CN108410962A - Archaeal dna polymerase strand-displacement activity detection method and kit - Google Patents
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Abstract
The invention discloses a kind of archaeal dna polymerase strand-displacement activity detection methods, including：It prepares amplification reaction system and carries out amplification reaction, obtain amplified production；The amplification reaction system includes amplification template strand, amplimer, archaeal dna polymerase to be measured, dNTP and amplification buffer；The end of amplification template strand from 3 ' to 5 ' includes the first single stranded zone, loop-stem structure area and the second single stranded zone successively；From 3 ' to the 5 ' direction of loop-stem structure area includes the first collochore, single-stranded ring region and the second collochore successively, and first collochore and the second collochore complete complementary match；Contain restriction enzyme digestion sites in second single stranded zone；The amplimer is incorporated in the first single stranded zone；Digestion is carried out to amplified production using corresponding restriction enzyme, obtains digestion products；Gel electrophoresis test is carried out to digestion products, determines the strand-displacement activity of archaeal dna polymerase according to testing result.The detection method of the present invention will not cause environmental pollution, and easy to operate, testing cost is low.
Description
Technical field
The present invention relates to molecular biology fields, more specifically to a kind of archaeal dna polymerase strand-displacement activity detection side
Method and kit.
Background technology
Archaeal dna polymerase（DNA polymerase）It is the important function enzyme of cellular replication DNA, chief active is catalytic dna
Synthesis.During DNA replication dna, it usually needs open DNA double chain helical structure in the case where unwindase collaboration participates in, then exist again
Archaeal dna polymerase effect is lower to carry out DNA synthesis.Archaeal dna polymerase of the part with strand-displacement activity is with opening DNA double chain hydrogen bond
Activity can also be carried out at the same time DNA double chain and open and synthesize new DNA double chain so that when being free of unwindase in system.
Existing technical literature discloses the strand-displacement activity that archaeal dna polymerase is detected using single stranded circle M13 phage DNAs,
Particularly specificity amplification primer is utilized to anneal with single stranded circle M13 phage DNAs, adds archaeal dna polymerase and expanded
Reaction, as shown in Figure 1, specificity amplification primer 2 is anchored on single stranded circle M13 phage DNAs 1, in archaeal dna polymerase
Effect is lower to occur synthesis reaction of DNA.When amplified reaction is carried out to the DNA polymerizations for when being held with primer 5 ', not having strand-displacement activity
The synthesis reaction of DNA of enzymatic terminates, and forms the first amplified production 3；Has the archaeal dna polymerase of strand-displacement activity with strand displacement
Mode continues synthetic DNA, the second amplified production 4 of formation.Wherein the first amplified production 3 and the second amplified production 4 are double-strand
Cyclic structure, contains specificity amplification primer sequence in the double-stranded circular structure of the first amplified production 3, the second amplified production it is double
Specificity amplification primer sequence is not contained in link-like structure.DATP in amplification reaction system is marked using isotopic element,
Then amplified production is tested using alkaline electrophoresis, the stripe size by detecting amplified production can determine whether that archaeal dna polymerase is
It is no that there is strand-displacement activity.But the method using isotope labelling detection archaeal dna polymerase strand-displacement activity is seriously polluted, to experiment
Condition requires high, testing cost height.
Therefore, it is necessary to it is a kind of it is pollution-free, archaeal dna polymerase strand-displacement activity low, that testing cost is low is required to experiment condition
Detection method.
Invention content
The purpose of the present invention is to provide a kind of archaeal dna polymerase strand-displacement activity detection method and kits, it is intended to solve
The detection of archaeal dna polymerase strand-displacement activity requires high, problem of high cost to reaction system in the prior art.
In order to realize goal of the invention, the present invention provides a kind of archaeal dna polymerase strand-displacement activity detection method, including it is following
Step：
A, it prepares amplification reaction system and carries out amplification reaction, obtain amplified production；The amplification reaction system includes amplification template
Chain, amplimer, archaeal dna polymerase to be measured, dNTP and amplification buffer；The end of amplification template strand from 3 ' to 5 ' includes successively
First single stranded zone, loop-stem structure area and the second single stranded zone；From 3 ' to the 5 ' direction of loop-stem structure area includes the first pairing successively
Area, single-stranded ring region and the second collochore, first collochore and the second collochore complete complementary pairing；Second single stranded zone
On contain restriction enzyme digestion sites；The amplimer is incorporated in the first single stranded zone；
B, digestion is carried out to amplified production using corresponding restriction enzyme, obtains digestion products；
C, gel electrophoresis test is carried out to digestion products, determines the strand-displacement activity of archaeal dna polymerase according to testing result.
