CN109321642A - Single tube nested PCR reaction system and amplification method - Google Patents
Single tube nested PCR reaction system and amplification method Download PDFInfo
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Abstract
The present invention relates to genetic engineering and molecular biology fields, provide a kind of single tube nested PCR reaction system, it includes double-stranded DNA template, outer primer to and inner primer pair, the outer primer includes complementary series and target area binding sequence from 5 ' ends to 3 ' ends, the base complementrity of the target area binding sequence end far from complementary series and the complementary series, the outer primer is for expanding the DNA profiling of denaturation unwinding under the first annealing temperature and obtaining intermediate product, the inner primer is for expanding the intermediate product of denaturation unwinding under the second annealing temperature and obtaining target product, the outer primer forms primer duplex structure under the second annealing temperature.The present invention also provides a kind of single tube nested PCR amplification method, single tube nested PCR reaction system of the invention and amplification method ensure that the purity of target product after Single tube amplification is higher.
Description
Technical field
The present invention relates to genetic engineering and molecular biology fields, more specifically to a kind of single tube nested PCR amplification
Method.
Background technique
Polymerase chain reaction (PCR) is the technology for the specific region of DNA amplification chain.DNA chain can be individually
Gene, the only only a part of gene or non-coding sequence.Most of PCR method usually expands up to 10k base-pair (kilo
Base pair, kb) DNA segment, but some technologies allow expand size up to 40kb segment (Cheng etc., 1994,
Proc Natl Acad Sci.91: 5695-5699).
The prior art provides a kind of nested PCR amplification method to improve PCR amplification specificity.Nested PCR amplification reaction
Two groups of different primers are used in two a sequence of reactions.Expand in reaction one, using the first primer to targeting regions DNA fragmentation
It carries out a stage PCR amplification and generates intermediate product, which may be also containing the product expanded from non-target region.Then
Intermediate product is used to originate two expansions reaction, described two, which expand reaction, carries out second order using the second primer pair targeting regions DNA fragmentation
Section PCR amplification generates target product.In general, the binding site of the second primer pair is located within the first primer pair.Nest-type PRC is usual
One is carried out in a test tube and expands reaction, and it is anti-that the aliquot of intermediate product is then transferred to two expansion of progress in the second test tube
It answers.The intermediate product of prior art nested PCR amplification method transfers between two test tubes will increase amplified production to environment
Pollution, even results in follow-up test result and severe deviations occurs.Therefore, it is badly in need of a kind of single tube nested PCR amplification method, for disappearing
The technical issues of except prior art nested PCR amplification method.
Summary of the invention
The purpose of the present invention is to provide a kind of single tube nested PCR amplification methods, for overcoming two-tube nested PCR amplification side
The technical issues of method.
A kind of single tube nested PCR reaction system comprising double-stranded DNA template, outer primer to and inner primer pair, it is described outer to draw
Object from 5 ' end to 3 ' end include complementary series and target area binding sequence, end of the target area binding sequence far from complementary series with
The base complementrity of the complementary series, the outer primer is for expanding the DNA profiling of denaturation unwinding under the first annealing temperature
And intermediate product is obtained, the inner primer is for expanding the intermediate product of denaturation unwinding under the second annealing temperature and obtaining mesh
Product is marked, the outer primer forms primer duplex structure under the second annealing temperature.
Further, the primer duplex structure includes primer hairpin structure or primer dimer structure.
Further, for the outer primer to including the first outer primer and the second outer primer, the primer duplex structure is the
The duplex structure formed between one outer primer and the second outer primer.
Further, to including the first outer primer and the second outer primer, the primer hairpin structure includes the outer primer
The duplex structure that first outer primer or the second outer primer itself are formed.
Further, the range of first annealing temperature can be 65-72 DEG C, and the range of second annealing temperature is
50-62℃。
Further, first annealing temperature is 65 degree, and second annealing temperature is 52 degree.
