CN107641635A - Recombinase polymeric enzymatic amplification kit, amplification method and amplifing reagent - Google Patents
Recombinase polymeric enzymatic amplification kit, amplification method and amplifing reagent Download PDFInfo
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Abstract
The present invention relates to a kind of recombinase polymeric enzymatic amplification kit, including：Ecoli RecA albumen；λ phage Orf albumen；Ecoli SSB albumen and archaeal dna polymerase；Present invention also offers a kind of recombinase polymeric enzymatic amplification method and amplifing reagent.The recombinase polymeric enzymatic amplification kit of the present invention, the amplification system to be acted synergistically using Ecoli RecA albumen, Ecoli SSB albumen and λ phage Orf albumen, extend the application of recombinase polymeric enzymatic amplification；Compared with traditional PCR technique, recombinase polymeric enzymatic amplification of the invention reaction can be carried out under constant temperature, circulated instrument without PCR instrument equitemperature, do not limited by lab space；The amplified reaction of the present invention can be completed at 10 to 60 minutes, far faster than round pcr or other isothermal amplification techniques.
Description
Technical field
The present invention relates to a kind of isothermal amplification technology, more specifically to a kind of recombinase polymeric enzymatic amplification（RPA）Examination
Agent box, amplification method and amplifing reagent.
Background technology
Traditional external DNA cloning mainly uses PCR（PCR）Method, extensive use at present
In fields such as biomedicines.PCR is made up of denaturation-fundamental reaction step of annealing-extension three：（1）The denaturation of template DNA,
After template DNA is heated to 90-95 DEG C of certain time, makes template DNA double-strand or the double-stranded DNA to be formed dissociation is expanded through PCR, make
Turn into it is single-stranded, so that it is combined with primer, be lower whorl react prepare；（2）The annealing of template DNA and primer, template DNA warp
Heat denatured is into after single-stranded, and temperature is down to 55-60 DEG C, and the primer complementary series pairing single-stranded with template DNA combines；（3）Primer
Extension, DNA profiling-primer conjugate is in the presence of archaeal dna polymerase, former as reaction using dNTP under the conditions of 70-75 DEG C
Material, target sequence is template, by base pairing and semi-conservative replication principle, synthesizes new half guarantor complementary with template DNA chain
Stay duplication chain, the repetitive cycling denaturation-process of annealing-extension three, so that it may more " semi-conservative replication chains " is obtained, and also it is this
New chain can turn into the template of circulation next time again.Often completing a circulation needs 2-4 minutes, and 2-3 hours will target gene be expanded with regard to energy
Millions of times of amplification amplification.The advantages of round pcr, is it is clear that but because the accurate temperature cycles instrument of its needs could be completed
Amplification procedure, higher to the degree of dependence of instrument, cost height, reaction time consumption length, these limitations make its application limit mostly in addition
It is formed in the good laboratory of condition, it is difficult to be widely used in Site Detection.
Therefore, it is necessary to a kind of amplification method provided independent of PCR instrument equitemperature circulation instrument.
The content of the invention
It is an object of the invention to provide a kind of recombinase polymeric enzymatic amplification kit, amplification method and amplifing reagent,
The efficient amplification to specific nucleic acid sequence is realized under constant temperature so that the process of nucleic acid amplification can be directly without PCR instrument
Completed under conditions of equitemperature circulation instrument.
The invention provides a kind of recombinase polymeric enzymatic amplification kit, the kit includes following components：Ecoli
RecA albumen, λ phage Orf albumen, Ecoli SSB albumen and archaeal dna polymerase.
Preferably, the kit also includes amplified reaction buffer solution, and the amplified reaction buffer solution buffers including Tris
Liquid, the amplified reaction buffer solution also include one kind or more in polyethylene glycol, dithiothreitol (DTT), phosphocreatine and creatine kinase
Kind.
Preferably, the kit also includes magnesium ion preparation.
Present invention also offers a kind of recombinase polymeric enzymatic amplification method, the described method comprises the following steps：
A, reaction system is configured, the reaction system includes：Template nucleic acid molecule, primer sets, Ecoli RecA albumen, λ
Phage Orf albumen, Ecoli SSB albumen, archaeal dna polymerase, dNTP, ATP and amplified reaction buffer solution, the amplified reaction delay
Fliud flushing includes Tris buffer solutions；
B, magnesium ion preparation is added into the reaction system；
C, amplified reaction is carried out under the conditions of 20 to 65 DEG C.
Preferably, the amplified reaction buffer solution also includes polyethylene glycol, dithiothreitol (DTT), phosphocreatine and creatine kinase
In one or more.
Preferably, the primer sets include pair of primers, and the length of the primer is 30 to 65bp.
Preferably, the Ecoli RecA protein concentrations are 20 to 250ng/ μ L；The λ phage Orf protein concentrations are
20 to 80ng/ μ L；The concentration of the Ecoli SSB albumen is 200 to 1000ng/ μ L.
Present invention also offers a kind of recombinase polymeric enzymatic amplification reagent, the reagent includes：Ecoli RecA albumen, λ
Phage Orf albumen, Ecoli SSB albumen and archaeal dna polymerase.