Preferably, the distance between 5 ' end of the restriction enzyme digestion sites and the second single stranded zone is at least
20bp。
Preferably, the described first single-stranded section length is 15 to 20bp, the amplimer and the first single stranded zone complete complementary
Pairing.
Preferably, the length of the single-stranded ring region is 3 to 10bp.
Preferably, second single stranded zone is (dA)a、(dT)a、(dC)a、(dG)aOr (U)a,, 20≤a≤80.
Preferably, further include bovine serum albumin(BSA) in the amplification reaction system.
Preferably, the gel electrophoresis test includes agarose gel electrophoresis test, the test of Page gel electrophoresis.
The present invention also provides a kind of archaeal dna polymerase strand-displacement activity detection kit, including amplification template strand, the expansions
It includes the first single stranded zone, loop-stem structure area and the second single stranded zone to increase the end of template strand from 3 ' to 5 ' successively；The loop-stem structure area from
3 ' to 5 ' directions include the first collochore, single-stranded ring region and the second collochore, first collochore and the second collochore successively
Complete complementary matches；Contain restriction enzyme digestion sites in second single stranded zone.
Preferably, the distance between 5 ' end of the restriction enzyme digestion sites and the second single stranded zone is at least
20bp。
Preferably, the kit further includes amplimer, and the amplimer is incorporated in first single stranded zone.
The present invention is adopted by expanding the design of template strand after carrying out amplification and corresponding digestion processing to amplification template strand
It can detect whether archaeal dna polymerase to be measured has strand-displacement activity with gel electrophoresis test, with traditional archaeal dna polymerase strand displacement
Activity test method is compared, and will not cause environmental pollution, easy to operate, testing cost is low.
Description of the drawings
Fig. 1 is the principle for using single stranded circle M13 phage DNAs detection archaeal dna polymerase strand-displacement activity in the prior art
Figure.
Fig. 2 is the structural schematic diagram that template strand is expanded in first embodiment.
Fig. 3 is that amplimer is anchored on the structural schematic diagram on amplification template strand in first embodiment.
Fig. 4 is the structural schematic diagram of amplified production when archaeal dna polymerase to be measured does not have strand-displacement activity in first embodiment.
Fig. 5 is the structural schematic diagram of amplified production when archaeal dna polymerase to be measured has strand-displacement activity in first embodiment.
Fig. 6 is the structural representation of the first digestion products when archaeal dna polymerase to be measured has strand-displacement activity in first embodiment
Figure.
Fig. 7 is the structural representation of the second digestion products when archaeal dna polymerase to be measured has strand-displacement activity in first embodiment
Figure.
Fig. 8 is the Page gel electrophoresis detection figures of digestion products in each reaction system of third embodiment of the invention.
Specific implementation mode
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.
Please refer to Fig. 2-3, first embodiment of the invention, it is proposed that a kind of archaeal dna polymerase strand-displacement activity detection method, packet
Include following steps：
A, it prepares amplification reaction system and carries out amplification reaction, obtain amplified production 300；The amplification reaction system includes amplification mould
Plate chain 100, amplimer 200, archaeal dna polymerase to be measured, dNTP and amplification buffer；The amplification template strand 100 from 3 ' extremely
5 ' ends include the first single stranded zone 110, loop-stem structure area 120 and the second single stranded zone 130 successively；The loop-stem structure area 120 from 3 '
Include the first collochore 121, single-stranded ring region 122 and the second collochore 123,121 He of the first collochore successively to 5 ' directions
Second collochore, 123 complete complementary pairing；Contain restriction enzyme digestion sites 131 in second single stranded zone 130；It is described
Amplimer 200 is incorporated in the first single stranded zone 110；
B, digestion is carried out to amplified production using corresponding restriction enzyme, obtains digestion products；
C, gel electrophoresis test is carried out to digestion products, determines the strand-displacement activity of archaeal dna polymerase according to testing result.