A kind of single tube nested PCR amplification method comprising:
Establish reaction system, include in the reaction system double-stranded DNA template, outer primer to and inner primer pair, the outer primer
It include complementary series and target area binding sequence, end and institute of the target area binding sequence far from complementary series from 5 ' ends to 3 ' ends
State the base complementrity of complementary series；
It carries out one and expands reaction, DNA profiling of the realization to denaturation unwinding is expanded and obtained by outer primer under the first annealing temperature
Obtain intermediate product；
It carries out two and expands reaction, target product to intermediate product amplification and is obtained by inner primer under the second annealing temperature, it is described
Outer primer forms primer duplex structure in the second annealing temperature.
Further, the primer duplex structure includes primer hairpin structure or primer dimer structure.
Further, for the outer primer to including the first outer primer and the second outer primer, the primer duplex structure is the
The duplex structure formed between one outer primer and the second outer primer.
Further, to including the first outer primer and the second outer primer, the primer hairpin structure includes the outer primer
The duplex structure that first outer primer or the second outer primer itself are formed.
Compared with the existing technology, in the reaction system of single tube nested PCR amplification method building of the present invention, 5 ' ends of outer primer
Including complementary series, when two expand the stage of reaction compared with such as 52 DEG C of low temperature thermal oxidation, primer double-strand mutually can be formed from connection
Structure, the primer duplex structure can be the dimer formed between primer hairpin structure, such as the first outer primer or second
The dimer formed between outer primer is also possible between dimer between primer, such as the first outer primer and the second outer primer
The dimer of formation.Therefore two expansion the stages of reaction only have inner primer to can just work normally to intermediate product carry out amplification form mesh
Mark product.Single tube nested PCR amplification method reaction system building of the invention is simple, and reaction process is not necessarily to intermediate product again
Packing, the technical issues of not only reducing prior art nested PCR amplification method, it is ensured that target product after Single tube amplification
Purity is higher.
Detailed description of the invention
Fig. 1 is the reaction principle figure of single tube single tube nested PCR amplification method of the present invention.
Fig. 2 is the PAGE glue electrophoretogram for testing a result.
Fig. 3 is the PAGE glue electrophoretogram for testing two results.
Fig. 4 is the PAGE glue electrophoretogram for testing three results.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.
Referring to FIG. 1, the present invention provides a kind of single tube nested PCR amplification method, comprising the following steps:
S1, establishes reaction system, the reaction system include double-stranded DNA template, outer primer to and inner primer pair.The outer primer
It include complementary series and target area binding sequence, end and institute of the target area binding sequence far from complementary series from 5 ' ends to 3 ' ends
State the base complementrity of complementary series.The target area binding sequence of the outer primer is used under the first annealing temperature to denaturation unwinding
DNA profiling expands to form intermediate product, and the inner primer is for expanding the intermediate product of denaturation unwinding under the second annealing temperature
Increasing forms target product, and the complementary series of the outer primer pair is used under the second annealing temperature draw with the formation of target area binding sequence
Object duplex structure, the primer duplex structure can be primer hairpin structure, be also possible to primer dimer structure.This implementation
In example, single tube nested PCR amplification kit can be made in the reaction system of S1 step building.
S2 carries out one and expands reaction, is expanded by outer primer to the DNA profiling of denaturation unwinding under the first annealing temperature,
Obtain intermediate product.In the present embodiment, first annealing temperature is higher than 60 degree, preferably, the model of first annealing temperature
It encloses and can be 65-72 DEG C.
S3 carries out two and expands reaction, by inner primer to the intermediate product template to denaturation unwinding under the second annealing temperature
Amplification, obtains target product, and the complementary series and target area binding sequence of the outer primer form primer under the second annealing temperature
Duplex structure.In the present embodiment, the range of second annealing temperature can be 50-62 DEG C, preferably, the second annealing temperature
Spend 10-15 DEG C lower than the first annealing temperature.In the present embodiment, complementary series and target area the binding sequence formation of the outer primer are drawn
The temperature range of object duplex structure can be 45-57 DEG C, preferably, the complementary series of the outer primer and target area binding sequence shape
It is 0-5 DEG C lower than the second annealing temperature of inner primer pair at the temperature of primer duplex structure.
