CN1087033C - Telomere repeated cloning-porous plate hybridization process to determine the activity of telomere enzyme - Google Patents
Telomere repeated cloning-porous plate hybridization process to determine the activity of telomere enzyme Download PDFInfo
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- CN1087033C CN1087033C CN98111646A CN98111646A CN1087033C CN 1087033 C CN1087033 C CN 1087033C CN 98111646 A CN98111646 A CN 98111646A CN 98111646 A CN98111646 A CN 98111646A CN 1087033 C CN1087033 C CN 1087033C
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Abstract
The present invention discloses a telomere repeated cloning-porous plate hybridization method to determine the activity of telomere enzyme. The method orderly comprises the following steps: a processing step of a sample, an amplification step, a coating step, a denaturation step, a hybridization step, a coloration step, etc. The present invention is used for determining the activity of telomere enzyme. The present invention has the advantages of simplicity, convenience, high speed, low cost, good repetitiveness, high sensitivity, etc. The present invention is especially suitable for judging benign and malignant thoracic and ascitic fluid.
Description
The present invention relates to the method for a kind of people's of mensuration tissue, body fluid Telomerase Activity.
The early stage method of measuring telomerase activation is to carry out radioautograph by measuring after cell extract is added to a synthetic oligonucleotide, polyacrylamide gel electrophoresis with the telomere repeated fragment, and this method susceptibility is extremely low, is eliminated.Kim in 1994 etc. set up telomeric repeat amplifcation protocol, and this method is used a large amount of amplifications of polymeric enzyme reaction (PCR) with the reaction product of Telomerase, make the mensuration susceptibility improve 10
4Doubly, be the method that is generally adopted by the whole world in recent years, but this method need be carried out radioautograph with amplified production behind polyacrylamide gel electrophoresis, method is very numerous and diverse.Domestic have the author to replace isotropic substance to develop with argentation, but method is still numerous and diverse, and detection sensitivity but descends to some extent.German Boehringer company in 1996 is with mark digoxigenin-probe detecting end granzyme amplified production, released Telomerase-ELISA detection kit, this method has greatly been simplified PCR product detecting operation step, can draw detected result in 4 hours, but in testing process, find digoxigenin labeled system instability, often occur between the different lot numbers of the test kit that provides, and the reagent price is very expensive, can't conventionally use than big-difference.
The object of the present invention is to provide a kind of have measure susceptibility and repeatability preferably, easy fast, the telomeric repeat amplification-micropore plate hybridization method of mensuration telomerase activation that cost is low.
The object of the present invention is achieved like this:
A kind of telomeric repeat amplification-micropore plate hybridization method of measuring telomerase activation, the difference of itself and prior art is: comprise the following steps: successively
1. sample process, dissolved cell: the tissue slice mill is even, and be suspended in freezing cracking in the cold lysate, supernatant is got in centrifugation;
2. reverse transcription, amplification: with CX primer reverse transcription, to the processing of increasing of above-mentioned supernatant liquor, the CX primer is 5 ' CCCT TACCCTTACCCTTACCCTTA with the PCR reaction solution that contains the Ts primer, and the Ts primer sequence is 5 ' Biotin AATCCGTCGAGCAGCGTT;
3. solid phase and hybridization:
A) bag quilt: Streptavidin is fixed on the microwell plate;
B) sex change: with amplified production NaOH denaturing treatment;
C) hybridization and colour generation: will on microwell plate, mix through the sex change mixed solution of denaturing treatment with the hybridization solution that contains the CF probe, carry out hybridization, add enzyme labelled antibody then, and with colour generation liquid colour generation, use the microplate reader colorimetric, probe CF is 5 ' FITC-CCCTAACCCTAACCCTAACCCTAAA.
During sample process, the cold lysate of employing is: 10mmol/L Tris-HCl, PH7.5,1mmol/L MgCl
2, 1mmol/L EGTA, 0.1mmol/L PMSF, 5mmol/L beta-mercaptoethanol, 5g/L CHAPS, 100g/L glycerine.
During amplification, used PCR reaction solution is 10XBuffer5 μ l, 1.5mmol/L MgCl
2, 0.1g/L gelatin, 200 μ mol/L dNTP, 0.23 μ mol/L TS primer, 2 μ Taq enzymes.
The concrete steps of bag quilt are: Streptavidin is dissolved in the PH10.0 carbonate buffer solution, and 10mg/L adds 100 μ l in the every hole of microwell plate, and 4 ℃ are spent the night, and PH7.4PBS washes, and to placing clean filter paper, makes driedly, and 4 ℃ of storages are standby.
