CN202671545U - Kit for fast detection of telomerase activity - Google Patents

Abstract

The utility model relates to a kit, in particular to a kit for real-time quantitative measurement of absolute average length of chromosome telomere in a sample by PCR (polymerase chain reaction). The kit is characterized by comprising a box, a pad, PCR solution, Taq polymerase, a 136B4 forward fragment, a 36B4 reverse fragment, a telomere forward fragment, a telomere reverse fragment, an 84-mer fragment and a 36B4 fragment. Containing cavities arranged on the pad are used for holding PCR solution, Taq polymerase, 136B4 forward, 36B4 reverse, telomere forward, telomere reverse and 84-mer. The kit can be used for measuring absolute average length of telomere fast and conveniently and is applicable to industrial production or detection.

Description

A kind of test kit of rapid detection telomerase activation

Background technology

Telomere (Telomere) is the section of DNA tumor-necrosis factor glycoproteins, is positioned at end of chromosome, can keep chromosomal integrity.Nineteen ninety, the length of the confirmation telomeres such as Harley can shorten gradually along with increasing of frequency dividing cell.Closely contact has been set up in this discovery between telomere and cell aging.The length of telomere repeat sequence may play a part a kind of molecular clock (molecular clock).The people's of different ages the somatic life-span is obviously different, and the length of its telomere is not identical yet.To shorten with advancing age.Can cultivate for 80-90 generations at subculture in vitro separately from neonatal somatocyte, cultivated for 20～30 generations from 70 years old old man's somatocyte external can only going down to posterity, and the tumor-necrosis factor glycoproteins length of telomere shorten also a lot.

Telomerase (Telomerase) is to be responsible for a kind of enzyme that telomere prolongs in the cell, is basic nucleoprotein reversed transcriptive enzyme, telomeric dna can be added to the eukaryotic cell end of chromosome.For keeping chromosome stability and cytoactive to play an important role, Telomerase can prolong the telomere (its cellular replication limited ability of the telomere of shortening) that shortens to telomere in the different plant species cell, thereby strengthens the multiplication capacity of cell in vitro.

But in the normal human cell, the activity of Telomerase is subject to quite tight regulation and control.Experiment showed, the activity that does not have Telomerase in the somatocyte, so the every division of somatocyte once, telomere also just shortens.Along with cell constantly divides, the length of telomere is shorter and shorter, and when reaching a critical length, cell dyeing is known from experience loss of stability, cell can not be divided again and enters apoptosis (apoptosis).The length of telomere has determined the life-span of cell, therefore can infer with the length of the telomere repeat sequence of losing the number of times of cell mitogen, so telomere is called as molecular clock or mitotic division clock (mitotic clock).Telomere is except relating to chromosome stability, and is outside the aging and death of cell, also closely related with the generation of tumour.In the various cells in human body, the chromosomal end of sexual cell and stem cell grows several thousand base pairs than the end of somatic chromosome, and can detect the activity of Telomerase in this two classes cell.In addition, all do not record the activity of Telomerase in other all somatocyte.But only exception is to derive from somatic malignant cell to have reappeared telomerase activation, bring into play the function of its synthetic telomere repeat sequence, lose to compensate normal telomeric sequence, make the tumor-necrosis factor glycoproteins of telomere can not reach the critical length that causes necrocytosis, thereby " immortality " of acquisition cell (immortality).Like this, malignant cell in vivo or external unrestrictedly division growth.

At present, telomerase activity the most frequently used method in detecting end is based on the TRAP method (telomeric repeat amplification protocol, TRAP) of PCR reaction.Take the human cell as example, this method uses PCR reaction amplification by 6 base repetitive sequences (TTAGGG) of Telomerase by the specific primer prolongation, makes the amount of this tumor-necrosis factor glycoproteins reach the degree that can be detected, thereby indirectly measures the activity of Telomerase.Although this method is widely used, the method still has its very important shortcoming: the step relative complex, length consuming time obtains false positive or false negative result etc. easily.Based on above reason, the method that need not more recently to reach by PCR reaction amplification telomere repeat sequence augmentation detection signal purpose is developed, and comprises and use multivalence to cross Nucleotide that nm gold particles has the molecular beacon of catalysis etc.But these technology all are not widely used, and possible reason is that the step of these technology is still very complicated, and the TRAP method comparison with original does not have very large advantage.