In one embodiment, for expanding the segment in the site mthfr gene rs1801133, three kinds of outer primers are provided respectively
To progress experiment contrast.
First outer primer is to the reaction system for prior art nest-type PRC comprising the first outer primer SEQ ID NO:
1, the second outer primer SEQ ID NO:2, the first outer primer SEQ ID NO:1 and the second outer primer SEQ ID of the first primer pair
NO:2 is not provided with complementary series.The first primer can refer to the following table 1 to primer sequence and work annealing temperature.
Second outer primer is to one embodiment for single tube nested PCR amplification of the present invention comprising the first outer primer SEQ
ID NO:3, the second outer primer SEQ ID NO:4, the first outer primer SEQ ID NO:3 and the second outer primer SEQ ID NO:4 exist
Primer dimer structure, the relevant primer sequence and work annealing of the second outer primer pair can be formed at second 49.5 DEG C of annealing temperature
Temperature can refer to the following table 2.The mode that the primer dimer structure of second outer primer pair is formed are as follows: the first outer primer SEQ ID
The complementary series GAGTGCTGAG of NO:3 is tied at 49.5 DEG C of the second annealing temperature with the target area of the second outer primer SEQ ID NO:4
Close sequence endCTCAGCACTCForm primer duplex structure.The complementary series of the second outer primer SEQ ID NO:4
Target area binding sequence end of the CTTGCACC at 49.5 DEG C of the second annealing temperature with the first outer primer SEQ ID NO:3GGTGCAAGForm primer duplex structure.
Third outer primer is to another embodiment for single tube nested PCR amplification of the present invention comprising the first outer primer SEQ
ID NO:5, the second outer primer SEQ ID NO:6, the first outer primer SEQ ID NO:5 and the second outer primer SEQ ID NO:6 exist
Primer hairpin structure, the relevant primer sequence of third outer primer pair can be respectively formed at second 53.8 DEG C of annealing temperature and 51.3 DEG C
And work annealing temperature can refer to the following table 3.The mode that the primer hairpin structure of third outer primer pair is formed are as follows: draw outside described first
The complementary series CTTGCAC of object SEQ ID NO:5 at 53.8 DEG C of the second annealing temperature with own sequence endGTGCAAGIt is formed
Primer hairpin structure.The complementary series GAGTGC of the second outer primer SEQ ID NO:6 is at 51.3 DEG C of the second annealing temperature
With the target area binding sequence end of itselfGCACTCForm primer hairpin structure.
Test one
Experiment purpose: the expanding effect of the above-mentioned three kinds of outer primers pair of verifying confirms above-mentioned three kinds of outer primers in high annealing item
Target gene segment can be normally expanded under part.
System configurations: with above-mentioned three kinds of outer primers to three reaction systems of configuration, each reaction in three test tubes respectively
For system configurations referring to the following table 4, each reaction system includes: long Taq mixed liquor (Shenzhen HYK Gene Technology Co., Ltd.
Production) 10 μ L；The 1.0 μ L of human whole blood DNA molecular of 25ng/ μ L；The 0.4 μ L of the first outer primer of 10 μm of ol/L； 10μmol/L
0.4 μ L of the second outer primer；8.2 μ L deionized waters；It mixes and is centrifuged, then centrifuge tube is placed in PCR instrument.
Reaction condition: continuing 4 minutes under the conditions of 94 DEG C, a circulation；It is for 20 seconds under the conditions of 94 DEG C, it is held under the conditions of 65 DEG C
Continue 30 seconds under the conditions of 20 seconds, 72 DEG C continuous, altogether 30 circulations；Continue 3 minutes circulations under the conditions of 72 DEG C, obtains target and produce
Object, target product is stored in 10 DEG C of environment, with specific reference to table 5.
