CN106011223A - Simple and rapid acid phosphatase activity determination method - Google Patents
Simple and rapid acid phosphatase activity determination method Download PDFInfo
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- CN106011223A CN106011223A CN201610596613.5A CN201610596613A CN106011223A CN 106011223 A CN106011223 A CN 106011223A CN 201610596613 A CN201610596613 A CN 201610596613A CN 106011223 A CN106011223 A CN 106011223A
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- acid phosphatase
- stranded dna
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- 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
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
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- 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
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
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- 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
- C12Q2304/00—Chemical means of detecting microorganisms
- C12Q2304/10—DNA staining
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Abstract
The invention discloses a simple and rapid acid phosphatase activity determination method, which specifically comprises the following steps: 1, obtaining a double-stranded DNA (deoxyribonucleic acid) with a strand of which terminal 3' is phosphorylated; 2, performing reaction in a reaction system in the presence of acid phosphatase by taking the double-stranded DNA as a substrate, and performing reaction on a reactant obtained by reaction and having the double-stranded DNA of which terminal 3' is phosphorylated in the presence of a sufficient amount of exonuclease; 3, after reaction is finished, determining the relative amounts of a single-stranded DNA and the double-stranded DNA in a reaction system, and deducing the activity of the acid phosphatase according to a detection result. Compared with the prior art, the simple and rapid acid phosphatase activity determination method has the advantages of simpler, more convenient and rapider operation steps, capability of implementing high-throughput automatic activity determination and detecting 15 microunits of acid phosphatase, high sensitivity and good using effects.
Description
Technical field
The present invention relates to technical field of biochemistry, the activity of acid phosphatase assay method of a kind of simple and fast.
Background technology
Acid phosphatase is a class hydrolytic enzyme, can get on dephosphorization acid groups at nucleotide, protein equimolecular, make target divide
Sub-dephosphorylation, maximally efficient under sour environment, therefore acid phosphatase of gaining the name.Acid phosphatase be technique for gene engineering and
A class toolenzyme critically important in molecular biology research.Non-specific catalytic dna and RNAr 5` and 3` terminal phosphate monoesters go
Phosphorylation, but non-degradable di-phosphate ester and three ester bonds.In molecular biology research, can be used to remove the phosphoric acid of DNA and RNA
Change end, in order to follow-up clone and labelling.Cloning procedure is possible to prevent after linear DNA molecule dephosphorylation from connecting.This enzyme
Acting on 3` end, no matter it is nose, flush end or female end.Acid phosphatase can also degrade PCR reaction in unnecessary
DNTPs, this PCR primer can be directly used for DNA sequencing and snp analysis.
The measuring method for activity of standard uses 4-NPP (pNPP) method, and its reaction equation is as follows:
PNPP (colourless)+H2O------ACP----> pNP (yellow)+HPO4 2-
By the content of spectrophotometry product pNP, and then calculate the activity of acid phosphatase.This based on colour developing
Method step is more, and the response time requires to control accurately, it is difficult to realizing high flux and automatization, this just brings not for user
Just.
Summary of the invention
It is an object of the invention to provide the activity of acid phosphatase assay method of a kind of simple and fast, to solve the above-mentioned back of the body
The problem proposed in scape technology.
For achieving the above object, the present invention provides following technical scheme:
The activity of acid phosphatase assay method of a kind of simple and fast, specifically comprises the following steps that
Step one, obtains the double-stranded DNA of the 3` end phosphorylation of a chain；
Step 2, reacts with this double-stranded DNA for substrate in the presence of acid phosphatase in reaction system, tool reaction obtained
The reactant of the dephosphorylized double-stranded DNA of 3` end reacts in the presence of enough exonucleases；
Step 3, after having reacted, measures single stranded DNA and the relative quantity of double-stranded DNA in reaction system, and is pushed away by this testing result
Derive the level of activity of acid phosphatase.
As the further scheme of the present invention: the relative quantity of double-stranded DNA or single stranded DNA is with fluorochrome label, passes through
Fluorescence method records, and the relative quantity fluorescence abundance of double-stranded DNA or single stranded DNA represents.
