA probes and the short cohesive end of hairpin structure DNA is a part of complementary
Pairing.
In the present invention, traditional two chain specific nucleic acid enzyme reaction condition, such as 50- is can be used in the step 1) enzyme digestion reaction
It is incubated for 15-60 minutes at 60 DEG C.
In the present invention, in step 2), this field popular response condition is can be used in rolling circle amplification condition, for example, at 20-40 DEG C
It is incubated for 60-120 minutes.
The third aspect of the present invention provides above-mentioned circulation microRNAs detection kit and/or the above method is followed in detection
Application in ring microRNAs.
MiRNAs is measured using kit and method of the invention, does not need any chemical modification, without complexity
Reaction process, do not need expensive instrument, miRNAs can be recycled in the detection architecture, easy to operate, when detection yet
Between short, at low cost, high sensitivity, specificity it is good, potential applicability in clinical practice is good.
Other features and advantages of the present invention will then part of the detailed description can be specified.
Detailed description of the invention
Exemplary embodiment of the invention is described in more detail in conjunction with the accompanying drawings, it is of the invention above-mentioned and its
Its purpose, feature and advantage will be apparent.
Fig. 1 is detection schematic diagram of the invention.
Fig. 2 shows 20% native polyacrylamide gel electrophoresis results of first step reaction product.
Fig. 3 shows 0.4% agarose gel electrophoresis results of entire detection architecture reaction product.
Fig. 4 A and Fig. 4 B show fluorescence spectrum (A) and fluorescence intensity (B) in the presence of various concentration target miR-21.Figure
In 4A, each spectral line represents control group from top to bottom, the concentration of miR-21 is followed successively by 1fM, 10fM, 100fM, 1pM, 10pM,
100pM、1nM。
Fig. 5 shows detection architecture specificity analysis result of the present invention.
Fig. 6 A and Fig. 6 B show detection architecture of the invention for detecting in breast cancer cell MDA-MB-231 and T47D
MiR-21 result, wherein each spectral line represents concentration as 0,1ng/ μ L, 2ng/ μ L, 5ng/ μ L, 10ng/ μ L from top to bottom.
Specific embodiment
The preferred embodiment of the present invention is described in more detail below.Although the following describe preferred implementations of the invention
Mode, however, it is to be appreciated that may be realized in various forms the present invention without that should be limited by the embodiments set forth herein.
By following embodiment, invention is further explained.The actual conditions person being not specified in embodiment, all presses
The condition suggested according to molecular biology usual operating conditions or manufacturer carries out, and various conventional reagents are commercially available.
Kit forms
4a) detect solution 1:20mM Tris-HCl buffer (100mM NaCl, 5mM MgCl2, pH 7.4) and (include hair
Card structure DNA probe HP:1nM-100nM), double-stranded specific nuclease (Duplex-specific nuclease, DSN)
0.1U/ μ L (buffer containing 10 × DSN master).
Solution 2:DNA cycling probe 1nM-100nM, Phi29DNA polymerase 0.5U/ μ L (reaction solution containing RCA) 4b) is detected,
DNTP:0.5mM.
4c) SYBR Gold Fluorescent nucleic acid stain.
Nucleic acid used in 1 present invention of table (containing DNA and miRNAs) sequencea
aThe base of underscore indicates complementary sequence in hairpin structure DNA, they will form the complementary region of hair fastener probe
(stem area).The base of overstriking in hairpin structure DNA indicates the complementary region of target miR-21.The base of overstriking in circular DNA probes
Indicate the complementary region of hairpin structure DNA.The base of underscore indicates the mutational site of miR-21 in M1, M2 and M3.
Operating procedure
(a) it is incubated for 30 minutes for 55 DEG C after mixing well the solution containing miRNA and detection solution 1.
(b) it will test solution 2 to be added in above-mentioned reaction solution (a), 30 DEG C are incubated for 90 minutes.
(c) SYBR Gold Fluorescent nucleic acid stain is added in above-mentioned reaction solution (b), reads fluorescence signal with microplate reader.