In the present embodiment, after amplimer 200 is incorporated in the first single stranded zone 110 on amplification template strand 100, to be measured
Under the action of archaeal dna polymerase, synthesis reaction of DNA can be occurred on template strand 100 by expanding.Due to the stem on amplification template strand 100
Containing the duplex structure formed by the first collochore 121 and the second collochore 123 in ring structure area, the item existing for no unwindase
Under part, if archaeal dna polymerase to be measured does not have strand-displacement activity, as shown in figure 4, synthesis reaction of DNA is carried out to the first single stranded zone 110
5 ' end when stop, obtaining third amplified production 300, double-strand cannot be synthesized in the second single stranded zone 130 of third amplified production 300
Structure, therefore digestion cannot be carried out to third amplified production 300 using corresponding restriction enzyme in stepb.If to be measured
Archaeal dna polymerase has strand-displacement activity, when synthesis reaction of DNA is carried out to the 3 ' end in loop-stem structure area, as shown in figure 5, waiting for
It surveys under the action of archaeal dna polymerase, due to the double-strand knot of complementary pairing formation between the first collochore 121 and the second collochore 123
Structure is opened, and synthesis reaction of DNA continues, and forms the 4th amplified production 400 with new duplex structure, and the 4th amplification
Product 400 includes restriction enzyme digestion sites 131；Using the 4th amplified production 400 of corresponding restriction enzyme pair
Digestion is carried out, the first digestion products 410 and the second digestion products 420 all have duplex structure.Therefore, electricity is carried out to digestion products
Swimming is tested, and is the band of third amplified production 300 when only having a kind of double-stranded DNA band in test result, shows that DNA to be measured is poly-
Synthase does not have strand-displacement activity, is the first digestion products 410 and the when occurring two kinds of double-stranded DNA bands in test result
The band of two digestion products 420 shows that archaeal dna polymerase to be measured has strand-displacement activity.
Synthesis reaction of DNA is carried out using the amplification template strand of the present embodiment, is tested using electrophoresis in subsequent process, you can inspection
Survey whether archaeal dna polymerase to be measured has strand-displacement activity, step is simple compared with using isotope detection method, and the present embodiment proposes
The detection method of archaeal dna polymerase strand-displacement activity will not pollute, experiment condition requires low, and testing cost is low.In addition, this
The detection method that embodiment provides, amplification template strand are single stranded nucleic acid molecule, and formation is double between expanding template strand own sequence
Chain structure, compared with forming duplex structure by two single stranded nucleic acid molecules, component is less in system, and annealing is convenient, reaction step
Simply.
In the present embodiment, the type of the digestion with restriction enzyme identification sequence is unlimited, only need to be in the second single stranded zone
Formed restriction enzyme site, the restriction enzyme digestion sites include but not limited to the I endonuclease digestion sites EcoR,
The I endonuclease digestion sites AatI, the I endonuclease digestion sites Acc65, the I endonuclease digestion sites Acc, I restriction endonuclease enzymes of Aci
Enzyme site.
Preferably, in expanding template strand, 5 ' end of the restriction enzyme digestion sites and the second single stranded zone away from
From at least 20bp.The amplification template strand that this programme provides detects archaeal dna polymerase strand-displacement activity to be measured, the digestion production obtained
The length of object can be detected clearly by gel electrophoresis test, to improve detection efficiency and accuracy.
Preferably, the described first single-stranded section length is 15 to 20bp.The amplification template strand that this programme provides, it is simple in structure,
Reduce design and synthesis difficulty.
Preferably, the amplimer is matched with the first single stranded zone complete complementary.The amplimer structure of this programme
Simply, it is easy to anneal between amplification template strand, amplification efficiency is high.
Preferably, the length of first collochore is 10 to 20bp.The amplification template strand that this programme provides is formed
Loop-stem structure area has stable duplex structure, while can also avoid making on amplification template strand since the first collochore is long
Form non-essential secondary structure.
Preferably, the length of the single-stranded ring region is 3 to 10bp.The single-stranded ring region is for connecting the first collochore and the
Two collochores, so that the amplification template strand is the single-stranded structure for including loop-stem structure area, the amplification template strand of this programme exists
In gel electrophoresis test, it is easy to distinguish with other duplex structures.