Preferably, the amplifing reagent also includes amplified reaction buffer solution, and the amplified reaction buffer solution delays including Tris
Fliud flushing；The amplified reaction buffer solution also include polyethylene glycol, dithiothreitol (DTT), phosphocreatine and creatine kinase in one kind or
It is a variety of.
Preferably, the Ecoli RecA protein concentrations are 20 to 250ng/ μ L；The Ecoli SSB protein concentrations are
200 to 1000ng/ μ L；The λ phage Orf protein concentrations are 20 to 80ng/ μ L.
The recombinase polymeric enzymatic amplification kit of the present invention, using Ecoli RecA albumen, Ecoli SSB albumen and λ
The amplification system of phage Orf albumen synergy, extend the application of recombinase polymeric enzymatic amplification；With normal PCR skill
Art is compared, and recombinase polymeric enzymatic amplification of the invention reaction can be carried out under constant temperature, without PCR instrument equitemperature circulating instrument
Device, do not limited by lab space；The present invention amplified reaction can be completed at 10 to 60 minutes, far faster than round pcr or other
Isothermal amplification technique.
Brief description of the drawings
Fig. 1 is the agarose gel electrophoresis inspection of recombinase polymeric enzymatic amplification and control PCR amplifications in the first specific embodiment
Mapping.
Fig. 2 is the capillary electrophoresis detection figure of recombinase polymeric enzymatic amplification in the second specific embodiment.
Fig. 3 is the agarose gel electrophoresis detection figure of recombinase polymeric enzymatic amplification in the 3rd specific embodiment.
Fig. 4 is the agarose gel electrophoresis detection figure of recombinase polymeric enzymatic amplification in the 4th specific embodiment.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, it is right below in conjunction with drawings and Examples
The present invention is further elaborated.
The present invention proposes first embodiment, a kind of recombinase polymeric enzymatic amplification kit, including following components：Ecoli
RecA albumen, λ phage Orf albumen, Ecoli SSB albumen and archaeal dna polymerase.
It should be noted that the recombinase polymeric enzymatic amplification of the present invention is the constant temperature based on biological In vivo homologous recombination principle
Amplification technique：Under preference temperature, recombinase Ecoli RecA albumen and λ phage Orf albumen collective effects, with primer knot
The complex for forming enzyme and primer is closed, complex navigates on the homologous target sequence of template nucleic acid molecule and forms strand displacement；It is single-stranded
Associated proteins are combined with the nucleotide chain being replaced, and prevent from further replacing；Start DNA in the presence of archaeal dna polymerase to close
Into being expanded to the target area on template nucleic acid molecule.
In recombinase polymeric enzymatic amplification kit of the present invention, Ecoli RecA albumen and Ecoli the SSB albumen sources
In Escherichia coli, λ phage Orf albumen sources are in bacteriophage lambda.The λ phage Orf albumen of the present invention and Ecoli RecA eggs
It is white different with the Species origin of Ecoli SSB albumen, but collective effect and recombinase polymerase can be realized in same system
Amplification, so as to greatly expand the application of recombinase polymeric enzymatic amplification technology.It should be noted that herein referred " comes
Source " only refers to its species, rather than the preparation source of finger protein.
In an embodiment of the present invention, the archaeal dna polymerase is preferably to have strand-displacement activity under the conditions of 20 to 65 DEG C
Archaeal dna polymerase.
In an embodiment of the present invention, the archaeal dna polymerase be preferably Sau archaeal dna polymerases, Bsu archaeal dna polymerases or
Bst archaeal dna polymerases.
In an embodiment of the present invention, the kit also includes dNTP, the dNTP be dTTP, dATP, dGTP and
DCTP equimolar compares mixed liquor；In amplification procedure, the dNTP is used as the substrate of DNA synthesis.
In an embodiment of the present invention, the kit also includes ATP；In amplification procedure, the ATP is template nucleic acid
Strand displacement process between molecule and complex provides energy.
In an embodiment of the present invention, the kit also includes amplified reaction buffer solution, the amplified reaction buffer solution
Including Tris buffer solutions；The Tris buffer solutions are used for the pH value of maintenance reaction system.
In an embodiment of the present invention, the amplified reaction buffer solution also includes polyethylene glycol；Added into reaction system
After polyethylene glycol, polyethylene glycol occupies the significant portion of volume in reaction system, while repel other as polymer substance
Component enters this space, so that other components more fully contact in amplification system, and then improves amplification efficiency.
In an embodiment of the present invention, the mean molecule quantity of the polyethylene glycol is 3000 to 20000；The molecular weight ranges
Interior polyethylene glycol, the repulsive interaction to other components in reaction system become apparent from.
In an embodiment of the present invention, the amplified reaction buffer solution also includes dithiothreitol (DTT)；The dithiothreitol (DTT)
Ecoli RecA albumen and archaeal dna polymerase can be effectively prevented to be oxidized.
