CN108642137A - A method of detecting Tumor biomarkers using palindrome padlock probe - Google Patents
A method of detecting Tumor biomarkers using palindrome padlock probe Download PDFInfo
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Abstract
The present invention provides a kind of method detecting Tumor biomarkers using palindrome padlock probe, devises palindromic sequence non-fluorescent label locking-type RCA probes,（1）Identify target part：3 ' the ends and 5 ' ends of padlock probe with target miRNA complete complementaries, can be located at；（2）Cleavage site part：Palindrome base fragment has been merged in half recognition site of restriction enzyme Nt.AlWI, this site.It is combined with nicking nickases, proposes N RCA concepts, and Two-way Chain displacement reaction occurs（D‑SDA）Combination is used for the detection of tumor markers miRNA.The efficient amplification detection to let 7a miRNA can be realized through the invention, and economical and practical, be expected to be used for the detection kit exploitation.
Description
Technical field
The invention belongs to biotechnologies, and in particular to a kind of to detect Tumor biomarkers using palindrome padlock probe
Method.
Background technology
Currently, many tradition amplifying techniques, including blotting, real-time quantitative PCR, cDNA microarray are widely used in miRNA inspections
It surveys.Although these methods have its advantage, meanwhile, they also have many limitations, such as the temperature control that sensitivity is insufficient, stringent
And need high experimental expenses.In order to overcome the above difficulty, we devise the locking-type RCA of palindromic sequence non-fluorescent label
Probe proposes N-RCA new concepts, and with two-way strand replacement reaction in conjunction with nicking nickases（D-SDA）Combination is used for tumour
The detection of marker miRNA.
Invention content
The purpose of the present invention is to provide a kind of methods detecting Tumor biomarkers using palindrome padlock probe, at present
There are no palindromic sequences to cooperate with insertion with nicking cleavage sites and the high-performance locking-type RCA probes of design, and the present invention is exactly profit
With this technological development automatic cutting-rolling circle amplification and the compound amplification of signal system of strand replacement reaction, it to be used for non-marked, Gao Ling
The tumor markers such as quick, high specific detection miRNA.
To achieve the above object, the present invention adopts the following technical scheme that：
A method of detecting Tumor biomarkers, the padlock probe using palindrome padlock probe：
（1）Identify target part：3 ' the ends and 5 ' ends of padlock probe with target miRNA complete complementaries, can be located at；
（2）Cleavage site part：Half recognition site of restriction enzyme Nt.AlWI.
（3）Contain palindromic sequence in the padlock probe：3’-CTAGCTAG-5’.
Contain the controllable restriction enzyme enzyme recognition site for incorporating palindromic sequence in the padlock probe： 3’-
CCTAGCTAGA-5’。
Specific method includes the following steps：
（1）It prepares and connect enzyme buffer solution with the T4 of the target miRNA of concentration is determined containing 1 μM of padlock probe；
（2）350 U/ μ L T4 ligases are added into above-mentioned solution, connection reaction in 2 hours is carried out at 16 DEG C, is closed
The padlock probe of conjunction；
（3）In 10 μ L steps（2）10 mM, 2 μ L dNTPs, 3 U polymerases, 5 U Nt.AlWI cuttings are added in mixture
Enzyme, 0.5 100 × BSA of μ L, 2 μ L 10 × phi29 buffer solutions and 4.7 μ L secondary water, be incubated under the conditions of 37 DEG C
Reaction 2 hours, it is ensured that N-RCA reacts abundant with D-SDA；
（4）In step（3）Mixture in 10 × Sybr Green I of 2 μ L are added, be incubated 30 minutes at room temperature；Then add
After adding pH 7.4 100 mM PBS to 200 μ L, fluoremetry is carried out at room temperature.
The advantage of the invention is that：
High sensitivity of the present invention, specificity are good.This method Monitoring lower-cut is low（5 pM）, the range of linearity it is wide（5-30 nM）, this is this
What system achieved N-RCA and D-SDA cooperates with enlarge-effect；The high reason of specificity is attributed to the fact that following 2 factors：(i) only
The target miRNA of complete complementary can just make the connection of padlock probe specificity, closed loop；(ii) nickase Nt.AlWI specificity is cut
5’-GGATCNNNN↓N-3’。
Description of the drawings
Fig. 1, experimental principle figure.
Fig. 2, feasibility study figure.
Fig. 3, specificity investigate figure.
The ability of target miRNA is detected in Fig. 4, complex environment.
