CN108642137B - Method for detecting tumor biomarkers by using palindromic padlock probes - Google Patents

Abstract

The invention provides a method for detecting tumor biomarkers by using a palindromic padlock probe, which designs a palindromic sequence non-fluorescence labeling padlock RCA probe and (1) identifies a target part: can be completely complementary with a target miRNA and is positioned at the 3 'end and the 5' end of the padlock probe; (2) cleavage site moiety: half recognition site of restriction endonuclease nt. AlWI, fused with palindromic base fragment. The probe is combined with nicking lyase, provides an N-RCA concept, and is used for carrying out bidirectional strand displacement reaction (D-SDA) combination for detecting a tumor marker miRNA. The invention can realize the high-efficiency amplification detection of the let-7a miRNA, is economical and practical, and is expected to be used for the development of clinical diagnosis kits.

Description

Method for detecting tumor biomarkers by using palindromic padlock probes

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for detecting a tumor biomarker by using a palindromic padlock probe.

Background

Currently, many traditional amplification techniques, including blotting, real-time quantitative PCR, microarray methods, are widely used for miRNA detection. Despite their advantages, these methods also have many limitations, such as insufficient sensitivity, tight temperature control, and high experimental costs. In order to overcome the difficulties, a palindromic sequence non-fluorescence labeled padlock RCA probe is designed, an N-RCA new concept is proposed by combining with nicking nickase, and the N-RCA new concept is combined with a bidirectional strand displacement reaction (D-SDA) for detecting a tumor marker miRNA.

Disclosure of Invention

The invention aims to provide a method for detecting tumor biomarkers by using a palindromic padlock probe, and a high-performance padlock RCA probe designed by cooperatively embedding a palindromic sequence and a nicking cleavage site does not exist at present.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for detecting a tumor biomarker using a palindromic padlock probe:

(1) identifying the target part: can be completely complementary with a target miRNA and is positioned at the 3 'end and the 5' end of the padlock probe;

(2) cleavage site moiety: half recognition site of restriction enzyme nt.

(3) The padlock probe contains a palindromic sequence: 3 '-CTAGCTAG-5'.

The padlock probe contains a controllable restriction enzyme recognition site fused with a palindrome sequence: 3' -CCTAGCTAGA-5’。

The method comprises the following steps:

(1) preparing a T4 ligase buffer solution containing 1 mu M padlock probes and target miRNA with determined concentration;

(2) adding 350U/. mu. L T4 ligase into the solution, and carrying out ligation reaction at 16 ℃ for 2 hours to obtain a closed padlock probe;

(3) adding 10 mM 2. mu.L dNTPs, 3U polymerase, 5U Nt. AlWI cleavage enzyme, 0.5. mu.L 100 XBSA, 2. mu.L 10 XPhi 29 buffer solution and 4.7. mu.L secondary water into 10. mu.L of the mixture in the step (2), and incubating the mixture at 37 ℃ for 2 hours to ensure that the N-RCA and the D-SDA are fully reacted;

(4) adding 2. mu.L of 10 XSybr Green I to the mixture of step (3) and incubating for 30 minutes at room temperature; then, after adding PBS (pH 7.4100 mM) to 200. mu.L, fluorescence measurement may be performed at room temperature.

The invention has the advantages that:

the invention has high sensitivity and good specificity. The method has low detection lower limit (5 pM) and wide linear range (5-30 nM), which is that the system obtains the synergistic amplification effect of the N-RCA and the D-SDA; the reason for the high specificity is attributed to the following 2 factors: (i) only the target miRNA which is completely complementary can make the padlock probe specifically connected and closed; (ii) the cleavage enzyme Nt. AlWI specifically cleaves 5 '-GGATCNNNN ↓.N-3'.

Drawings

Fig. 1, experimental schematic.

Fig. 2, feasibility examination diagram.

FIG. 3 is a specific examination chart.

Figure 4, ability to detect target mirnas in complex environments.

Detailed Description

The detection system provided by the invention only relates to a DNA probe-padlock probe. The padlock probe consists of two parts: (1) identifying the target part: it can be completely complementary with target miRNA and is positioned at the 3 'end and the 5' end of the padlock probe; (2) cleavage site moiety: half recognition site of restriction enzyme nt. AlWI can specifically recognize (5 '-GGATCNNNN ↓ _ N-3'/3 '-CCTAGNNNNN-5') in double-stranded DNA, cut the upstream strand in the double strand, and the random base "N" can be designed according to the need. In this invention, we specifically designed the random base strand to contain a palindromic sequence (3 '-CTAGCTAG-5') and insert a padlock probe. We explored the target let-7a miRNA as a model. The reaction comprises the following 9 processes (one-pot reaction): firstly, hybridizing a padlock probe with a target let-7a miRNA. Meanwhile, in the presence of DNA ligase, the 5 'end and the 3' end gap of the padlock probe are connected and closed to form an annular padlock probe; secondly, when polymerase, nickase and dNTP exist, let-7a miRNA is used as a polymerization primer, so that a nickase-induced rolling circle reaction (N-RCA) is initiated, and a double-stranded rolling circle product containing the Nt.AlWI recognition site is formed; ③ on the circular double-chain rolling ring product, the chain displacement can be continuously generated through the polymerization reaction/cutting reaction to generate the cutting fragment (NF 1). It is noted that the 3 ' end of these NF1s contains the palindromic sequence (5 ' -GATCGATC-3 '); fourthly, every two NF1 are close to each other, and the 3' ends are hybridized with each other; in the presence of polymerase, the NF1 hybridized two strands are used as templates to extend bidirectionally to generate extended double-stranded DNA; sixthly, under the action of the cutting enzyme, strand displacement reaction (D-SDA) can occur on the newly extended double strand, and a new cutting fragment (NF 2) is generated; seventhly, regenerating the extended double-stranded DNA; the hybridization of NF2 and NF1 can also form double-chain DNA; and ninthly, the double-stranded DNA generated by the above is embedded by a fluorescent dye (Sybr Green I) and a fluorescent signal is output. The invention can realize the high-efficiency amplification detection of the let-7a miRNA, is economical and practical, and is expected to be used for the development of clinical diagnosis kits.