Test case 1
20% native polyacrylamide gel electrophoresis is carried out to the product (i.e. step (a) products therefrom) of first step reaction
Test, as a result as shown in Figure 2.Cannot be started when containing only miR-21 (1), (2) hairpin structure DNA in detection architecture
The reaction of one step, and containing miR-21 and double-stranded specific nuclease (3) in the detection architecture or containing hairpin structure DNA and
When double-stranded specific nuclease (4), double-stranded specific nuclease is to target miR-21 and hairpin structure DNA without degradation.When
(5) can only start the part reaction of the first step when containing target miR-21 and hairpin structure DNA in detection architecture.Only when
It, could be completely when hairpin structure DNA in detection architecture, double-stranded specific nuclease and target miR-21 are existed simultaneously (6)
Start the reaction of the first step.These experimental phenomenas are consistent with testing principle shown in Fig. 1, it is strong demonstrate the technical solution can
Row.
Test case 2
0.4% agarose gel electrophoresis survey is carried out to the reaction product (i.e. step (c) products therefrom) of entire detection architecture
Examination, as a result as shown in Figure 3.When in the detection architecture without target miR-21, which not can be carried out double-stranded specific core
Target circular response and the rolling circle amplification reaction (1) that sour enzyme mediates, could be into when detection architecture miR-21 containing target
The nuclease-mediated target circular response of row double-stranded specific and rolling circle amplification reaction (2), the detection of this phenomenon and the system
Principle is consistent, and further demonstrates the feasibility of the technical solution.
Test case 3
The test case is used to test the sensitivity of the detection architecture, specificity and the application in complex biological sample.Knot
Fruit as shown in figure 4, with target miR-21 concentration raising, corresponding fluorescence intensity is also gradually increased.The concentration of lgCmiR-21
It can be drawn standard calibration curve (B) in the fluorescence intensity of 1-4, which is fluorescence intensity (λ ex=485nm, λ
Em=544nm)=297.5+36.21 × lgCmiR-21 (linearly dependent coefficient R2=0.991), the detection architecture is to miR-21
Detection be limited to 1fM, illustrate that the sensitivity of the method for the present invention is high.
Test case 4
The test case is used to illustrate the specificity of detection architecture of the present invention.
As shown in figure 5, the relative intensity of fluorescence of the sample of the miR-21 containing target is 1,2 and 3 base mispairing respectively
3 times, 6.6 times and 11.4 times of miR-21 relative intensity of fluorescence.This results show specific pole of the technical solution
It is high.
Test case 5
The test case is used to illustrate the detection architecture for detecting the miR- in breast cancer cell MDA-MB-231 and T47D
21。
Total miRNAs in breast cancer cell MDA-MB-231 and T47D is extracted, with this method miRNAs total to various concentration
In miR-21 detected, as shown in fig. 6, illustrate that this method can detecte the miRNA in cell, there is certain clinic to answer
Use prospect.
Test case 6
The test case is used to illustrate the assessment of detection effect when the detection architecture is applied to actually detected sample.As a result such as table
Shown in 2, its testing result is had no effect on when in the detection architecture containing 10% normal human serum, this is the result shows that the skill
Art scheme can carry out the detection of miRNAs in complicated biological sample (serum), have extraordinary application prospect and practical
Value.
Table 2 detects miR-21 (n=3) under the conditions of containing 10% normal human serum
| Sample | It is actually added into value (pM) | Average computation value (pM)b | Average recoveries rate (%)c | Relative standard deviation (%)d |
| 1 | 10 | 12.25 | 122.57 | 4.29 |
| 2 | 50 | 56.41 | 56.41 | 2.26 |
| 3 | 100 | 117.23 | 117.23 | 0.88 |
bdIt is independent three times to test the value measured
cAverage recoveries rate (%)=100 × (CAverage computation value/CIt is actually added into value)
Various embodiments of the present invention are described above, above description is exemplary, and non-exclusive, and
It is not limited to disclosed each embodiment.Without departing from the scope and spirit of illustrated each embodiment, for this skill
Many modifications and changes are obvious for the those of ordinary skill in art field.
