CN113481279A - Method for detecting telomerase activity - Google Patents

Method for detecting telomerase activity Download PDF

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CN113481279A
CN113481279A CN202110745677.8A CN202110745677A CN113481279A CN 113481279 A CN113481279 A CN 113481279A CN 202110745677 A CN202110745677 A CN 202110745677A CN 113481279 A CN113481279 A CN 113481279A
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telomerase
detecting
activity
magnetic beads
telomerase activity
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苏玲玲
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Guangzhou Bolais Biotechnology Co ltd
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Guangzhou Bolais Biotechnology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/91245—Nucleotidyltransferases (2.7.7)
    • G01N2333/9125—Nucleotidyltransferases (2.7.7) with a definite EC number (2.7.7.-)
    • G01N2333/9128—RNA-directed DNA polymerases, e.g. RT (2.7.7.49)

Abstract

The invention discloses a method for detecting telomerase activity, which comprises the following steps: firstly, performing extension reaction on streptavidin modified magnetic beads and biotinylated telomerase substrate primers to prolong and generate a telomere repeat sequence of TTAGGG; coupling the Azide modified DNA probe with a DBCO group on dUTP through a click chemistry reaction to generate a plurality of DNA probes with side chains; triggering another two hairpin DNA probes H1 and H2 to assemble to generate a long-chain assembly product; and step four, detecting the fluorescence on the surface of the magnetic beads through a fluorescence detector to realize the detection of the activity of the telomerase. The invention has accurate and reliable detection result and high sensitivity.