Example 1

The verification process of the invention takes 20 microliter as a reaction system, and the spectral measurement takes 200 microliter as an example. The sequences used are as follows:

2. feasibility study of palindromic padlock probes for miRNA detection:

to demonstrate the feasibility of this invention, we set up an experimental group (with target miRNA) and a control group (without target miRNA), respectively. First, the ratio between palindromic padlock probe (final concentration 1 μ M) and miRNA (final concentration 1 μ M) was varied according to 1: 1 was added to T4 ligase buffer and incubated for 2 hours at 16 ℃ with 350U/. mu. L T4 DNA ligase to allow ligation of the padlock probes.

Then, 10. mu.L of 10 mM 2. mu.L dNTPs, 3U phi29 polymerase, 5U Nt. AlWI cleavage enzyme, 0.5. mu.L 100 XBSA, 2. mu.L of 10 XPhi 29 buffer solution and 4.7. mu.L of secondary water were added to 10. mu.L of the above mixture, and the mixture was incubated at 37 ℃ for 2 hours to ensure sufficient reaction between N-RCA and D-SDA; to the above product, 2. mu.L of 10 XSybr Green I was added so that the final concentration of Sybr Green I was 1X, and incubated at room temperature for 30 minutes. Then, after adding pH 7.4100 mM PBS to 200. mu.L, it was finally measured by fluorescence spectroscopy. In the absence of the target miRNA, the procedure is identical to that described above.

The parameters were set (excitation wavelength 480 nm, emission wavelength: 500 nm-600 nm; maximum voltage set at 500V; slit width: 5 nm.

As can be seen from fig. 2, in the absence of the target miRNA, the fluorescence is weaker; and the fluorescence signal of the target miRNA is obviously improved due to the existence of the target miRNA in the system. Indicating that this method is feasible for miRNA detection.

The palindromic padlock probe is used for specific detection of miRNA:

to verify the specific recognition ability of the palindromic padlock probes in this invention, we added possible coexisting mirnas (miRNA-122, miRNA-141, miRNA-21 and miRNA-26a) to the target miRNA, and compared their relative fluorescence values by fluorescence spectroscopy. The specific operation steps are as follows: first, palindromic padlock probes (final concentration 1 μ M) and mirnas (final concentration 1 μ M) (let-7a mirnas, miRNA-122, miRNA-141, miRNA-21 and miRNA-26a) were each prepared according to the ratio of 1: 1 in the mass ratio of T4 ligase buffer solution, adding 350U/. mu. L T4 ligase into the solution, carrying out ligation reaction for 2 hours at 16 ℃ to obtain a closed padlock probe, then adding 10 mM 2. mu.L dNTPs, 3U phi29 polymerase, 5U Nt. AlWI cutting enzyme, 0.5. mu.L 100 xBSA, 2. mu.L of 10 xphi 29 buffer solution and 4.7. mu.L of secondary water into 10. mu.L of the mixture, and carrying out incubation reaction for 2 hours at 37 ℃ to ensure that the N-RCA and the D-SDA are fully reacted; to the above product, 2. mu.L of 10 XSybr Green I was added, at a final concentration of 1 XSybr Green I. Finally, 180. mu.l of PBS was added to the above product to perform spectrometry. The specific parameters are the same as the feasibility investigation.

As can be seen from fig. 3: non-target miRNAs such as miRNA-122, miRNA-141, miRNA-21 and miRNA-26a which possibly coexist have no obvious interference, and the average signal changes by about 12%.

And the palindromic padlock probe is used for detecting miRNA in complex environment:

to investigate whether the palindromic padlock probes proposed by us can be applied to the detection potential of practical samples, we applied the palindromic padlock probes to detect target miRNAs in complex environments (fetal bovine serum). The specific operation process is as follows: first a solution containing (0, 1%, 5%) fetal bovine serum was added to a 20 microliter system, followed by a palindromic probe (final concentration of 1 μ M) and target miRNA (final concentration of 1 μ M) at a ratio of 1: 1, adding a T4 ligase buffer solution, incubating at 16 ℃ for 2 hours under the action of T4 DNA ligase (350U), adding 3U polymerase, 5U Nt. The mixture was then incubated in Sybr Green I for 30 minutes, then 180 μ l PBS was added, and then the fluorescence assay was performed, with the parameters specified as "feasibility verification".

As can be seen from fig. 4, the fluorescence values of the target mirnas detected at the same concentrations in the sera of the cows containing 0%, 1% and 5% respectively did not change significantly. The method shows that the target miRNA can be accurately detected even if the environment is complex. Meanwhile, the method is proved to have potential application value in clinical diagnosis.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

SEQUENCE LISTING

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Claims (1)

1. A palindromic padlock probe for detecting a tumor biomarker, comprising: the lock-type probe comprises:

(1) identifying the target part: can be completely complementary with a target miRNA and is positioned at the 3 'end and the 5' end of the padlock probe;

(2) cleavage site moiety: half recognition site of restriction endonuclease nt. alwi;

(3) palindromic base sequence: 3 '-CTAGCTAG-5'; method for producing a composite material

The padlock probe contains a controllable restriction enzyme recognition site fused with a palindrome sequence: 3 '-CCTAGCTAGA-5'.

Images