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Claims (10)
1. a kind of circulation microRNAs detection kit, which is characterized in that circulation microRNAs detection kit include with
Lower component:
(1) a part and target microRNA with the hairpin structure DNA, the hairpin structure DNA of cohesive end are at least
A part of complementary pairing；Hairpin structure DNA is unfolded in the complementary pairing, and it is miscellaneous to form miRNA-DNA with target microRNA
Hand over body；
(2) double-stranded specific nuclease and double-stranded specific nucleic acid enzyme reaction buffer solution；
(3) circular DNA probes, a part of the circular DNA probes and a part of complementary pairing of hairpin structure DNA；
(4) optional one of following component:
(4-1) SYBR Gold Fluorescent nucleic acid stain；
(4-2) archaeal dna polymerase；
(4-3) DNA polymerase reaction buffer；
(4-4)4×dNTPs。
2. circulation microRNAs detection kit according to claim 1, wherein the hairpin structure DNA's is close long
A part of cohesive end and at least part complementary pairing of target microRNA；Preferably, the length of the hairpin structure DNA
A part and target microRNA complete complementary that cohesive end starts are matched；It is further preferred that the hairpin structure DNA
The base number of at least part complementary pairing of a part and target microRNA of long cohesive end is 15-24.
3. circulation microRNAs detection kit according to claim 2, wherein a part of the circular DNA probes
With a part of complementary pairing of the short cohesive end of hairpin structure DNA.
4. circulation microRNAs detection kit according to claim 1, wherein the hairpin structure DNA is with hair fastener knot
The form of the buffer of structure DNA provides, concentration 1nM-100nM；
The circular DNA probes are provided in the form of the rolling circle amplification reaction solution of circular DNA probes, concentration 1nM-100nM.
5. circulation microRNAs detection kit according to claim 1, wherein the target microRNA has such as
Sequence shown in SEQ ID NO:1；The hairpin structure DNA has the sequence as shown in SEQ ID NO:2；The circular DNA probes
With the sequence as shown in SEQ ID NO:3.
6. a kind of method of specific detection circulation microRNAs, which is characterized in that method includes the following steps:
1) determinand is mixed with hairpin structure DNA, double-stranded specific nuclease is added, carry out enzyme digestion reaction；
2) circular DNA probes are added into enzyme digestion reaction product, carry out rolling circle amplification；
3) rolling circle amplification product is detected；
Wherein,
The hairpin structure DNA has cohesive end, at least part complementary pairing of part of it and target microRNA；Institute
Stating complementary pairing is unfolded hairpin structure DNA, forms miRNA-DNA hybrid with target microRNA；
A part of the circular DNA probes and a part of complementary pairing of hairpin structure DNA.
7. according to the method described in claim 6, wherein, detecting rolling circle amplification product using SYBR Gold Fluorescent nucleic acid stain.
8. according to the method described in claim 6, wherein, a part and target of the long cohesive end of the hairpin structure DNA
At least part complementary pairing of microRNA；Preferably, the long cohesive end of the hairpin structure DNA starts a part with
The pairing of target microRNA complete complementary；It is further preferred that a part and mesh of the long cohesive end of the hairpin structure DNA
The base number for marking at least part complementary pairing of microRNA is 15-24.
9. according to the method described in claim 8, wherein, a part and the short viscosity of hairpin structure DNA of the circular DNA probes
A part of complementary pairing of end.
10. appointing in circulation microRNAs detection kit described in any one of claim 1-5 and/or claim 6-9
Application of the method described in meaning one in detection circulation microRNAs.
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| CN110004214B (en) * | 2019-04-11 | 2023-01-31 | 合肥工业大学 | Method for detecting microRNA-21 by double DNA machine |
| CN112941154A (en) * | 2021-02-21 | 2021-06-11 | 中南大学 | Molecular beacon probe, application of molecular beacon probe in preparation of circBART2.2 detection preparation and kit |
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