The utility model content

The utility model purpose:

This patent relates to a kind of based on exonuclease III(exonuclease III) can progressively remove the characteristic that double-stranded DNA 3 ' is put down end or the terminal single core thuja acid of 3 ' indentation, the method for telomerase activation in the measure sample cell.

A kind of test kit of rapid detection telomerase activation is characterized in that comprising box body, 4 Reagent Tubes; Described Reagent Tube is equipped with respectively the extension mixture, the molecular beacon sequence of mark, ExoIII, 1x NEB Buffer 1.

Described reaction mixture composition is: 20 mM Tris-HCl, and pH 8.3; 1.5 mM MgCl ₂63 mM KCl; 0.05% Tween 20 (w/v); 1 mM EGTA; 10 μ M dATP; 10 μ M dGTP; 10 μ M dCTP; 10 μ M dUTP; 300 nM extension primers; 0.1 mg/ml BSA; Wherein said extension primer sequence is: 5 '-AAT CCG TCG AGC AGA GTT-3 '; The molecular beacon sequence of described mark is: 5 '-(fluorophor)- TAG GGT TACTAA CCC TAA CCC TAA CCC T-(quenching group)-AA CCC TAA CCT TT-3 ', wherein tilted letter representative lock nucleic acid LNA.

Described fluorophor comprises: 5 ' 6-FAM, 5 ' TET, 5 ' HEX, 5 ' TAMRA, 5 ' CY5, middle 6-FAM-dT, 3 ' 6-FAM, 3 ' TAMRA, middle TAMRA-dT, 5 ' ROX, 3 ' ROX, 5 ' Texas Red, 5 ' Cy3, middle Cy3,3 ' Cy3,3 ' Cy5,5 ' Cy5,5 ' Cy5.5,5 ' AMCA, 3 ' AMCA, 5 ' JOE, 3 ' JOE, Alexa Fluor 488,5 ' Methylene Blue, 3 ' Methylene Blue, FR610; Described quenching group comprises: 3 ' BHQ-1,3 ' BHQ-2,3 ' Dabcyl, 5 ' Dabcyl, 3 ' Eclipse.

A kind of application of test kit of rapid detection telomerase activation is characterized in that realizing by step:

A. from cell, extract the sample that contains Telomerase;

B. contain at 2 μ L and add respectively 48 μ L extension mixtures in the Telomerase sample, hatch 1 h for 30 ℃, obtain extension products; Wherein said extension primer sequence is: 5 '-AAT CCG TCG AGC AGA GTT-3 ';

C. with above-mentioned extension products, add respectively and contain 1x NEB Buffer 1,100 U ExoIII, the molecular beacon sequence of 2 μ M marks, reaction mixture placed 37 ℃ of 2 h after, use the visible spectrophotometer reading result; The molecular beacon sequence of wherein said mark is: 5 '-(fluorophor)- TAG GGT TACTAA CCC TAA CCC TAA CCC T-(quenching group)-AA CCC TAA CCT TT-3 ', wherein tilted letter representative lock nucleic acid LNA.

The principle explanation:

1, molecular beacon (MBs) is combined with target sequence, produces the principle (Fig. 1) of detection signal:

But MBs is a kind of hair clip type stem ring double-tagging oligonucleotide fluorescent probe of specific recognition nucleotide sequence, have high specific, hypersensitivity, easy and simple to handle, can be used for the advantages such as quantitative analysis.The nucleic acid array complementation pairing at two ends, so mark fluorophor at one end is tightly close with the quenching group that is marked at the other end.Fluorophor is excited the photon of rear generation by the quencher cancellation, and the energy that is produced by fluorophor discharges with infrared rather than visible light form.Under the renaturation temperature, untie because template forms the loop-stem structure two strands when existing, and match with template sequence.After the template pairing, molecular beacon will become chain but not hairpin, so that fluorophor separates with quencher, fluorophor is excited, and because the cancellation effect is disengaged, sends excitation photon, thereby can be detected by the detector.