As the further scheme of the present invention: fluorescent dye uses single double-stranded DNA diversity fluorescent dye.
As the further scheme of the present invention: dyestuff uses goldview dyestuff.
As the further scheme of the present invention: double-stranded DNA template is with salmon sperm dna for substrate at forward primer and reversely
Expanded the linear dsdna obtained in the presence of primer by PCR, both 3` ends of primer wherein used have phosphorylation
Group.
As the further scheme of the present invention: forward primer uses 5`-aagtaccgtaggccagtcatgca-3`, reversely
Primer uses 5`-gtacgccagtcagctagcgatagc-3`.
Compared with prior art, the invention has the beneficial effects as follows: the invention discloses the acid phosphatase of a kind of simple and fast
Activity determination method, the method is simpler than prior art operation step, convenient and quick, can realize high flux and automatization
Determination of activity, and highly sensitive, can detect that the acid phosphatase of 15mU, using effect is good.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the technical scheme of this patent is described in more detail.
Embodiment 1
The activity of acid phosphatase assay method of a kind of simple and fast, specifically comprises the following steps that
Step one, employing salmon sperm dna is substrate, and employing 5`-aagtaccgtaggccagtcatgca-3` is forward primer, adopts
It is that reverse primer carries out pcr amplification reaction, PCR reactive component used and body with 5`-gtacgccagtcagctagcgatagc-3`
Long-pending as shown in table 1.
The component of table 1 PCR reaction and volume
| Component | Volume |
| 10xTaq buffer | 5 μ l |
| dNTP(10mM) | 1 μ l |
| Primers F (10 μMs) | 2μl |
| Primer R (10 μMs) | 2μl |
| Template | 10ng |
| Taq enzyme | 1U |
| dd H2O | To 50 μ l |
The course of reaction of PCR is shown in Table 2
The course of reaction of table 2 PCR
Step 2, reacts at 37 ° of C in the presence of acid phosphatase with this double-stranded DNA for substrate and is maintained at 4 ° of C half an hour,
The reactant with the dephosphorylized double-stranded DNA of 3` end reaction obtained is anti-at 37 ° of C in the presence of enough exonucleases
4 ° of C should be maintained at half an hour.
The component of acid phosphatase and volume are shown in Table 3, and component and the volume of exonuclease are shown in Table 4.
The component of table 3 acid phosphatase and volume
| Component | Volume |
| 10x ACP buffer | 2μl |
| Phosphorylation PCR primer | 100ng |
| ACP | 0-1024mU |
| dd H2O | To 20 μ l |
The component of table 4 exonuclease and volume
| Component | Volume |
| 10x exonuclease buffer | 5μl |
| Acid phosphatase reaction product | 20μl |
| Exonuclease | 10U |
| dd H2O | To 50 μ l |
Step 3, after having reacted, adds 1 μ l goldview dyestuff in product；Detect with fluorescence microplate reader, excite
Wavelength is 430nm, and a length of 470nm of transmitted wave, testing result is shown in Table 5
Table 5 testing result
| Acid phosphatase enzyme amount (mU) | Fluorescent value |
| 1024 | 1485 |
| 512 | 1486 |
| 256 | 1511 |
| 128 | 1514 |
| 64 | 1406 |
| 32 | 1052 |
| 16 | 662 |
| 8 | 448 |
| 4 | 348 |
| 2 | 301 |
| 1 | 278 |
The double-stranded DNA of phosphorylation can be converted into unphosphorylated double-stranded DNA by acid phosphatase. and exonuclease can be special
Property ground phosphorylation double-stranded DNA is degraded to single stranded DNA, and non-degradable unphosphorylated double-stranded DNA.Goldview dyestuff combines
Double-stranded DNA produces green fluorescence under burst of ultraviolel, and single stranded DNA combination dye manifests red fluorescence.The activity of acid phosphatase
Being directly proportional with the amount of dephosphorylation double-stranded DNA in certain limit, therefore its activity just can use goldview green fluorescence intensity
It is measured.
The result of table 5 shows, the double-stranded DNA of phosphorylation can be removed phosphoric acid by acid phosphatase, and goes phosphoric acid degree to present agent
Amount dependency, therefore can characterize activity of acid phosphatase by fluorescence intensity.