Description

Method for detecting telomerase activity
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a method for detecting telomerase activity.
Background
Telomerase is a special reverse transcriptase consisting of RNA and protein, involved in the synthesis of telomeres (a specific nucleotide sequence and structure) at the DNA terminus of eukaryotic cells. The telomere length of normal somatic cells is gradually shortened along with the division of the cells, the activity of telomerase is enhanced, the telomere length can be kept not to be shortened, and the cells are permanently proliferated to become cancerous. Therefore, telomerase detection and inhibitors thereof can be used for tumor diagnosis and treatment.
The detection of telomerase is suitable for diagnosis and differential diagnosis of various tumor diseases. For example, small cell lung cancer has increased telomerase activity in its early stage, and non-small cell lung cancer has more activity changes than in its middle and late stages.
Telomerase has been of interest in recent years due to its association with studies of longevity, cancer, and the like. The telomerase can copy and synthesize a telomere sequence by taking a template region of self RNA as a template. The telomerase activity is realized in cells with active proliferation such as embryonic cells, the telomerase is inactivated in normal mature somatic cells, and meanwhile, most of tumor cells are found to be telomerase positive, while the positive rate of the telomerase in tissues beside cancers and normal tissues is low, so that the telomerase is presumed to be a wide tumor marker.
At present, methods for detecting telomerase mainly include an isotope method, a staining method, a fluorescence method and an ELISA method.
1. Isotope method. Isotopic methods are most used. There are many reports of incorporation using [ α -32P ] dGTP or dCTP, but some scholars label the 5' end of the TS primer with [ γ -32P ] dATP and T4 kinase, and find that low levels of telomerase are more easily detected and more easily quantified. The gel was run on a PAGE gel and then examined for 3h by autoradiography on an X-ray film or by scanning with a Phosphoimager. The isotope method has the advantages of high sensitivity, detection of 100 immortalized cells for 27 cycles, radioactive pollution, and long time for autoradiography which takes more than 8 hours to two days.
2. And (3) dyeing. After electrophoresis, the gel is stained with SYBR green or EB, and then observed under an ultraviolet lamp or measured by a CCD image system. EB has the best effect under a 302nm ultraviolet transilluminator and an orange-red ultraviolet filter, and can obtain sensitivity similar to SYBR green. The staining method is simple and rapid, the sensitivity is 100 immortalized cells for 30 cycles, and the signal intensity of the staining band can not accurately reflect the number of molecules (the large fragment stains more strongly), so the staining method can only judge the relative telomerase activity, but can not determine the exact number of the telomerase extension product.
3. Fluorescence method. Add 10pmol fluorescein labeled (such as FAM, FITC) TS and/or CX primer amplification, through 8% denaturing PAGE gel electrophoresis, DNA sequencer automatically read the value, fragment management system software automatically calculate the scanning curve peak height and peak area, from the loading to the results only need 90 min. The fluorescence system is extremely sensitive, in order to avoid the unreliable result possibly given by the excessive amplification product, the low protein content of 0.5-1.0 mu g is used, the 27 cycles of amplification are carried out, and the fragment management system can automatically detect very weak fluorescence, so that the activity of the low-level telomerase can not be accurately measured [ 8-11 ]. Another application of fluorescence is in situ-TRAP assays, where telomerase activity can be detected at the cellular level, thus allowing one to determine which cells, how many cells, have telomerase activity, and the source of cells for which activity cannot be known by lytic extraction. In situ analysis was performed on a siliconized slide and the results were observed under a fluorescent microscope. At present, the method is only used for fresh specimens, the experiment for freezing and storing pathological specimens is not successful, and the improvement method is needed to prevent the dispersion of intracellular telomerase in freeze thawing, increase the permeability of the specimens and the like.
4. Marking biotin at the 5' end of the TS primer by ELISA method, after PCR amplification, denaturing the product, adding a digoxin-labeled probe which can be specifically combined with the repeated segment of the amplification product, combining the biotin on the hybridization product with the avidin fixed on the micropore plate, combining the digoxin on the probe with peroxidase-labeled anti-digoxin antibody, then adding a substrate, and measuring by using an enzyme-labeling instrument after color development. The ELISA method was used in a Boehringer's kit, and a ladder band differing by 6bp was not observed because of the absence of electrophoresis.
The isotope method has high sensitivity and is most applied, but radioactive pollution exists, the time is long, the dyeing method is simple and convenient to operate, the time is short, but only relative telomerase activity can be detected, and the fluorescence method is only used for fresh samples, so that a novel telomerase detection method is required to be provided, the accuracy of a detection result can be ensured, and the method is safe and reliable.
Disclosure of Invention
Based on this, it is therefore a primary object of the present invention to provide a method for detecting telomerase activity, which is capable of safely and reliably detecting telomerase activity, and which has high detection sensitivity and accuracy.
Another object of the present invention is to provide a method for detecting telomerase activity, which produces an assembly product with a plurality of fluorophores, so that fluorescence on the surface of magnetic beads can be detected by a fluorescence detector, thereby realizing accurate detection of telomerase activity.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a method for detecting telomerase activity, the method comprising:
firstly, performing extension reaction on streptavidin modified magnetic beads and biotinylated telomerase substrate primers to prolong and generate a telomere repeat sequence of TTAGGG;
coupling the Azide modified DNA probe with a DBCO group on dUTP through a click chemistry reaction to generate a plurality of DNA probes with side chains;
triggering another two hairpin DNA probes H1 and H2 to assemble to generate a long-chain assembly product;
and step four, detecting the fluorescence on the surface of the magnetic beads through a fluorescence detector, and further realizing the detection of the activity of telomerase.