The concrete steps of sex change are: get 0.6mol/LNaOH and amplified production balanced mix, room temperature 10 minutes.
Hybridization solution is 0.25gFicol1400,0.25gBSA, 0.25gPVP, 0.0625g ox DNA, adds 20XSSC31.25ml, 1mol/L HCl375ml, adds H
2O to 1000ml contains CF probe 50mmol/L.
Hybridization with the concrete steps of colour generation is: in microwell plate, add 100 μ l hybridization solutions, 25 μ l sex change mixed solutions, 37 ℃ 1 hour, PH7.4PBS gives a baby a bath on the third day after its birth time, add enzyme labelled antibody 100 μ l, 37 ℃ 30 minutes, wash plate, add TMB colour generation liquid 100 μ l, 37 ℃ 10 minutes, add 2mol/L H
2SO
4100 μ l are respectively at microplate reader 450nm and 690nm colorimetric.
The present invention has set up the micropore plate hybridization method with fluorescein-anti-luciferin system, is used for telomerase activity and has easy quick, with low cost, good reproducibility susceptibility advantages of higher, is particularly useful for judging good, pernicious ascites pleural fluid.
The invention will be further described below in conjunction with embodiment:
A kind of telomeric repeat amplification-micropore plate hybridization method of measuring telomerase activation comprises the following steps: successively
1. sample process: get tissue 50～100mg and thinly slice, grind evenly with knife blade, fragment of tissue is suspended in cold lysate (10mmol/L Tris-HCl, PH7.5,1mmol/L MgCl
2, 1mmol/L EGTA, 0.1mmol/L PMSF, 5mmol/L beta-mercaptoethanol, 5g/L CHAPS, 100g/L glycerine) in, ice-water bath 30 minutes, centrifugal 30 minutes of 12000rpm, gets supernatant and puts-30 ℃ rapidly by 4 ℃.
2. reverse transcription, amplification: at the bottom of the 0.2ml centrifuge tube, add 5.75 μ mol/L CX primers (5 ' CCCTTACCCTTACCCTTACCCTTA), 2 μ l, add 10 μ l low melt point paraffins, (Paraffin wax, Aldrich), after solidifying, paraffin adds 46 μ l PCR reaction solutions, its final concentration is 10XBuffer5 μ l, 1.5mmol/L MgCl
2, 0.1g/L gelatin, 200 μ mol/L dNTP, 0.23 μ mol/L TS primer, 2 μ Taq enzymes.Wherein the TS primer sequence is 5 ' Biotin AATCCGTCGAGCAGCGTT.Add sample process supernatant liquor 2 μ l during mensuration, 25 ℃ 30 minutes, 94 ℃ 5 minutes, 94 ℃ 30 seconds, 50 ℃ 30 seconds, 72 ℃ 90 seconds, 30 circulations, 72 ℃ 10 minutes.
3. solid phase and hybridization:
A) bag quilt: Streptavidin is dissolved in the PH10.0 carbonate buffer solution, and 10mg/L adds 100 μ l in the every hole of microwell plate, and 4 ℃ are spent the night, and PH7.4PBS gives a baby a bath on the third day after its birth time, is inverted in clean filter paper, makes driedly, and 4 ℃ of storages are standby.
B) sex change: get 0.6mol/LNaOH and amplified production balanced mix, room temperature 10 minutes.
C) hybridization and colour generation: (0.25gFicoll400,0.25gBSA, 0.25gPVP, 0.0625g ox DNA add 20XSSC31.25ml, 1mol/L HCl375ml, add H to add 100 μ l hybridization solutions in microwell plate
2O to 1000ml, contain CF probe 50mmol/L, probe CF is 5 ' FITC-CCCTAACCCTAACCCTAACCCTAAA), 25 μ l sex change mixed solutions, 37 ℃ 1 hour, PH7.4PBS gives a baby a bath on the third day after its birth time, adds enzyme labelled antibody (face with the preceding PH7.4PBS1 of using: 1000 dilute) 100 μ l, 37 ℃ 30 minutes, wash plate three times, add TMB colour generation liquid 100 μ l, 37 ℃ 10 minutes, add 2mol/L H
2SO
4100 μ l are respectively at microplate reader 450nm and 690nm colorimetric.
