CN110878335A

CN110878335A - Method for detecting brain glioma marker based on combination of TdT enzyme and NtbspQI nicking enzyme

Info

Publication number: CN110878335A
Application number: CN201910918646.0A
Authority: CN
Inventors: 常津; 崔警予; 宫晓群; 朴佳芳
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2019-09-26
Filing date: 2019-09-26
Publication date: 2020-03-13

Abstract

The invention discloses a method for detecting a brain glioma marker based on signal amplification of terminal deoxynucleotidyl transferase (TdT) and NtbspQI nickase combined use. Firstly, a specific site of NtbspQI nickase is utilized to cut DNA, dTTP is used as a substrate, the function of growing polyT at the end 3 without a template of the TdT is utilized, the traditional miRNA detection is converted into the detection of the polyT by utilizing copper ions, the high-efficiency, quick, high-sensitivity and strong-specificity detection of the miRNA-153 is realized, the method has good specificity, sensitivity and anti-interference capability, and a new direction is provided for establishing a sensitive and specific miRNA detection method.

Description

Method for detecting brain glioma marker based on combination of TdT enzyme and NtbspQI nicking enzyme

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for detecting a glioma marker based on the combined use of TdT enzyme and NtbspQI nicking enzyme.

Background

In recent years, many microRNAs (miRNAs) have become potential tumor markers for early diagnosis of cancer. Of the known miRNAs, 50% are associated with a variety of human tumors, and the abnormal expression of these miRNAs is closely related to the occurrence and development of tumors. Among them, brain glioma (glioblastoma) accounts for about 46% of intracranial tumors. The world health organization 1998 publishes that the malignant glioma is the 2 nd cause of death of patients with tumors under the age of 34 and the 3 rd cause of death of patients between the ages of 35-54. Mortality rates are all at the forefront.

Because the number of cancer patients in China is rapidly increased at present, scientific prevention, early diagnosis, timely treatment and postoperative monitoring of cancer become research subjects and directions with great significance. Many biological studies have proved that early diagnosis of cancer can be realized, the survival rate of cancer can be effectively improved, and sensitive detection of miRNA can play an important role in the occurrence, development, metastasis and prognosis of tumors. The miRNA to be detected is miRNA-153, which is an important molecular biological marker in brain glioma.

The traditional detection methods mainly comprise Northern blotting, microarray and reverse transcriptase real-time quantitative PCR (RT-PCR). Among these methods, the real-time quantitative PCR technique is simple to operate and has better sensitivity and specificity than the other two techniques. However, because of the short sequence, low expression level and relatively poor stability of miRNA, reverse transcription real-time quantitative PCR techniques usually require complicated primer design or primer modification, which is disadvantageous for sensitive and universal detection of miRNA.

In the present invention, a method for signal amplification by a combination of terminal deoxynucleotidyl transferase (TdT) and NtbspQI nickase was devised. The method utilizes NtbspQI nickase to cut at a enzyme cutting site, so that 3-terminal hydroxyl is released, TdT extends at the 3 terminal by taking dTTP as a substrate, the grown polyT is cut by the nickase, the length of the polyT is controlled by controlling the proportion of the two enzymes, the cut polyT and added copper ions are reduced into 0-valent copper under the action of a reducing agent, the copper ions are combined on the polyT, and a fluorescence value is detected by an enzyme labeling instrument, so that the amount of miRNA is determined, and the quantitative detection of trace miRNA is achieved. Meanwhile, the scheme has good specificity, sensitivity and anti-interference capability, and provides scientific basis for further application of the scheme.

Disclosure of Invention

We developed a method for detecting a brain glioma marker miRNA-153 based on signal amplification of a combination of terminal deoxynucleotidyl transferase (TdT) and NtbspQI nickase.

Firstly, a specific site of NtbspQI nickase is utilized to cut DNA, dTTP is used as a substrate, the function of growing polyT at the end 3 without a template of the TdT is utilized, the traditional miRNA detection is converted into the detection of the polyT by utilizing copper ions, the high-efficiency, quick, high-sensitivity and strong-specificity detection of the miRNA-153 is realized, the method has good specificity, sensitivity and anti-interference capability, and a new direction is provided for establishing a sensitive and specific miRNA detection method.

The technical scheme of the invention realizes the high-efficiency, quick, sensitive and specific detection of miRNA-153, and the specific detection steps are as follows:

(1) (sequence one) and DNA probe (sequence two) are combined with miRNA-153

Diluting the stem-loop structure and the DNA probe from 100uM to 10uM, adding the diluted stem-loop structure and the DNA probe into 20ul of the system to ensure that the final concentration is 1uM, controlling the adding amount of different miRNA-153, mixing and reacting for 1h at normal temperature, and causing the three structures to generate conformational change.

(2) Adding enzymes

TdT enzyme and NtbspQI nickase were added, and the reaction was carried out at 37 ℃ for 1h and then inactivated at 85 ℃ for 25 min.

(3) Adding copper ions and a reducing agent

Adding copper ions and a reducing agent, reacting at room temperature for 20min, measuring a fluorescence value by using an enzyme-labeling instrument, and representing the content of miRNA according to the fluorescence value.

(4) Experiment optimization and data processing

And respectively screening the probe sequence, the buffer solution type and the enzyme ratio in the whole process, and optimizing parameters such as probe dosage, reaction time, temperature and the like to finally obtain the optimal conditions of the method.