Preferably, second single stranded zone is (dA)a、(dT)a、(dC)a、(dG)aOr (U)a, the amplification template strand of this programme
Design is simple, additionally it is possible to the second single stranded zone be avoided to form non-essential two level knot with amplification template strand other sequences complementary pairing
Structure.
Preferably, 20≤a≤120.Detection method in this programme is obtained when archaeal dna polymerase to be measured has strand-displacement activity
The second digestion products be easy detected by gel electrophoresis test.
Further, 20≤a≤80.Compared with said program, the amplification template strand of this programme is easily designed, synthesis cost
It is low.
Preferably, further include bovine serum albumin(BSA) in the amplification reaction system, the bovine serum albumin(BSA) can improve
The stability of zymoprotein.
Preferably, the gel electrophoresis test includes agarose gel electrophoresis test, the test of Page gel electrophoresis.This programme
The requirement of detection method experiment condition is low, and testing cost is low, pollution-free.
Second embodiment of the invention proposes a kind of archaeal dna polymerase strand-displacement activity detection kit, the kit packet
Include amplification template strand；The end of amplification template strand from 3 ' to 5 ' includes that the first single stranded zone, loop-stem structure area and second are single-stranded successively
Area；From 3 ' to the 5 ' direction of loop-stem structure area successively include the first collochore, single-stranded ring region and the second collochore, described first
Collochore and the second collochore complete complementary pairing.
Preferably, the kit further includes amplimer, and the amplimer is incorporated in first single stranded zone.
Kit using the present invention is detected the strand-displacement activity of archaeal dna polymerase to be measured, and amplimer is anchored on
In first single stranded zone, under the action of archaeal dna polymerase to be measured, expands and synthesis reaction of DNA occurs on template strand, due to expanding template
Containing the duplex structure formed by the first collochore and the second collochore in the loop-stem structure area of chain, the item existing for no unwindase
Under part, if archaeal dna polymerase to be measured does not have strand-displacement activity, synthesis reaction of DNA stops when carrying out to the 3 ' end in loop-stem structure area,
Duplex structure cannot be synthesized in second single stranded zone, therefore amplification cannot be produced using corresponding restriction enzyme in stepb
Object carries out digestion.If archaeal dna polymerase to be measured has strand-displacement activity, when synthesis reaction of DNA is carried out to the 3 ' ends in loop-stem structure area
When, under the action of archaeal dna polymerase to be measured, due to the double-strand knot of complementary pairing formation between the first collochore and the second collochore
Structure is opened, and synthesis reaction of DNA continues, and aggregates into the amplified production with new duplex structure, and amplified production includes
Restriction enzyme digestion sites carry out digestion using corresponding restriction enzyme to amplified production, and obtaining both of which has
The digestion products of duplex structure.Therefore, by carrying out electrophoresis test to digestion products, only has a kind of double-stranded DNA in test result
When band, show that archaeal dna polymerase to be measured does not have strand-displacement activity, when occurring two double-stranded DNA bands in test result, table
Bright archaeal dna polymerase to be measured has strand-displacement activity.
Archaeal dna polymerase strand-displacement activity to be measured is detected using the kit of the present embodiment, it is easy to operate, experiment condition is wanted
Ask low, it is at low cost, pollution-free.
Preferably, the kit further includes amplification buffer.
Preferably, the kit further includes bovine serum albumin(BSA), and the bovine serum albumin(BSA) can improve zymoprotein
Stability.
The third embodiment of the present invention proposes a kind of archaeal dna polymerase strand-displacement activity detection method.
Establish three amplification reaction systems：Amplification reaction system one is used to detect the strand-displacement activity of Bsu archaeal dna polymerases；
Amplification reaction system two is used to detect the strand-displacement activity of T4 archaeal dna polymerase sums；Archaeal dna polymerase is free of in blank control group.
A, amplification reaction system：2 μ 5 × amplification buffers of L, the amplification template strand that a concentration of 10 μM of 1 μ L, 1 μ L are a concentration of
10 μM of amplimer, the bovine serum albumin(BSA) of 0.2 a concentration of 100mM of μ L, 0.5 μ L archaeal dna polymerases to be measured add water to 10 μ L.