In an embodiment of the present invention, the amplified reaction buffer solution also includes phosphocreatine and creatine kinase；The phosphorus
Creatine acid and creatine kinase are used to be catalyzed ATP regeneration, and this process can be repeated constantly, thereby may be ensured that efficient amplification.
It should be noted that Tris buffer solutions in kit of the present invention, polyethylene glycol, dithiothreitol (DTT), phosphocreatine and
Creatine kinase can also separate store, kit using when mixed again.
In an embodiment of the present invention, the kit also includes magnesium ion preparation, and the magnesium ion preparation is used to activate
Archaeal dna polymerase, so as to shorten the reaction time.
It should be noted that magnesium ion preparation separately stores with other components in kit, will during kit use
After other components are sufficiently mixed in kit, then magnesium ion preparation is added thereto, can avoid the occurrence of non-specific amplification.
In an embodiment of the present invention, the magnesium ion preparation is magnesium acetate or magnesium chloride.
The present invention proposes second embodiment, a kind of recombinase polymeric enzymatic amplification method, comprises the following steps：
A, reaction system is configured, the reaction system includes：Template nucleic acid molecule, primer sets, Ecoli RecA albumen, λ
Phage Orf albumen, Ecoli SSB albumen, archaeal dna polymerase, dNTP, ATP and amplified reaction buffer solution, the amplified reaction delay
Fliud flushing includes Tris buffer solutions；
B, magnesium ion preparation is added into the reaction system.
C, amplified reaction is carried out under the conditions of 20 to 65 DEG C.
The template nucleic acid molecule can be double chain DNA molecule, can also be the cDNA molecules synthesized by RNA reverse transcriptions.
Tris buffer solutions of the present invention are used for the pH value of maintenance reaction system, the pH scopes of the present embodiment 7.0 to
8.0。
In an embodiment of the present invention, the amplified reaction buffer solution also includes polyethylene glycol；Added into reaction system
After polyethylene glycol, polyethylene glycol occupies the significant portion of volume in reaction system, while repel other as polymer substance
Component enters this space, so that other components more fully contact in amplification system, and then improves amplification efficiency.
In an embodiment of the present invention, the mean molecule quantity of the polyethylene glycol is 3000 to 20000；The molecular weight ranges
Interior polyethylene glycol, the repulsive interaction to other components in reaction system become apparent from.
In an embodiment of the present invention, the amplified reaction buffer solution also includes dithiothreitol (DTT)；The dithiothreitol (DTT)
The Ecoli RecA albumen in mixed liquor and archaeal dna polymerase can be effectively prevented to be oxidized.
In an embodiment of the present invention, the amplified reaction buffer solution also includes phosphocreatine and creatine kinase；The phosphorus
Creatine acid and creatine kinase are used to be catalyzed ATP regeneration, and this process can be repeated constantly, thereby may be ensured that the height of nucleic acid molecules
Effect amplification.
The magnesium ion preparation is used to activate archaeal dna polymerase, and so as to shorten the reaction time, prepared by the reaction system completes
Add magnesium ion preparation thereto again afterwards, non-specific amplification can be avoided the occurrence of.In an embodiment of the present invention, the magnesium from
Sub- preparation is magnesium acetate or magnesium chloride.
In an embodiment of the present invention, the template nucleic acid molecule is double chain DNA molecule.
In an embodiment of the present invention, template nucleic acid molecule is used as using human whole blood DNA.
In an embodiment of the present invention, the primer sets include pair of primers；The length 30 of the primer is our to 65bp
Case can ensure the specificity identified during strand displacement.
In an embodiment of the present invention, respectively with actinRPA-0_F（SEQ ID NO:1）、actinRPA-0_R（SEQ
ID NO:2）；actinRPA-1_F（SEQ ID NO:3）、actinRPA-1_R（SEQ ID NO:4）；actinRPA-2_F（SEQ
ID NO:5）、actinRPA-2_R（SEQ ID NO:6）；Or actinRPA-3_F（SEQ ID NO:7）、actinRPA-3_R
（SEQ ID NO:8）As primer sets, recombinase polymeric enzymatic amplification is carried out to the template nucleic acid molecule.
In an embodiment of the present invention, the archaeal dna polymerase is that the DNA for having strand-displacement activity under the conditions of 20 to 65 DEG C gathers
Synthase, the archaeal dna polymerase of this programme are applied to during recombinase polymeric enzymatic amplification so that amplified reaction at normal temperatures can be fast
Speed is carried out.
In an embodiment of the present invention, the archaeal dna polymerase is Sau archaeal dna polymerases, Bsu archaeal dna polymerases or Bst
Archaeal dna polymerase.
In an embodiment of the present invention, the concentration of the template nucleic acid molecule is 10pg/ μ L to 100ng/ μ L；In this programme
Concentration range in, the amplified reaction of template nucleic acid molecule can be carried out efficiently.
In an embodiment of the present invention, the concentration of every kind of primer is 0.1 to 1.0 μM in the primer sets；In this programme
In concentration range, the amplified reaction of template nucleic acid molecule can be carried out efficiently.