Specific implementation mode
Detection architecture proposed by the present invention pertains only to a kind of DNA probe --- padlock probe.Padlock probe is by two parts structure
At：（1）Identify target part：It with target miRNA complete complementaries, can be located at the 3 ' ends and 5 ' ends of padlock probe；（2）Cleavage
Point part：Half recognition site of restriction enzyme Nt.AlWI.Nt.AlWI can be in specific recognition double-stranded DNA（5’-
GGATCNNNN↓N-3’/3’-CCTAGNNNNN-5’）, by the upstream chain cutting in double-strand, and randomized bases " N " can be according to need
It is designed.In the invention, randomized bases chain is specifically designed to containing palindromic sequence by we（3’-CTAGCTAG-5’）, and
Embedded padlock probe.We probe into using target let-7a miRNA as model.The reaction includes following 9 processes（One pot anti-
It answers）：1. padlock probe hybridizes with target let-7a miRNA first.Meanwhile in the presence of DNA ligase, this padlock probe 5 '
End is connected with 3 ' end notches and is closed, and forms cyclic annular padlock probe；2. there are when polymerase, nickase, dNTP, let-7a
MiRNA is as polymerization primer, to cause the rolling ring reaction of nickase induction（N-RCA）, formation includes Nt.AlWI identifications position
The annular double-strand rolling ring product of point；3. on annular double-strand rolling ring product, can constantly be sent out by polymerisation/cleavage reaction
Raw strand displacement generates cutting segment（NF1）.It should be noted that 3 ' the ends of these NF1s include palindromic sequence（5’-
GATCGATC-3’）；4. each two NF1 is close to each other, 3 ' end phase mutual crosses；5. in the presence of polymerase, the NF1 of hybridization two
It is each other the two-way extension of template, generates the double-stranded DNA of extension；6. in the case where cutting enzyme effect, the meeting in the new double-strand for extending and generating
Strand replacement reaction occurs（D-SDA）, generate new cutting segment（NF2）；7. meanwhile regenerating the double-stranded DNA of extension；⑧
NF2 hybridizes with NF1 can also form double-stranded DNA；9. last, the double-stranded DNA of the above generation is by fluorescent dye（Sybr Green I）
It is embedded, export fluorescence signal.The efficient amplification detection to let-7a miRNA can be realized through the invention, and economical and practical,
It is expected to be used for the detection kit exploitation.
Embodiment 1
The invention verification process is with 20 microlitres for reaction system, and spectroscopic assay is for 200 microlitres.Sequence used such as following table：
2, palindrome padlock probe is used for the feasibility study of miRNA detections：
To prove the feasibility of the invention, we are respectively provided with experimental group（There is target miRNA）With control group（Without target
miRNA）.First, palindrome padlock probe（1 μM of final concentration）According to 1 between miRNA (1 μM of final concentration)：The amount of 1 substance
Ratio is added in T4 ligase buffer solutions, and under the effect of 350 U/ μ L T4 DNA ligases, and 16 DEG C are incubated 2 hours so that
Connection reaction occurs for padlock probe.
Then, 10 mM, 2 μ L dNTPs, 3 U phi29 polymerases, 5 U are added in 10 μ L said mixtures
Nt.AlWI nickases, 0.5 100 × BSA of μ L, 2 μ L 10 × phi29 buffer solutions and 4.7 μ L secondary water, at 37 DEG C
Under the conditions of incubation reaction 2 hours, it is ensured that N-RCA reacts abundant with D-SDA；In the above product, 10 × Sybr of 2 μ L is added
Green I so that the ultimate density of Sybr Green I be 1 ×, room temperature be incubated 30 minutes.Then, pH 7.4 is then added
After 100 mM PBS to 200 μ L, measured finally by Fluorescence Spectrometer.When without target miRNA, step and the above one
It causes.
Parameter is set as（480 nm of excitation wavelength, launch wavelength：500 nm- 600 nm；Maximum voltage is set as：500
V；Slit width：5 nm.
As can be seen from Figure 2 in no target miRNA, fluorescence is weaker；And there are target miRNA, fluorescence in system
Signal significantly improves.It is feasible to illustrate that the method is detected for miRNA.