By reaction system mixing, it is incubated 30 minutes in 30 DEG C；After reaction, system temperature is heated to 75 DEG C and is incubated 20 minutes,
So that archaeal dna polymerase to be measured is inactivated, obtains amplified production.Wherein, amplification template chain-ordering such as SEQ ID NO:Shown in 1, amplification is drawn
Object sequence such as SEQ ID NO:Shown in 2；Archaeal dna polymerase to be measured is Bsu archaeal dna polymerases, amplified reaction in amplification reaction system one
Archaeal dna polymerase to be measured is T4 archaeal dna polymerases in system two；Archaeal dna polymerase, remaining component and amplification are free of in blank control group
Reaction system component is identical.
B, 1 μ L EcoR, I restriction endonucleases are added into the amplified production of above-mentioned each reaction system respectively, in 37 DEG C of items after mixing
It is incubated under part 1 hour and carries out endonuclease reaction, obtain digestion products.
C, electrophoresis detection is carried out to digestion products using 12% Page glue, testing result is as shown in Figure 4, wherein swimming lane 0
For molecular size marker；Swimming lane 1 is blank control group, occurs molecular band at 230bp, for the anchoring containing loop-stem structure
The amplification template strand of amplimer；Swimming lane 2 is the digestion products of amplification reaction system one, occurs molecule item at 45bp and 75bp
Band, for the digestion products of the two kinds of different lengths obtained after digestion；Swimming lane 3 is the digestion products of amplification reaction system two,
Occurs molecular band at 230bp, for the amplified production containing loop-stem structure.This illustrates that Bsu archaeal dna polymerases have strand-displacement activity,
T4 archaeal dna polymerases do not have strand-displacement activity.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
All any modification, equivalent and improvement etc., should all be included in the protection scope of the present invention made by within refreshing and principle.
SEQUENCE LISTING
<110>Guangzhou Kang Xin Rui Jiyin health Science and Technology Ltd.
<120>Archaeal dna polymerase strand-displacement activity detection method and kit
<130>
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 120
<212> DNA
<213>Artificial sequence
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tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 60
tttttttttt ggaattcgaa ggtcttttga ccttcgaatt ggcgtcttgg tcggtatggg 120
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
cccataccga ccaagacgcc 20
Claims (10)
1. a kind of archaeal dna polymerase strand-displacement activity detection method, which is characterized in that include the following steps：
A, it prepares amplification reaction system and carries out amplification reaction, obtain amplified production；The amplification reaction system includes amplification template
Chain, amplimer, archaeal dna polymerase to be measured, dNTP and amplification buffer；The end of amplification template strand from 3 ' to 5 ' includes successively
First single stranded zone, loop-stem structure area and the second single stranded zone；From 3 ' to the 5 ' direction of loop-stem structure area includes the first pairing successively
Area, single-stranded ring region and the second collochore, first collochore and the second collochore complete complementary pairing；Second single stranded zone
On contain restriction enzyme digestion sites；The amplimer is incorporated in the first single stranded zone；
B, digestion is carried out to amplified production using corresponding restriction enzyme, obtains digestion products；
C, gel electrophoresis test is carried out to digestion products, determines the strand-displacement activity of archaeal dna polymerase according to testing result.
2. archaeal dna polymerase strand-displacement activity detection method according to claim 1, which is characterized in that described restricted interior
The distance between 5 ' end of enzyme cutting restriction enzyme site and the second single stranded zone is at least 20bp.
3. archaeal dna polymerase strand-displacement activity detection method according to claim 1, which is characterized in that described first is single-stranded
Section length is 15 to 20bp, and the amplimer and the first single stranded zone complete complementary match.
4. archaeal dna polymerase strand-displacement activity detection method according to claim 1, which is characterized in that the single-stranded ring region
Length be 3 to 10bp.
5. archaeal dna polymerase strand-displacement activity detection method according to claim 1, which is characterized in that described second is single-stranded
Area is (dA)a、(dT)a、(dC)a、(dG)aOr (U)a,, 20≤a≤80.
6. archaeal dna polymerase strand-displacement activity detection method according to claim 1, which is characterized in that the amplified reaction
It further include bovine serum albumin(BSA) in system.