Claims (1)
1, a kind of telomeric repeat amplification-micropore plate hybridization method of measuring telomerase activation is characterized in that: comprise the following steps: successively
1. sample process, dissolved cell: the tissue slice mill is even, and be suspended in freezing cracking in the cold lysate, supernatant is got in centrifugation;
2. reverse transcription, amplification: with CX primer reverse transcription, with the PCR reaction solution that contains the Ts primer above-mentioned supernatant liquor is increased, the CX primer is 5 ' CCCTTACCCTTACCCTTACCCTTA, and the Ts primer sequence is 5 ' Biotin AATCCGTCGAGCAGCGTT;
3. solid phase and hybridization:
A) bag quilt: Streptavidin is fixed on the microwell plate;
B) sex change: with amplified production NaOH denaturing treatment;
C) hybridization and colour generation: will on microwell plate, mix through the sex change mixed solution of denaturing treatment with the hybridization solution that contains the CF probe, carry out hybridization, add enzyme labelled antibody then, and with colour generation liquid colour generation, use the microplate reader colorimetric, probe CF is 5 ' FITC-CCCTAACCCTAACCCTAACCCTAAA;
Above-mentioned steps 1. in, during sample process, the cold lysate of employing is: 10mmol/L Tris-HCl, PH7.5,1mmol/L.MgCl
2, 1mmol/L EGTA, 0.1mmol/L PMSF, 5mmol/L beta-mercaptoethanol, 5g/L CHAPS, 100g/L glycerine;
Above-mentioned steps 2. in, during amplification, used PCR reaction solution is 10XBuffer 5 μ l, 1.5mmol/L MgCl
2, 0.1g/L gelatin, 200 μ mol/L dNTP, 0.23 μ mol/LTS primer, 2. μ Taq enzyme;
Above-mentioned steps 3. in, the concrete steps of bag quilt are: Streptavidin is dissolved in the PH10.0 carbonate buffer solution, and 10mg/L adds 100 μ l in the every hole of microwell plate, and 4 ℃ are spent the night, and PH7.4PBS washes, and to placing clean filter paper, makes driedly, 4 ℃ of storages are standby;
Above-mentioned steps 3. in, the step of sex change is: get 0.6mol/L NaOH and amplified production balanced mix, room temperature 10 minutes; Hybridization solution is 0.25gFicol1400,0.25gBSA, 025gPVP, 0.0625g ox DNA, adds 20XSSC31.25ml, 1mol/L HCl375ml, adds
H2O to 1000ml contains CF probe 50mmol/L; Hybridization with the concrete steps of colour generation is: in microwell plate, add 100 μ l hybridization solutions, 25 μ l sex change mixed solutions, 37 ℃ 1 hour, PH7.4PBS gives a baby a bath on the third day after its birth time, add enzyme labelled antibody 100 μ l, 37 ℃ 30 minutes, wash plate, add TMB colour generation liquid 100 μ l, 37 ℃ 10 minutes, add 2mol/LH
2SO
4100 μ l are respectively at microplate reader 450nm and 690nm colorimetric.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN98111646A CN1087033C (en) | 1998-12-29 | 1998-12-29 | Telomere repeated cloning-porous plate hybridization process to determine the activity of telomere enzyme |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN98111646A CN1087033C (en) | 1998-12-29 | 1998-12-29 | Telomere repeated cloning-porous plate hybridization process to determine the activity of telomere enzyme |
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| CN1230598A CN1230598A (en) | 1999-10-06 |
| CN1087033C true CN1087033C (en) | 2002-07-03 |
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| CN98111646A Expired - Fee Related CN1087033C (en) | 1998-12-29 | 1998-12-29 | Telomere repeated cloning-porous plate hybridization process to determine the activity of telomere enzyme |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101705277B (en) * | 2009-11-13 | 2012-05-23 | 南开大学 | A class of primers for detecting telomerase activity |
| CN106811524B (en) * | 2017-01-19 | 2020-04-24 | 陕西师范大学 | Telomerase activity colorimetric detection method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10215900A (en) * | 1997-02-04 | 1998-08-18 | Dai Ichi Seiyaku Co Ltd | Measurement of enzyme activity |
| CN1202935A (en) * | 1995-11-28 | 1998-12-23 | 伯伦格曼海姆有限公司 | Method of detecting telomerase activity |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1202935A (en) * | 1995-11-28 | 1998-12-23 | 伯伦格曼海姆有限公司 | Method of detecting telomerase activity |
| JPH10215900A (en) * | 1997-02-04 | 1998-08-18 | Dai Ichi Seiyaku Co Ltd | Measurement of enzyme activity |
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