Specifically, in the first step, streptavidin modified magnetic beads and biotinylated telomerase substrate primers (TS primers) are connected through a streptavidin-biotin reaction, and the TS primers perform an extension reaction in the presence of telomerase to prolong the telomere repeat sequences of TTAGGG.
When 5-DBCO-PEG4-dUTP, dATP and dGTP are added in the extension reaction process, the repeated sequence of TTUGGG is extended. The U base in the extension product can generate click chemistry reaction because the DBCO group (the DBCO group is dibenzocyclooctyne: a reaction group of click chemistry) is modified, so that the coupling reaction with a DNA probe can be conveniently generated.
In the second step, an Azide (Azide) modified DNA probe is designed to be coupled with the DBCO group on dUTP through a click chemistry reaction, so that the telomerase extension product can click on the DNA probes with a plurality of side chains. This DNA strand can be designed as a trigger strand for a hybridization chain reaction.
In step three, the assembly of two additional hairpin DNA probes (H1 and H2) is triggered, resulting in a long-chain assembly product. When FAM (a fluorophore) is labeled on the other two hairpin DNA probes, the assembly product can have many fluorophores.
In the fourth step, redundant probes H1 and H2 can be washed away through a magnetic separation experiment, and then fluorescence on the surface of the magnetic beads is detected through a fluorescence detector, so that the detection of the activity of telomerase is realized.
Further, in the method, the probe sequence is
Figure BDA0003144265000000041
The invention has the beneficial effects that:
the method comprises the steps of firstly enabling streptavidin modified magnetic beads and biotinylated telomerase substrate primers to perform extension reaction, extending and generating telomere repetitive sequences of TTAGGG, then performing coupling to generate DNA probes with a plurality of side chains, so that telomerase extension products can click on the DNA probes with the plurality of side chains, and then generating long-chain assembly products on the basis of the DNA probes, wherein the assembly products have a plurality of fluorescent groups, and the fluorescence on the surfaces of the magnetic beads is detected by a fluorescence detector, so that the detection of the activity of telomerase is realized.
The detection method has accurate and reliable detection result and high sensitivity.
Drawings
FIG. 1 is a flow chart of a method implemented by the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
As shown in FIG. 1, the present invention is a schematic diagram of a baculovirus fluorescent nanoprobe realized by labeling baculovirus envelope glycoprotein GP64 by self-biotinylation and then labeling its genome with ruthenium complex using replication and self-assembly process of baculovirus [ Ru (phen ]2dppz]2+And (3) preparing.
And connecting the streptavidin modified magnetic beads with biotinylated telomerase substrate primers (TS primers) through a streptavidin-biotin reaction, wherein the TS primers generate an extension reaction under the condition that telomerase exists, and the telomere repetitive sequence of TTAGGG is generated by extension. When 5-DBCO-PEG4-dUTP, dATP and dGTP are added in the extension reaction process, the repeated sequence of TTUGGG is extended. The U base in the extension product can undergo click chemistry reaction due to the modification of DBCO group (dibenzocyclooctyne: a reaction group of click chemistry). An Azide (Azide) modified DNA probe is designed to be coupled with DBCO groups on dUTP through a click chemistry reaction, so that a telomerase extension product can click on DNA probes with a plurality of side chains. This DNA strand can be designed as a trigger strand for hybridization chain reaction, which can trigger the assembly of two other hairpin DNA probes (H1 and H2) to generate a long-chain assembly product. When FAM (a fluorophore) is labeled on the other two hairpin DNA probes, the assembly product can have many fluorophores. Redundant H1 and H2 probes can be washed away through a magnetic separation experiment, and then fluorescence on the surfaces of the magnetic beads is detected through a fluorescence detector, so that the activity of telomerase is detected.
Wherein the probe sequence is
Figure BDA0003144265000000051
The experimental procedure was as follows:
first, 10. mu.L of magnetic beads were added, the supernatant was removed by attracting the magnetic beads with a magnet, 10. mu.L of 1 XPBS was added, 5. mu.L of TS primer (10. mu.M) was added, and the mixture was allowed to stand at room temperature for 30 min. The TS primers not bound to the magnetic beads were removed by magnet, 25. mu.L of telomerase substrate primer extension system (1 XPCR buffer 22.5. mu. L, dATP (2.5mM, 0.5. mu.L), dAGP (2.5mM, 0.5. mu.L), 5-DBCO-PEG4-dAUP (2.5mM, 0.5. mu.L), 1. mu.L of telomerase, extension at 37 ℃ for 1H, supernatant removed by magnet, 1 XSSC 10. mu.L, T1 (10. mu.M, 5. mu.L), reaction at 30 ℃ for 1H, supernatant removed by magnet, H1 (10. mu.M) and H2 (10. mu.M) each 10. mu.L, hybridization reaction at 30 ℃ for 2H, magnetic beads washed with 1 XPCR PBS multiple times, and the results were observed under a fluorescent microscope.
② the extension of telomerase is firstly carried out. Telomerase diluted with lysis solution was added to a telomerase substrate primer extension system (TS primer (10. mu.M) 5. mu.L, 1 XPCR buffer 22.5. mu. L, dATP (2.5mM, 0.5. mu.L), dAGP (2.5mM, 0.5. mu.L), 5-DBCO-PEG4-dAUP (2.5mM, 0.5. mu.L), extended at 37 ℃ for 1H, 10. mu.L of magnetic beads were added, left to stand at room temperature for 30min, the supernatant was aspirated with a magnet, 1 XSSC 10. mu.L was added, T1 (10. mu.M, 5. mu.L) was added, the supernatant was aspirated with a magnet, 10. mu.L each of H1 (10. mu.M) and H2 (10. mu.M) was added, the hybridization reaction was performed at 30 ℃ for 2H, the magnetic beads were washed with 1 XPBS several times, and the results were observed with a fluorescence detector (fluorescence spectrometer, fluorescence microscope, etc.).
In a word, the streptavidin-modified magnetic beads and biotinylated telomerase substrate primers are subjected to extension reaction to generate a telomere repetitive sequence of TTAGGG by extension, and then are coupled to generate DNA probes with a plurality of side chains, so that the telomerase extension products can click on the DNA probes with the plurality of side chains, and then long-chain assembly products are generated on the basis of the DNA probes, wherein the assembly products have a plurality of fluorescent groups, and the fluorescence on the surfaces of the magnetic beads is detected by a fluorescence detector, so that the activity of telomerase is detected.
The detection method has accurate and reliable detection result and high sensitivity.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims (7)