7. archaeal dna polymerase strand-displacement activity detection method according to claim 1, which is characterized in that the gel electrophoresis
Test includes agarose gel electrophoresis test, the test of Page gel electrophoresis.
8. a kind of archaeal dna polymerase strand-displacement activity detection kit, which is characterized in that including expanding template strand, the amplification mould
The end of plate chain from 3 ' to 5 ' includes the first single stranded zone, loop-stem structure area and the second single stranded zone successively；The loop-stem structure area from 3 ' extremely
5 ' directions include successively the first collochore, single-stranded ring region and the second collochore, and first collochore and the second collochore are complete
Complementary pairing；Contain restriction enzyme digestion sites in second single stranded zone.
9. archaeal dna polymerase strand-displacement activity detection kit according to claim 8, which is characterized in that described restricted
The distance between endonuclease digestion site and 5 ' end of the second single stranded zone are at least 20bp.
10. archaeal dna polymerase strand-displacement activity detection kit according to claim 8, which is characterized in that the kit
Further include amplimer, the amplimer is incorporated in first single stranded zone.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003052138A2 (en) * | 2001-12-19 | 2003-06-26 | Molecular Light Technology Research Limited | Methods of detecting modification of genetic material |
| EP2298935A1 (en) * | 1999-10-29 | 2011-03-23 | Hologic Inc. | Methods for detection of a target nucleic acid sequence |
| US20120115145A1 (en) * | 2010-11-05 | 2012-05-10 | Oxitec Ltd | Strand displacement activity of modified polymerases and uses thereof |
| CN104357543A (en) * | 2014-09-28 | 2015-02-18 | 南京诺唯赞生物科技有限公司 | Method for determining activity of PAP (poly(A)polymerase) |
| CN105112540A (en) * | 2015-09-17 | 2015-12-02 | 山东大学 | Method for detecting DAN methyltransferase activity based on strand displacement amplification and DNAzyme amplification |
| EP2419538B1 (en) * | 2009-04-16 | 2016-09-14 | Padma Arunachalam | Methods and compositions to detect and differentiate small rnas in rna maturation pathway |
| CN106319032A (en) * | 2015-06-24 | 2017-01-11 | 深圳华大基因研究院 | Method for measuring activity of Phi29 DNA polymerase and rolling circle sequencing method |
-
2017
- 2017-02-09 CN CN201710071711.1A patent/CN108410962A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2298935A1 (en) * | 1999-10-29 | 2011-03-23 | Hologic Inc. | Methods for detection of a target nucleic acid sequence |
| WO2003052138A2 (en) * | 2001-12-19 | 2003-06-26 | Molecular Light Technology Research Limited | Methods of detecting modification of genetic material |
| EP2419538B1 (en) * | 2009-04-16 | 2016-09-14 | Padma Arunachalam | Methods and compositions to detect and differentiate small rnas in rna maturation pathway |
| US20120115145A1 (en) * | 2010-11-05 | 2012-05-10 | Oxitec Ltd | Strand displacement activity of modified polymerases and uses thereof |
| CN104357543A (en) * | 2014-09-28 | 2015-02-18 | 南京诺唯赞生物科技有限公司 | Method for determining activity of PAP (poly(A)polymerase) |
| CN106319032A (en) * | 2015-06-24 | 2017-01-11 | 深圳华大基因研究院 | Method for measuring activity of Phi29 DNA polymerase and rolling circle sequencing method |
| CN105112540A (en) * | 2015-09-17 | 2015-12-02 | 山东大学 | Method for detecting DAN methyltransferase activity based on strand displacement amplification and DNAzyme amplification |
Non-Patent Citations (4)
| Title |
|---|
| CAIFENG DING, XIANGLING LI, YING GE, AND SHUSHENG ZHANG: "Fluorescence Detection of Telomerase Activity in", 《ANALYTICAL CHEMISTRY》 * |
| GIOVANNI MAGA，ULRICH HUBSCHER: "Okazaki fragment processing: Modulation of the", 《PNAS》 * |
| ROBERT M. BROSH JR.: "Special Methods collection on DNA helicases", 《METHODS》 * |
| 栾瑞波，郭秋平: "一种简便快速的聚合酶活性实时检测新方法", 《生命科学研究》 * |
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