Above the better embodiment of this patent is explained in detail, but this patent is not limited to above-mentioned embodiment party
Formula, in the ken that one skilled in the relevant art is possessed, it is also possible on the premise of without departing from this patent objective
Make a variety of changes.
Claims (6)
1. the activity of acid phosphatase assay method of a simple and fast, it is characterised in that specifically comprise the following steps that
Step one, obtains the double-stranded DNA of the 3` end phosphorylation of a chain；
Step 2, reacts with this double-stranded DNA for substrate in the presence of acid phosphatase in reaction system, tool reaction obtained
The reactant of the dephosphorylized double-stranded DNA of 3` end reacts in the presence of enough exonucleases；
Step 3, after having reacted, measures single stranded DNA and the relative quantity of double-stranded DNA in reaction system, and is pushed away by this testing result
Derive the level of activity of acid phosphatase.
The activity of acid phosphatase assay method of simple and fast the most according to claim 1, it is characterised in that described double-strand
The relative quantity of DNA or single stranded DNA is with fluorochrome label, is recorded by fluorescence method, and double-stranded DNA or single stranded DNA
Relative quantity fluorescence abundance represents.
The activity of acid phosphatase assay method of simple and fast the most according to claim 2, it is characterised in that described fluorescence
Dyestuff uses single double-stranded DNA diversity fluorescent dye.
The activity of acid phosphatase assay method of simple and fast the most according to claim 3, it is characterised in that described dyestuff
Use goldview dyestuff.
The activity of acid phosphatase assay method of simple and fast the most according to claim 1, it is characterised in that described double-strand
DNA profiling is to be expanded, with salmon sperm dna, the Linear Double obtained in the presence of forward primer and reverse primer by PCR for substrate
Chain DNA, both 3` ends of primer wherein used have phosphate group.
The activity of acid phosphatase assay method of simple and fast the most according to claim 5, it is characterised in that described forward
Primer uses 5`-aagtaccgtaggccagtcatgca-3`, and reverse primer uses 5`-
gtacgccagtcagctagcgatagc-3`。
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| CN201610596613.5A CN106011223A (en) | 2016-07-27 | 2016-07-27 | Simple and rapid acid phosphatase activity determination method |
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| CN201610596613.5A CN106011223A (en) | 2016-07-27 | 2016-07-27 | Simple and rapid acid phosphatase activity determination method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108896520A (en) * | 2018-06-13 | 2018-11-27 | 南昌大学 | The ratio fluorescent method of principle is inhibited to detect As (V) based on activity of acid phosphatase |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102459650A (en) * | 2009-06-08 | 2012-05-16 | 韩国生命工学研究院 | Method for screening and quantifying various enzyme activities using a genetic enzyme screening system |
| CN103380213A (en) * | 2011-02-23 | 2013-10-30 | 东洋纺株式会社 | Alkaline phosphatase |
| CN104263831A (en) * | 2014-09-28 | 2015-01-07 | 南京诺唯赞生物科技有限公司 | Method for simply, conveniently and quickly determining activity of alkaline phosphatase |
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- 2016-07-27 CN CN201610596613.5A patent/CN106011223A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102459650A (en) * | 2009-06-08 | 2012-05-16 | 韩国生命工学研究院 | Method for screening and quantifying various enzyme activities using a genetic enzyme screening system |
| CN103380213A (en) * | 2011-02-23 | 2013-10-30 | 东洋纺株式会社 | Alkaline phosphatase |
| CN104263831A (en) * | 2014-09-28 | 2015-01-07 | 南京诺唯赞生物科技有限公司 | Method for simply, conveniently and quickly determining activity of alkaline phosphatase |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108896520A (en) * | 2018-06-13 | 2018-11-27 | 南昌大学 | The ratio fluorescent method of principle is inhibited to detect As (V) based on activity of acid phosphatase |
| CN108896520B (en) * | 2018-06-13 | 2021-01-01 | 南昌大学 | Ratiometric fluorescence detection of As (V) based on the principle of inhibition of acid phosphatase Activity |
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