2, by exonuclease III(ExoIII) remove 3 ' end single core thuja acid, reach the principle (Fig. 2) that fluorescent signal is constantly regenerated, strengthened:

ExoIII have from 3 of double-stranded DNA '-the terminal degraded of OH generates 3 ' → 5 ' 5 prime excision enzyme activity of 5 '-mononucleotide.This enzyme has high degree of specificity to double-stranded DNA, and the smooth end of degrading, 3 ' indentation are terminal and the DNA of otch is arranged, but can not degrade 3 '-protruding terminus.Utilize the characteristic of ExoIII, after hybridizing with the Telomerase extension products and activating fluorophor, MBs 3 ' terminal nucleotide is progressively removed by the ExoIII in the reaction system, final so that the MBs that is degraded separates with target sequence, this is so that target sequence can be again and complete MBs hybridization, and activates its fluorophor.This target sequence is hybridized-is activated the step that fluorophor-ExoIII degraded MBs-target sequence separates with MBs and constantly circulates with MBs, thereby solved the limitation that target sequence and MBs can only be combined with the ratio of 1:1, so that fluorescent signal strengthened greatly, until can be detected by the detector.

Beneficial effect:

1, the utility model can be realized the extraction of Telomerase by the design Auele Specific Primer.

2, Telomerase is the known the strongest biomarker of malignant tumour specificity, and its enzymic activity all has very large potential value in the early diagnosis of tumour and prognosis evaluation.Simultaneously, in the non-tumor cells such as sexual cell and hemopoietic stem cell, also telomerase activation be can detect, thereby normal cell and tumour cell are difficult to distinguish only according to having or not of telomerase activation.So the quantitative analysis of telomerase activation is important means in cancer diagnosis and the prevention.The Telomerase activity method based on ExoIII 3 '-5 ' 5 prime excision enzyme activity that this patent relates to, simple in steps, weak point consuming time, the characteristics such as accuracy is high, and cost is low have very large extensive and commercial applications potentiality.

3, core of the present utility model is to utilize exonuclease III(exoneclease III) can progressively remove the single core thuja acid that double-stranded DNA 3 ' is held, and to the characteristic of single stranded DNA non-activity, so that with molecular beacon (the molec μ Lar beacons of Telomerase extension sequence specific combination, MBs) constantly be activated-remove, then new MBs again be combined with target sequence-activate-remove, thereby make MBs signal (fluorescence, color reaction etc.) constantly increased, reached the degree that can be detected.What of Telomerase extension products are the intensity of detection signal directly reflected, thereby indirectly reflected the power of telomerase activation in the cell.This technological step is simple, weak point consuming time, and possible false positive or the false negative result of having avoided the PCR reaction to bring.In addition, this technology

4, the required instrument consumptive material of this test kit is cheap, has the some commercial potential for extensive detecting end telomerase activity.

Description of drawings:

Fig. 1. use MBs carries out quantitative analysis to target sequence principle.In the situation without target sequence, MBs is the hair clip type structure, its fluorophor under the effect of quenching group, emitting fluorescence not; After hybridizing with target sequence, the fluorophor of MBs separates with quenching group, the fluorescent signal that generation can be detected.

Fig. 2. use the principle of the Characteristics Detection telomerase activation of ExoIII.1. MBs and target sequence, i.e. Telomerase extension products hybridization produces fluorescent signal, and it is terminal to form 3 ' indentation at its 3 '-OH end, thereby becomes the substrate of ExoIII 5 prime excision enzyme activity; 2. after ExoIII progressively removed the oligonucleotide of MBs 3 '-OH end, MBs separated with extension products; Extension products after the separation can be combined with complete MBs again, and the step before repeating, so that fluorescent signal is enhanced.

Fig. 3. the HeLa Cell Telomerase Activity.The fluorescent signal strong by about 40% (P＜0.05) that the corresponding fluorescence intensity ratio blank of sample Telomerase Activity (Lysis buffer) that extracts from about 8000 HeLa cells or the HeLa cell sample after the deactivation are corresponding.

Fig. 4. different cell Detection of Telomerase Activity results.With blank (Lysis buffer) or known WI-38 cell without telomerase activation strength of signal compare, strength of signal significantly strengthens (P＜0.05) in HeLa cell or the HeLa+WI-38 cell sample.