In an embodiment of the present invention, the Ecoli RecA protein concentrations are 20 to 250ng/ μ L；The λ phage
Orf protein concentrations are 20 to 80ng/ μ L；The Ecoli SSB protein concentrations are 200 to 1000ng/ μ L；In the concentration of this programme
In the range of, Ecoli RecA albumen, Ecoli SSB albumen and λ phage Orf albumen can preferably play synergy, from
And make it that amplification efficiency is higher.
In an embodiment of the present invention, the archaeal dna polymerase concentration is 5 to 120ng/ μ L；In the concentration range of this programme
Interior, the polymerisation of template nucleic acid molecule can be carried out efficiently.
In an embodiment of the present invention, the equimolar that the dNTP is dTTP, dATP, dGTP and dCTP compares mixed liquor；Institute
The concentration for stating dNTP is preferably 0.1 to 3mM；The dNTP is used as the substrate of polymerisation synthetic DNA chain.
In an embodiment of the present invention, the concentration of the ATP is 1 to 10mM；In amplification procedure, the ATP is template
Strand displacement process between nucleic acid molecules and complex provides energy, in the concentration range of this programme, may advantageously facilitate amplification
The progress of reaction.
In an embodiment of the present invention, the Tris buffer concentrations are 20 to 500mM；The pH scopes of this programme are 7.0
To 8.0；This programme is advantageous to the progress of amplified reaction.
In an embodiment of the present invention, the concentration of the polyethylene glycol is 2.5 to 10mM；In the concentration range of this programme
It is interior, be advantageous to the progress of amplified reaction.
In an embodiment of the present invention, the phosphocreatine concentration is 20 to 100mM；The creatine kinase concentration be 10 to
1000ng/μL；In the concentration range of this programme, ATP regeneration may advantageously facilitate.
In an embodiment of the present invention, the concentration of the dithiothreitol (DTT) is 2.5 to 10mM；In the concentration range of this programme
It is interior, be advantageous to avoid Ecoli RecA albumen and archaeal dna polymerase from being oxidized.
Preferably, concentration of the magnesium ion preparation in reaction mixture is 5 to 30mM；In the concentration range of this programme
It is interior, be advantageous to activate archaeal dna polymerase effect.
The present invention proposes 3rd embodiment, a kind of recombinase polymeric enzymatic amplification reagent, including following components：Ecoli
RecA albumen；λ phage Orf albumen；Ecoli SSB albumen；Archaeal dna polymerase.
In an embodiment of the present invention, the reagent also includes dNTP；The dNTP is dTTP, dATP, dGTP and dCTP
Equimolar than mixed liquor, the dNTP is used as the substrate of DNA synthesis.In an embodiment of the present invention, the reagent also wraps
Include ATP；Strand displacement processes of the ATP between template nucleic acid molecule and complex provides energy.
In an embodiment of the present invention, the reagent also includes amplified reaction buffer solution, the amplified reaction buffer solution bag
Include Tris buffer solutions；The Tris buffer solutions are used for the pH value of maintenance reaction system, the pH scopes of the present embodiment 7.0 to
8.0。
In an embodiment of the present invention, the amplified reaction buffer solution also includes phosphocreatine and creatine kinase；The phosphorus
Creatine acid and creatine kinase are used to be catalyzed ATP regeneration, and this process can be repeated constantly, thereby may be ensured that the height of nucleic acid molecules
Effect amplification.
In an embodiment of the present invention, the amplified reaction buffer solution also includes polyethylene glycol；Added into reaction system
After polyethylene glycol, polyethylene glycol occupies the significant portion of volume in reaction system, while repel other as polymer substance
Component enters this space, so that other components more fully contact in amplification system, and then improves amplification efficiency.
In an embodiment of the present invention, the mean molecule quantity of the polyethylene glycol is 3000 to 20000；The molecular weight ranges
Interior polyethylene glycol, the repulsive interaction to other components in reaction system become apparent from.
In an embodiment of the present invention, the amplified reaction buffer solution also includes dithiothreitol (DTT)；The dithiothreitol (DTT)
Ecoli RecA and archaeal dna polymerase can be effectively prevented to be oxidized.
In an embodiment of the present invention, the reagent also includes primer sets, and the primer sets include pair of primers；It is described to draw
The length of thing is 30 to 65bp, and this programme can ensure the specificity identified during strand displacement.
In an embodiment of the present invention, the Ecoli RecA protein concentrations are 20 to 250ng/ μ L；The λ phage
Orf protein concentrations are 20 to 80ng/ μ L；The Ecoli SSB protein concentrations are 200 to 1000ng/ μ L.
In an embodiment of the present invention, the archaeal dna polymerase concentration is 5 to 120ng/ μ L.
In an embodiment of the present invention, the concentration of every kind of primer is 0.1 to 1.0 μM in the primer sets.
In an embodiment of the present invention, the concentration range of the dNTP is 0.1 to 3mM.
In an embodiment of the present invention, the concentration of the ATP is 1 to 10mM.
In an embodiment of the present invention, the Tris buffer concentrations are 20 to 500mM.
In an embodiment of the present invention, the concentration of the polyethylene glycol is 2.5 to 10mM.