, palindrome padlock probe be used for miRNA specific detection：
To verify the specific recognition capability of palindrome padlock probe in the invention, we add it in target miRNA and may be total to
The miRNA deposited（MiRNA-122, miRNA-141, miRNA-21 and miRNA-26a）, it is opposite that it is compared by fluorescent spectrometry
Fluorescent value.Concrete operation step is as follows：Palindrome padlock probe first（1 μM of final concentration）With miRNA (1 μM of final concentration)
(let-7a miRNA, miRNA-122, miRNA-141, miRNA-21 and miRNA-26a) is respectively according to 1：1 amount of substance
Ratio is added in T4 ligase buffer solutions, and 350 U/ μ L T4 ligases are added into above-mentioned solution, are carried out 2 hours at 16 DEG C
Connection reaction, 10 mM, 2 μ L dNTPs, 3 U are then added in the padlock probe being closed in 10 μ L said mixtures
10 × phi29 the buffer solutions and 4.7 μ L of phi29 polymerases, 5 U Nt.AlWI nickases, 0.5 100 × BSA of μ L, 2 μ L
Secondary water, the incubation reaction 2 hours under the conditions of 37 DEG C, it is ensured that N-RCA reacts abundant with D-SDA；In the above product, add
Enter the 10 × Sybr Green I of 2 μ L, final concentration of 1 × Sybr Green I.Finally 180 are added in above-mentioned product
Microlitre PBS, carry out spectroscopic assay.Design parameter is the same as " feasibility study ".
As can be seen from Figure 3：MiRNA-122, miRNA-141, miRNA-21 and miRNA-26a etc. can be compatible non-
Target miRNA is not interfered significantly, average signal variation about 12% or so.
, detection of the palindrome padlock probe for miRNA in complex environment：
Whether the palindrome padlock probe that we propose by investigation can be applied to the detection potentiality of actual sample, we lock the palindrome
Formula probe application is in complex environment（Fetal calf serum）Middle detection target miRNA.Specific operation process is as follows：First at 20 microlitres
Addition contains in system（0,1%, 5%）Fetal calf serum solution, subsequent palindrome padlock probe（Final concentration of 1 μM）With target
miRNA（Final concentration of 1 μM）According to 1：In 1 addition T4 ligase buffer solutions, and in T4 DNA ligases（350 U）Work
Under, 16 DEG C are incubated 2 hours, and then 3 U polymerases and 5 U Nt.AlWI nickases are added, dNTP, BSA, polymerize enzyme buffer
Solution is incubated 2 hours under the conditions of 37 DEG C.Then, it after said mixture is incubated 30 minutes in Sybr Green I, is added
180 microlitres of PBS then carries out fluoremetry, the parameter being specifically arranged is the same as " feasibility verification ".
Figure 4, it is seen that containing 0%, 1% respectively, the target of same concentrations is detected in 5% fetal calf serum
MiRNA, fluorescent value change unobvious.Illustrate that, even if environment complexity, our method is still accurate to target miRNA detections.Together
When, it was demonstrated that the method has potential application in clinical diagnosis.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification should all belong to the covering scope of the present invention.
SEQUENCE LISTING
<110>University of Fuzhou
<120>A method of detecting Tumor biomarkers using palindrome padlock probe
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Claims (3)
1. a kind of method detecting Tumor biomarkers using palindrome padlock probe, it is characterised in that：The padlock probe：
（1）Identify target part：3 ' the ends and 5 ' ends of padlock probe with target miRNA complete complementaries, can be located at；
（2）Cleavage site part：Half recognition site of restriction enzyme Nt.AlWI；
（3）Palindrome base sequence：3’-CTAGCTAG-5’。
2. a kind of method detecting Tumor biomarkers using palindrome padlock probe according to claim 1, feature
It is：Contain the controllable restriction enzyme enzyme recognition site for incorporating palindromic sequence in the padlock probe： 3’-CCTAGCTAGA-
5’。
3. a kind of method detecting Tumor biomarkers using palindrome padlock probe according to claim 1, feature
It is：Described method includes following steps：
（1）It prepares and connect enzyme buffer solution with the T4 of the target miRNA of concentration is determined containing 1 μM of padlock probe；
（2）350 U/ μ L T4 ligases are added into above-mentioned solution, connection reaction in 2 hours is carried out at 16 DEG C, is closed
The padlock probe of conjunction；
（3）In 10 μ L steps（2）2 μ L, 10 mM dNTPs, 3 U polymerases, 5 U Nt.AlWI cuttings are added in mixture
Enzyme, 0.5 100 × BSA of μ L, 2 μ L 10 × phi29 buffer solutions and 4.7 μ L secondary water, be incubated under the conditions of 37 DEG C
Reaction 2 hours, it is ensured that N-RCA reacts abundant with D-SDA；
（4）In step（3）Mixture in 10 × Sybr Green I of 2 μ L are added, be incubated 30 minutes at room temperature；Then add
After adding pH 7.4 100 mM PBS to 200 μ L, fluoremetry is carried out at room temperature.
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Cited By (2)
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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 |
| CN114350751A (en) * | 2022-01-14 | 2022-04-15 | 福州大学 | CHA-PHCR detection system based on cross-linked network structure and application thereof |
| CN114350751B (en) * | 2022-01-14 | 2023-11-10 | 福州大学 | CHA-PHCR detection system based on cross-linked network structure and application thereof |
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