Fig. 5. use traditional TRAP method detecting end telomerase activity result.Wherein A is 1-3 swimming lane electrophoresis, and B is the 4-5 electrophorogram; In blank (Lysis buffer), deactivation HeLa cell and the WI-38 cell without telomerase activation ( swimming lane 2,3,5), do not observe the telomere repeat sequence fragment band that the Telomerase extension produces, and in the known HeLa cell or HeLa+WI-38 cell sample that telomerase activation arranged (swimming lane 1,4), can observe the telomere repeated fragment band of indirect reaction telomerase activation.

Fig. 6 is the utility model synoptic diagram, and wherein 5 is box body, and 1-4 is respectively first to fourth Reagent Tube.

Embodiment

ExoIII and NEB Buffer 1 be available from New England Biolabs company, the MBs(LNA-DNA of all fluorescence groups/cancellation group mark) and Oligonucleolide primers synthetic by giving birth to worker's biotechnology (Shanghai) company, by the HPLC purifying, and checked order by mass spectrum.Wherein fluorophor is CAL Fluor Red 610(FR610), quenching group is black hole quencher(BHQ).LNA-DNA mosaic MB sequence for detection of telomerase activation is: 5 '-(FR610)- TAG GGT TACTAA CCC TAA CCC TAA CCC T-(BHQ)-AA CCC TAA CCT TT-3 ' (tilted letter representative lock nucleic acid LNA); Telomerase extension primer is: 5 '-AAT CCG TCG AGC AGA GTT-3 '.The cultural method that tumour cell (the HeLa cell strain is available from ATCC) and human embryonic lung diploid fibroblast (the WI-38 cell strain is available from ATCC) culture condition provide with reference to ATCC.

Embodiment 1.

A kind of test kit of rapid detection telomerase activation is characterized in that comprising 5,4 Reagent Tubes of box body; Wherein the first Reagent Tube 1 is equipped with the extension mixture, and the second Reagent Tube 2 fills markd molecular beacon sequence, and the 3rd Reagent Tube 3 is equipped with ExoIII, and the 4th Reagent Tube 4 is equipped with 1x NEB Buffer 1.

A kind of application of test kit of rapid detection telomerase activation, realize as follows:

One, the extraction of cell sample telomerase

Collect respectively tumour cell (the HeLa cell strain is available from ATCC) and human embryonic lung diploid fibroblast (the WI-38 cell strain is available from ATCC), centrifugal after with cell by 200 μ L/10 ⁶The ratio Eddy diffusion of cell leaves standstill 30 min on ice in lysate (1x CHAPS Lysis buffer, ATCC); Afterwards in 12000 g, 4 ℃ of centrifugal 20 min; Collect supernatant in-80 ℃ of preservations, obtain two kinds and contain the Telomerase supernatant, be i.e. cleer and peaceful WI-38 cell Telomerase supernatant on the HeLa cell Telomerase.

Two, Telomerase extension

Contain the about 5 μ g/ μ L of total protein at 2 μ L() two kinds contain and add respectively 48 μ L extension mixtures in the Telomerase supernatant sample to be measured, hatch 1 h for 30 ℃, obtain extension products, i.e. HeLa cell extension products and WI-38 cell extension products;

Wherein the extension mix ingredients is: 20 mM Tris-HCl, and pH 8.3; 1.5 mM MgCl ₂63 mM KCl; 0.05% Tween 20 (w/v); 1 mM EGTA; 10 μ M dATP; 10 μ M dGTP; 10 μ M dCTP; 10 μ M dUTP; 300 nM extension primers, wherein the primer nucleotides sequence is classified 5 '-AAT CCG TCG AGC AGA GTT-3 ' as; 0.1 mg/ml BSA.

Telomerase activation detects

Reaction system is 50 μ L, and with above-mentioned extension products, interpolation contains 1x NEB Buffer 1,100 U ExoIII, the molecular beacon sequence of 2 μ M marks respectively; After reaction mixture placed 37 ℃ of 2 h, use visible spectrophotometer reading result: λ _Ex=590 nm, the fluorescent signal strong by about 40% (P＜0.05) corresponding to HeLa cell sample of (be after the heat treated after the deactivation) seen Fig. 3 after the corresponding fluorescence intensity ratio blank of the sample Telomerase Activity of extracting from the HeLa cell (Lysis buffer) or the deactivation;

λ _Em=610 nm, with blank (Lysis buffer) or known WI-38 cell without telomerase activation strength of signal compare, strength of signal significantly strengthens (P＜0.05) and sees (Fig. 4) in HeLa cell or the HeLa+WI-38 cell sample.