In an embodiment of the present invention, the phosphocreatine concentration is 20 to 100mM；The creatine kinase concentration be 10 to
1000ng/μL。
In an embodiment of the present invention, the concentration of the dithiothreitol (DTT) is 2.5 to 10mM.
The present invention is described in further detail below by way of specific embodiment.
First specific embodiment of the invention, there is provided a kind of recombinase polymeric enzymatic amplification method, the present embodiment are anti-in configuration
Answering also includes the step A0 of extraction nucleic acid molecules before system, amplification step is as follows：
A0, extraction nucleic acid molecules, the present embodiment extract human whole blood DNA molecular by the method for poba gene group extracts kit；
A, reaction system is configured：Template nucleic acid molecule, primer sets, Ecoli RecA albumen, λ phage Orf albumen, Ecoli
SSB albumen, archaeal dna polymerase, dNTP, ATP and Tris buffer solution.Specifically, configured in the present embodiment following concentration 2 × it is pre-
Mixed liquid：200ng/ μ L Ecoli RecA albumen, 50ng/ μ L λ phage Orf albumen, 508ng/ μ L Ecoli SSB albumen,
160ng/ μ L Bsu albumen, 5mM ATP, 4mM dNTP, 100mM Tris buffer solutions（pH 7.5）, 10mM dithiothreitol (DTT)s,
100mM phosphocreatines, 200ng/ μ L creatine kinases, 10.92% (w/v) polyethylene glycol.
It is separately added into 200 μ L centrifuge tubes：1 μ L concentration is 9 μ g/ μ L human whole blood DNA；Above-mentioned primer sets：5 μ L are dense
Spend the sense primer actinRPA-4_F for 10 μM（SEQ ID NO:9）, 5 μ L concentration be 10 μM of anti-sense primer actinRPA-
4_R（SEQ ID NO:10）；25 μ 2 × premixed liquids of L；9 μ L deionized waters；Mix and centrifuge.
B, magnesium ion preparation is added into above-mentioned reaction system：Specifically, in the present embodiment into step A reaction system
The magnesium acetate solution that 5 μ L concentration are 140mM is added, fully mixes and centrifuges.
C, amplified reaction is carried out under the conditions of 20 to 65 DEG C：Centrifuge tube is placed in 37 DEG C of constant-temperature metal bath by the present embodiment
It is incubated 4min；Reaction tube is taken out, is fully turned upside down 4-5 times, is mixed and centrifuge, then carries out the incubation of similarity condition, is incubated
20min。
After reaction terminates, centrifuge tube is taken out, sampling is gone forward side by side row agarose gel electrophoresis detection, 5-8 in testing result such as Fig. 1
Shown in swimming lane.
As control experiment, the present invention has also carried out PCR amplifications to human whole blood DNA molecular, and amplification step is as follows：
A ', the method extraction human whole blood DNA molecular by poba gene group extracts kit；
B ', it is separately added into 200 μ L centrifuge tubes：1 μ L concentration is 9 μ g/ μ L human whole blood DNA；5 μ L concentration are 10 μM upper
Swim primer actinRPA-4_F（SEQ ID NO:9）；5 μ L concentration are 10 μM of anti-sense primer actinRPA-4_R（SEQ ID
NO:10）；With 9 μ L deionized waters；25 μ L 2 × Taq mix, mix and centrifuge；
C ', centrifuge tube is placed in PCR instrument, response procedures are set：Continue 1 minute under the conditions of 95 DEG C, continue 30 under the conditions of 60 DEG C
Second, continue 30 seconds under the conditions of 72 DEG C, altogether 30 circulations, start PCR reactions；
After reaction terminates, reaction tube is taken out, sampling is gone forward side by side row agarose gel electrophoresis detection, 1-4 swimming lanes in testing result such as Fig. 1
It is shown.
Swimming lane 1-3 is PCR amplification situations in Fig. 1, and swimming lane 4 is the negative control of PCR amplifications, and swimming lane 5 is restructuring enzymatic polymerization
The negative control of enzymatic amplification, swimming lane 6-8 are recombinase polymeric enzymatic amplification situation, and 0 is molecular size label.From experimental result
It is more or less the same as can be seen that the recombinase polymeric enzymatic amplification of the present embodiment expands obtained yield with PCR.This illustrates this implementation
The recombinase polymeric enzymatic amplification method of example can be expanded effectively to template nucleic acid molecule；But compared with Standard PCR technology, behaviour
Make simplicity, substantially reduce proliferation time, it is not necessary to which accurate temperature Instrument can be achieved.
Second specific embodiment of the invention, the difference with the first specific embodiment are that the present embodiment is drawn with upstream respectively
Thing actinRPA-0_F（SEQ ID NO:1）With anti-sense primer actinRPA-0_R（SEQ ID NO:2）；Sense primer
actinRPA-1_F（SEQ ID NO:3）With anti-sense primer actinRPA-1_R（SEQ ID NO:4）；Sense primer
actinRPA-2_F（SEQ ID NO:5）With anti-sense primer actinRPA-2_R（SEQ ID NO:6）；Sense primer
actinRPA-3_F（SEQ ID NO:7）With anti-sense primer actinRPA-3_R（SEQ ID NO:8）As primer sets, four are formed
Individual reaction system.Recombinase polymeric enzymatic amplification is carried out to human whole blood DNA.Amplified reaction samples after terminating and carries out the second capillary
Electrophoresis tube detects, as a result as shown in Figure 2.