Drawings

FIG. 1 is a schematic reaction diagram;

FIG. 2 is a representation of the agarose electrophoresis of the most suitable buffer;

FIG. 3 design of DNA probes and stem loops;

FIG. 4 NtbspQI nickase cleavage site;

FIG. 5 is a representation of the agarose electrophoresis of the optimum enzyme ratio.

Detailed Description

The invention will be further illustrated in the following examples, without however being restricted thereto.

The method comprises the following specific steps: FIG. 1 is a reaction scheme of the present invention.

(1) Feasibility experiments: in order to successfully perform the experiment, it is necessary to search for the most suitable buffers for the two enzymes, RB 10X and CoCl for the buffer of TdT₂The buffer of the NtbspQI nicking enzyme is NEBuffer3.1, so the following research is carried out in order to select the most suitable buffer, a) both the buffers are added into the reaction system; b) adding TDT buffer only; c) adding only NtbspQI nicking enzyme buffer; d) adding CoCl₂And NEBuffer 3.1; e) add RB10 × and nebuffer 3.1. Can be finally judged to be CoCl₂Is the buffer necessary for TdT, but the presence of NEBuffer3.1 can make the buffer of TDT ineffective, while the NtbspQI nickase can be normally used in the buffer of TdT, so it is finally concluded that the most suitable buffer is RB 10X and CoCl when two enzymes are used together₂. As shown in fig. 2.

(2) Selection of Stem-Loop and DNA probes

For better sensitivity and stability of the experimental results, we designed a stem-loop and a DNA probe:

stem-loop structure 5'-GATCACTTTTGCTCTTCAAAAG-3'

DNA Probe 5'-TTGAAGAGTGACTATGCAA-3'

mi-RNA-153 5’-UUGCAUAGUCACAAAAGUGAUC-3’

As can be seen from FIG. 3, the stem-loop structure and the DNA probe have a part complementary to miRNA-153, respectively, and a part complementary to miRNA-153 and the DNA probe also have a part complementary to miRNA-153, and the NtbspQI nicking enzyme has a cleavage site as shown in FIG. 4, which is present on the stem of the stem-loop structure, and when no target is present, since the stem-loop is stable enough and cannot be complementary to the DNA probe, there is no way to cleave even if the enzyme is present, and the 3-terminal is blocked, so that both enzymes do not function. When the target is present, the stem loop is opened, and the cleavage site of the NtbspQI nickase is exposed, so that cleavage can be performed, and then TdT can be extended at the exposed 3-terminal due to the presence of the substrate.

(3) Optimum TdT and NtbspQI nicking enzyme ratio

In order to avoid that the TdT is cut at a long point after the cleavage with the NtbspQI nickase and the 3-terminus is exposed, and thus a longer polyT cannot be generated, the concentration of the NtbspQI nickase should be reduced, and therefore the most suitable ratio of TdT to NtbspQI nickase should be investigated. TdT and NtbspQI nickases were adjusted to: the results are shown in FIG. 5 for 1:1, 2:1, 5:1, and 10: 1.

Sequence listing

<110> Tianjin university

<120> group-based method for detecting brain glioma marker by combining TdT enzyme and NtbspQI nicking enzyme

<160>3

<170>SIPOSequenceListing 1.0

<210>1

<211>22

<212>RNA

<213> brain glioma (Artificial Sequence)

<400>1

uugcauaguc acaaaaguga uc 22

<210>2

<211>19

<212>DNA

<213> human source (Artificial Sequence)

<400>2

ttgaagagtg actatgcaa 19

<210>3

<211>22

<212>DNA

<213> human source (Artificial Sequence)

<400>3

gatcactttt gctcttcaaa ag 22

Claims

1. The method for detecting the brain glioma marker based on the combination of TdT enzyme and NtbspQI nicking enzyme is characterized by comprising the following steps:

(1) the stem-loop structure and the DNA probe are fully combined with miRNA-153 to generate conformational change, and a NtbspQI nicking enzyme cutting site is provided; (2) adding NtbspQI nicking enzyme and TdT at the same time, and cutting and extending; (3) copper ions and a reducing agent are added into the generated polyT, and then excitation light of 335nm is given after the copper and the polyT are combined, so that emission light of 565nm can be given.

2. The method of claim 1, wherein the microRNA is detected using a combination of TdT and NtbspQI nickases.

3. The method of claim 1, wherein only buffer carried by TdT is used when TdT and NtbspQI nickases are used in combination to detect microRNAs, and wherein the presence of NEBuffer.3.1 carried by the NtbspQI nickases affects the activity of TdT, wherein the cleavage and extension comprises the steps of:

preparation of a composition containing 250. mu.M CoCl₂(terminal deoxynucleotidyl transferase kit provided), 1 Xterminal deoxynucleotidyl transferase reaction buffer (terminal deoxynucleotidyl transferase kit provided) and 4mM dTTP, adding the micro RNA to be detected, the stem-loop structure, the DNA probe, 20U terminal deoxynucleotidyl transferase and 5UNtbspQI nickase, reacting at constant temperature of 37 ℃ for 60min, and inactivating at 85 ℃ for 25 min. Then, copper ions (200uM) and a reducing agent are added, and finally, the fluorescence value is detected on a microplate reader.

4. The method according to claim 1, wherein the optimum ratio of the TdT and NtbspQI nickases for detecting microRNA is 5:1 when TdT and NtbspQI nickases are used in combination.

5. The detection method according to claim 1, characterized in that: design of stem-loops and DNA probes: both the stem-loop and the DNA probe have a sequence complementary to miRNA-153, and the stem-loop and the DNA probe also have a complementary sequence.