Described molecular beacon sequence is: 5 '-(fluorophor)- TAG GGT TACTAA CCC TAA CCC TAA CCC T-(quenching group)-AA CCC TAA CCT TT-3 ' (tilted letter representative lock nucleic acid LNA); Wherein the fluorophor in this example is FR610, and quenching group is 3 ' BHQ-1.

Electrophoresis uses 10% non-denaturing PAGE glue, and deposition condition is 400 V, 1.5 h.Use Ethidium Bromide solution-dyed after electrophoresis is finished, observations under ultraviolet lamp; In blank (Lysis buffer), deactivation HeLa cell and the WI-38 cell without telomerase activation ( swimming lane 2,3,5), do not observe the telomere repeat sequence fragment band that the Telomerase extension produces, and in the known HeLa cell or HeLa+WI-38 cell sample that telomerase activation arranged (swimming lane 1,4), can observe the telomere repeated fragment band (Fig. 5) of indirect reaction telomerase activation

Comparative Examples: TRAP assay

The method is used Trapeze Telomerase Detection Kit(Millipore).

1. under the condition that the primer TS of particular sequence Primer (5 '-AAT CCG TCG AGC AGA GTT-3 ') exists, obtain Telomerase extension product, and use this extension product of pcr amplification.

The reaction mixture composition of telomerase gene extension and follow-up pcr amplification extension product is as follows:

10x TRAP Reaction Buffer	5 μL
		50x dNTPs Mix	1 μL
TS Primer
		1 μL
TRAP Primer Mix			1 μL
	Taq polymerase (5U/μL)	0.4 μL
Embodiment
	1 obtains Telomerase and extracts supernatant	2 μL
dH 2O			39.6 μL

The PCR reaction conditions is: 30 ℃, and 30 min; 33 circulations (94 ℃, 30 s; 59 ℃, 30 s; 72 ℃, 1 min)

2. electrophoretic analysis pcr amplification product

Electrophoresis uses 10% non-denaturing PAGE glue, and deposition condition is 400 V, 1.5 h.Use Ethidium Bromide solution-dyed after electrophoresis is finished, observations under ultraviolet lamp, (Fig. 5).Wherein the WI-38 cell strain is human embryonic lung diploid fibroblast, as normal somatocyte, has been proved to be and does not contain telomerase activation, so here as negative control.

The Telomerase activity method based on the ExoIII 5 prime excision enzyme activity of using this patent to relate to, we observe the known resulting fluorescent signal of HeLa cell sample of telomerase activation that exists and compare with blank Lysis buffer or WI-38 cell strain as negative control, and fluorescent signal has remarkable enhancing (Fig. 3,4).In addition, use through the HeLa of thermal treatment deactivation cell in contrast, find the fluorescence intensity that obtains and the fluorescence intensity basic identical (Fig. 3) of using blank Lysis buffer to obtain, the impact that this has just got rid of other factors proves and uses the measured fluorescent signal of normal HeLa cell extract really corresponding to its telomerase activation.When using in addition WI-38 cell and HeLa cytomixis extract, we find the strength of signal that obtains and use separately HeLa cell extract basically identical (Fig. 4) that this has also proved the accuracy of the method mensuration Cell Telomerase Activity.Corresponding, when using traditional TRAP method detecting end telomerase activity, the HeLa cell sample can be observed and represent telomerase activation telomere repeat sequence band (Fig. 5, swimming lane 1), and these bands do not observe (Fig. 5, swimming lane 2,3,5) in negative control.This has proved that from the side the Telomerase activity method acquired results that this patent relates to is true and reliable.

Claims (1)

1. the test kit of a rapid detection telomerase activation is characterized in that comprising box body, 4 Reagent Tubes; Wherein the first Reagent Tube is equipped with the extension mixture, and the second Reagent Tube fills markd molecular beacon sequence, and the 3rd Reagent Tube is equipped with ExoIII, and the 4th Reagent Tube is equipped with 1x NEB Buffer 1.

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