Swimming lane 1-3 is the amplification situation that actin RPA-0 are primer in Fig. 2, and swimming lane 4-6 is that actin RPA-01 are primer
Amplification situation, swimming lane 7-9 is the amplification situation that actin RPA-02 are primer, and swimming lane 10-12 is actin RPA-03 to draw
The amplification situation of thing.As a result show, the present embodiment actin RPA-0, actin RPA-01, actin RPA-02, actin
RPA-03 can be expanded smoothly, obtain purpose product.This example demonstrates that the recombinase polymeric enzymatic amplification method energy of the present embodiment
It is enough that effectively human whole blood DNA molecular is expanded.
3rd specific embodiment of the invention, amplification step are as follows：
A0, extraction nucleic acid molecules, the present embodiment extract human whole blood DNA molecular by the method for poba gene group extracts kit；
A, three reaction systems for including different primers group are configured：Template nucleic acid molecule, primer sets, Ecoli RecA albumen, λ
Phage Orf albumen, Ecoli SSB albumen, archaeal dna polymerase, dNTP, ATP and Tris buffer solution.Specifically, in the present embodiment
Configure 2 × premixed liquid of following concentration：40ng/ μ L Ecoli RecA albumen, 160ng/ μ L λ phage Orf albumen, 400ng/
μ L Ecoli SSB albumen, 10ng/ μ L Sau albumen, 0.2mM dNTP, 2mM ATP, 40mM Tris buffer solutions, 40mM phosphoric acid
Creatine, 20ng/ μ L creatine kinases, 4% (w/v) polyethylene glycol.
It is separately added into 200 μ L centrifuge tubes：1 μ L concentration is 0.45pg/ μ L human whole blood DNA；Above-mentioned different primers
Group：The anti-sense primer that sense primer that 5 μ L concentration are 0.9 μM, 5 μ L concentration are 0.9 μM；25 μ 2 × premixed liquids of L；9 μ L deionizations
Water, mix and centrifuge；
Above-mentioned different primers group is respectively sense primer CFTRRPA-5_F（SEQ ID NO:11）With anti-sense primer CFTRRPA-5_
R（SEQ ID NO:12）；Sense primer CFTRRPA-6_F（SEQ ID NO:13）With anti-sense primer CFTRRPA-6_R（SEQ ID
NO:14）；Sense primer CFTRRPA-7_F（SEQ ID NO:15）With anti-sense primer CFTRRPA-7_R（SEQ ID NO:16）.
B, magnesium ion preparation is added into above-mentioned reaction system：Specifically, in the present embodiment into step A reaction system
The magnesium chloride solution that 5 μ L concentration are 270 mM is added, fully mixes and centrifuges.
C, amplified reaction is carried out under the conditions of 20 to 65 DEG C：Centrifuge tube is placed in 20 DEG C of constant-temperature metal bath and is incubated
4min；Reaction tube is taken out, is fully turned upside down 4-5 times, is mixed and centrifuge, then carries out the incubation of similarity condition, is incubated
20min。
After reaction terminates, centrifuge tube is taken out, samples row agarose gel electrophoresis detection of going forward side by side.
Agarose gel electrophoresis is detected as shown in figure 3, swimming lane 0 is molecular size label in Fig. 3, and swimming lane 1-2 is
CFTRRPA-5 is the amplification situation of primer, and swimming lane 3-4 is the amplification situation that CFTRRPA-6 is primer, and swimming lane 5-6 is
CFTRRPA-7 is the amplification situation of primer.As a result show, the recombinase polymeric enzymatic amplification method of the present embodiment can be effectively to people
Class whole blood DNA molecule is expanded.
4th specific embodiment of the invention, there is provided a kind of recombinase polymeric enzymatic amplification method, amplification step are as follows：
A0, extraction nucleic acid molecules, the present embodiment extract human whole blood DNA by the method for poba gene group extracts kit；
A, six reaction systems for including different primers group are configured：Template nucleic acid molecule, primer sets, Ecoli RecA albumen, λ
Phage Orf albumen, Ecoli SSB albumen, archaeal dna polymerase, dNTP, ATP and Tris buffer solution.Specifically, in the present embodiment
Configure 2 × premixed liquid of following concentration：500ng/ μ L Ecoli RecA albumen；40ng/ μ L λ phage Orf albumen；
2000ng/ μ L Ecoli SSB albumen；240ng/ μ L Bst albumen；6mM dNTP；20mM ATP；1000mM Tris are buffered
Liquid；40mM phosphocreatines；2000ng/ μ L creatine kinases；20% (w/v) polyethylene glycol.