1. A method for detecting telomerase activity, the method comprising:
firstly, performing extension reaction on streptavidin modified magnetic beads and biotinylated telomerase substrate primers to prolong and generate a telomere repeat sequence of TTAGGG;
coupling the Azide modified DNA probe with a DBCO group on dUTP through a click chemistry reaction to generate a plurality of DNA probes with side chains;
triggering another two hairpin DNA probes H1 and H2 to assemble to generate a long-chain assembly product;
and step four, detecting the fluorescence on the surface of the magnetic beads through a fluorescence detector, and further realizing the detection of the activity of telomerase.
2. The method for detecting telomerase activity as claimed in claim 1, wherein in step one, streptavidin-modified magnetic beads are linked to biotinylated telomerase substrate primers via a streptavidin-biotin reaction, and the TS primer undergoes an extension reaction in the presence of telomerase to extend the telomeric repeats that produce TTAGGG.
3. The method for detecting telomerase activity as claimed in claim 2, wherein the 5-DBCO-PEG4-dUTP, dATP, dGTP, is designed to extend beyond the TTUGGG repeat sequence during the extension reaction.
4. The method for detecting telomerase activity as claimed in claim 1, wherein in step two, an Azide (Azide) modified DNA probe is designed to couple with the DBCO group on dUTP by click chemistry, and the telomerase extension product will click on the DNA probe with many side chains.
5. The method for detecting telomerase activity as claimed in claim 1, wherein in step three, when FAM is labeled on the other two hairpin DNA probes, the assembly product has a plurality of fluorophores.
6. The method for detecting telomerase activity as claimed in claim 1, wherein in step four, excess H1 and H2 probes are washed away by magnetic separation assay, and fluorescence on the surface of the magnetic beads is detected by a fluorescence detector.
7. The method for detecting telomerase activity of claim 1, wherein the probe sequence is:
Bio-TS primer 5’-Biotin-TTTTTTAATCCGTCGAGCAGAGTT-3’；
T1(Azide-probe)5’-N3-AGTCTAGGATTCGGCGTGGGTTAA-3’；
H1 5’-FAM-TTAACCCACGCCGAATCCTAGACTCAAAGTAGTCTAGGATTCGGCGTG-3’
H2 5’-FAM-AGTCTAGGATTCGGCGTGGGTTAACACGCCGAATCCTAGACTACTTTG-3’。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106811524A (en) * 2017-01-19 2017-06-09 陕西师范大学 A kind of telomerase activation colorimetric detection method
CN109517880A (en) * 2018-11-15 2019-03-26 中山大学 The method of gold nanoparticle detection urine Telomerase Activity based on chain replacement reaction and DNA modification
CN112410400A (en) * 2019-08-22 2021-02-26 深圳市第二人民医院 Telomerase activity detection kit and telomerase activity detection method

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106811524A (en) * 2017-01-19 2017-06-09 陕西师范大学 A kind of telomerase activation colorimetric detection method
CN109517880A (en) * 2018-11-15 2019-03-26 中山大学 The method of gold nanoparticle detection urine Telomerase Activity based on chain replacement reaction and DNA modification
CN112410400A (en) * 2019-08-22 2021-02-26 深圳市第二人民医院 Telomerase activity detection kit and telomerase activity detection method

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
俸家富等, 四川科学技术出版社 *

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