It is separately added into 200 μ L centrifuge tubes：1 μ L concentration is 4.5mg/ μ L human whole blood DNA；Above-mentioned different primers
Group：The anti-sense primer that sense primer that 5 μ L concentration are 0.9 μM, 5 μ L concentration are 0.9 μM；25 μ 2 × premixed liquids of L；9 μ L deionizations
Water, mix and centrifuge.
Wherein above-mentioned different primers group is respectively sense primer lambdaRPA-8_F（SEQ ID NO:17）And anti-sense primer
lambdaRPA-8_R（SEQ ID NO:18）；Sense primer lambdaRPA-9_F（SEQ ID NO:19）And anti-sense primer
lambdaRPA-9_R（SEQ ID NO:20）；Sense primer lambdaRPA-10_F（SEQ ID NO:21）And anti-sense primer
lambdaRPA-10_R（SEQ ID NO:22）；Sense primer lambdaRPA-11_F（SEQ ID NO:23）And anti-sense primer
lambdaRPA-11_R（SEQ ID NO:24）；Sense primer lambdaRPA-12_F（SEQ ID NO:25）And anti-sense primer
lambdaRPA-12_R（SEQ ID NO:26）；Sense primer lambdaRPA-13_F（SEQ ID NO:27）And anti-sense primer
lambdaRPA-13_R（SEQ ID NO:28）.
B, magnesium ion preparation is added into above-mentioned reaction system：Specifically, the present embodiment adds into step A reaction system
Enter the magnesium chloride solution that 5 μ L concentration are 45 mM, fully mix and centrifuge.
C, amplified reaction is carried out under the conditions of 20 to 65 DEG C：Centrifuge tube is placed in 65 DEG C of constant-temperature metal bath and is incubated
4min；Reaction tube is taken out, is fully turned upside down 4-5 times, is mixed and centrifuge, then carries out the incubation of similarity condition, is incubated
20min。
After reaction terminates, centrifuge tube is taken out, samples row agarose gel electrophoresis detection of going forward side by side.
Agarose gel electrophoresis is detected as shown in figure 4, swimming lane 0 is molecular size label in Fig. 4, and swimming lane 1-2 is
LambdaRPA-8 is the amplification situation of primer, and swimming lane 3-4 is the amplification situation that lambdaRPA-9 is primer, and swimming lane 5-6 is
LambdaRPA-10 is the amplification situation of primer, and swimming lane 7-8 is the amplification situation that lambdaRPA-11 is primer, and swimming lane 9-10 is
LambdaRPA-12 is the amplification situation of primer, and swimming lane 11-12 is the amplification situation that lambdaRPA-13 is primer.As a result table
Bright, the recombinase polymeric enzymatic amplification method of the present embodiment can be expanded effectively to human whole blood DNA molecular.
As a result showing the recombinase polymeric enzymatic amplification method of the present embodiment effectively can expand human whole blood DNA molecular
Increase.
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 made within refreshing and principle etc., should be included in the scope of the protection.
SEQUENCE LISTING
<110>Guangzhou Kang Xin Rui Jiyin health Science and Technology Ltd.
<120>Recombinase polymeric enzymatic amplification kit, amplification method and amplifing reagent
<130>
<160> 28
<170> PatentIn version 3.3
<210> 1
<211> 30
<212> DNA
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<400> 1
cggtggtcct gccggaggcg tagagggaca 30
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<211> 30
<212> DNA
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<400> 2
tcagaaagat tcctacgtgg gcgacgaggc 30
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<211> 40
<212> DNA
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<400> 3
tagagggaca gcacggcctg gatggccacg tacatggcgg 40
<210> 4
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<212> DNA
<213>Artificial sequence
<400> 4
ggcgtcatgg tcggtatggg tcagaaagat tcctacgtgg 40
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<211> 50
<212> DNA
<213>Artificial sequence
<400> 5
cctgccggag gcgtagaggg acagcacggc ctggatggcc acgtacatgg 50
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gtcatggtcg gtatgggtca gaaagattcc tacgtgggcg acgaggctca 50
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<212> DNA
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<400> 7
tcggtatggg tcagaaagat tcctacgtgg gcgacgaggc tcagagcaag agaggtatcc 60
tga 63
<210> 8
<211> 63
<212> DNA
<213>Artificial sequence
<400> 8
cagcacggcc tggatggcca cgtacatggc gggcacgttg aaggtctcaa acatgatctg 60
ggt 63
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tacatggcgg gcacgttgaa ggtctcaaac 30
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cctacgtggg cgacgaggct cagagcaaga 30
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cctactacag ggcagtatct tttgtaagtt cttagatatt agcac 45
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accaactgtc ataaagctgc cctaaaatta ttactattag tca 43
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cttcaattca acaatgctat gccagtacaa acccatacgt tc 42
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gtcacaaaat tgagccagat aggttaatgt tcttcaacca tc 42
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catgtgtcct tttggcttta attaatatct ctattttgat gacc 44
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gtgtagaaag ccaaacaaag acaaaatcat gaaggacctg 40
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aacatggtga tgattctgct gcttgataaa ttttcaggta ttcgt 45
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gcgatgcatt ccgagaggca atagctgaag gccatataac aac 43
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accagaaata aacccaagcc aatcccaaaa gaatctgacg ta 42
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ctcgacgctt tcttgttcgt aacttcgatt ttggtcaatc acct 44
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aaacaaacgc aacgaggctc tacgaatcga gagtg 35
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aagcagcatt gagaactttg gaatccagtc cct 33
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<400> 23
acatggtttg ttgttatatg gccttcagct attgcctctc g 41
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<400> 24
atgctccact tgaagacatc accacaaaag aaattgcg 38
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aaacaaacgc aacgaggctc tacgaatcga gagtg 35
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<400> 26
aagcagcatt gagaactttg gaatccagtc cct 33
<210> 27
<211> 33
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<400> 27
atactcacct gcatcctgaa cccattgacc tcc 33
<210> 28
<211> 34
<212> DNA
<213>Artificial sequence
<400> 28
tcgccatcct gggaagactc ctgttatcaa gcac 34
Claims (10)
1. a kind of recombinase polymeric enzymatic amplification kit, it is characterised in that the kit includes following components：Ecoli RecA
Albumen, λ phage Orf albumen, Ecoli SSB albumen and archaeal dna polymerase.
2. recombinase polymeric enzymatic amplification kit according to claim 1, it is characterised in that the kit also includes expanding
Increase reaction buffer, the amplified reaction buffer solution includes Tris buffer solutions, and the amplified reaction buffer solution also includes poly- second two
One or more in alcohol, dithiothreitol (DTT), phosphocreatine and creatine kinase.
3. recombinase polymeric enzymatic amplification kit according to claim 2, it is characterised in that the kit also includes magnesium
Ion formulations.
A kind of 4. recombinase polymeric enzymatic amplification method, it is characterised in that the described method comprises the following steps：
A, reaction system is configured, the reaction system includes：Template nucleic acid molecule, primer sets, Ecoli RecA albumen, λ
Phage Orf albumen, Ecoli SSB albumen, archaeal dna polymerase, dNTP, ATP and amplified reaction buffer solution, the amplified reaction delay
Fliud flushing includes Tris buffer solutions；
B, magnesium ion preparation is added into the reaction system；
C, amplified reaction is carried out under the conditions of 20 to 65 DEG C.
5. recombinase polymeric enzymatic amplification method according to claim 4, it is characterised in that the amplified reaction buffer solution is also
Including the one or more in polyethylene glycol, dithiothreitol (DTT), phosphocreatine and creatine kinase.
6. recombinase polymeric enzymatic amplification method according to claim 4, it is characterised in that the primer sets are drawn including a pair
Thing, the length of the primer is 30 to 65bp.
7. recombinase polymeric enzymatic amplification method according to claim 4, it is characterised in that the Ecoli RecA albumen is dense
Spend for 20 to 250ng/ μ L；The λ phage Orf protein concentrations are 20 to 80ng/ μ L；The Ecoli SSB protein concentrations are
200 to 1000ng/ μ L.
8. a kind of recombinase polymeric enzymatic amplification reagent, it is characterised in that the reagent includes：Ecoli RecA albumen, λ phage
Orf albumen, Ecoli SSB albumen and archaeal dna polymerase.
9. recombinase polymeric enzymatic amplification reagent according to claim 8, it is characterised in that the amplifing reagent also includes expanding
Increase reaction buffer, the amplified reaction buffer solution includes Tris buffer solutions；The amplified reaction buffer solution also includes poly- second two
One or more in alcohol, dithiothreitol (DTT), phosphocreatine and creatine kinase.
10. recombinase polymeric enzymatic amplification reagent according to claim 8, it is characterised in that the Ecoli RecA albumen
Concentration is 20 to 250ng/ μ L；The concentration of the Ecoli SSB albumen is 200 to 1000ng/ μ L；The λ phage Orf eggs
White concentration is 20 to 80ng/ μ L.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610587482.4A CN107641635A (en) | 2016-07-22 | 2016-07-22 | Recombinase polymeric enzymatic amplification kit, amplification method and amplifing reagent |
| PCT/CN2017/093088 WO2018014799A1 (en) | 2016-07-22 | 2017-07-17 | Recombinase polymerase amplification reagent kit, amplification method, and amplification reagent |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610587482.4A CN107641635A (en) | 2016-07-22 | 2016-07-22 | Recombinase polymeric enzymatic amplification kit, amplification method and amplifing reagent |
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| WO (1) | WO2018014799A1 (en) |
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| CN111154739A (en) * | 2020-02-06 | 2020-05-15 | 广州普世利华科技有限公司 | Novel recombinase-dependent amplification method and kit |
| CN113454237A (en) * | 2018-12-20 | 2021-09-28 | 阿尔韦奥科技公司 | Isothermal amplification with electrical detection |
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| CN115948515A (en) * | 2022-12-30 | 2023-04-11 | 圣湘生物科技股份有限公司 | Recombinase-mediated total freeze-drying constant temperature amplification system and its preparation method and application |
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| WO2018014799A1 (en) | 2018-01-25 |
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