Electrophoresis result: swimming lane 1,3,5 as shown in Figure 2 is the blank of each primer pair, and swimming lane 2,4,6 is the base of each primer pair
Because of a group amplified production.SEQ ID NO:1 and SEQ ID NO:2:439bp；SEQ ID NO:3 and SEQ ID NO:4:457bp；
SEQ ID NO:5 and SEQ ID NO:6:452bp, Fig. 2 electrophoresis result illustrate three kinds of outer primers under high annealing all
The segment in the site mthfr gene rs1801133 can be normally expanded, the target product after obtaining corresponding gene fragment amplification.
Test two
Experiment purpose: interference of each outer primer to reaction is expanded to two under the second annealing temperature is verified.
System configurations: taking nest-type PRC one to expand the template that the dilution of the product (intermediate product) of reaction expands reaction as two,
Be added in different test tubes the outer primer of various concentration to and fixed concentration inner primer to constructing multiple reaction systems, each reaction
System the difference is that only outer primer to concentration difference, concrete configuration is referring to the following table 6.
Reaction system one, five, nine is outer primer blank control, respectively includes long Taq mixed liquor (Shenzhen Hua Yinkang
Gene Tech. Company Limited's production) 10 μ L；Product dilution (100 times of dilution) 1.0 μ L of reaction are expanded in blank control one；10μ
The 0.4 μ L of the first outer primer SEQ ID NO:1 of mol/L；The 0.4 μ L of the second outer primer SEQ ID NO:2 of 10 μm of ol/L；10μ
The 0.4 μ L of the first inner primer SEQ ID NO:7 of mol/L；The 0.4 μ L of the second inner primer SEQ ID NO:8 of 10 μm of ol/L； 7.4
μ L deionized water；It mixes and is centrifuged, then centrifuge tube is placed in PCR instrument.
Reaction system two includes long Taq mixed liquor (Shenzhen HYK Gene Technology Co., Ltd.'s production) 10 μ L；One
Expand product dilution (100 times of dilution) 1.0 μ L of reaction；The 0.4 μ L of the first outer primer SEQ ID NO:1 of 10 μm of ol/L；10μ
The 0.4 μ L of the second outer primer SEQ ID NO:2 of mol/L；The 0.4 μ L of the first inner primer SEQ ID NO:7 of 10 μm of ol/L；10μ
The 0.4 μ L of the second inner primer SEQ ID NO:8 of mol/L；7.4 μ L deionized waters；It mixes and is centrifuged, be then placed in centrifuge tube
In PCR instrument.
Reaction system three includes long Taq mixed liquor (Shenzhen HYK Gene Technology Co., Ltd.'s production) 10 μ L；One
Expand product dilution (100 times of dilution) 1.0 μ L of reaction；The 0.4 μ L of the first outer primer SEQ ID NO:1 of 1 μm of ol/L；1μ
The 0.4 μ L of the second outer primer SEQ ID NO:2 of mol/L；The 0.4 μ L of the first inner primer SEQ ID NO:7 of 10 μm of ol/L；10μ
The 0.4 μ L of the second inner primer SEQ ID NO:8 of mol/L；7.4 μ L deionized waters；It mixes and is centrifuged, be then placed in centrifuge tube
In PCR instrument.
Reaction system four includes long Taq mixed liquor (Shenzhen HYK Gene Technology Co., Ltd.'s production) 10 μ L；One
Expand product dilution (100 times of dilution) 1.0 μ L of reaction；The 0.4 μ L of the first outer primer SEQ ID NO:1 of 0.1 μm of ol/L；0.1
The 0.4 μ L of the second outer primer SEQ ID NO:2 of μm ol/L；The 0.4 μ L of the first inner primer SEQ ID NO:7 of 10 μm of ol/L；10μ
The 0.4 μ L of the second inner primer SEQ ID NO:8 of mol/L；7.4 μ L deionized waters；It mixes and is centrifuged, be then placed in centrifuge tube
In PCR instrument.
Reaction system six includes long Taq mixed liquor (Shenzhen HYK Gene Technology Co., Ltd.'s production) 10 μ L；One
Expand product dilution (100 times of dilution) 1.0 μ L of reaction；The 0.4 μ L of the first outer primer SEQ ID NO:3 of 10 μm of ol/L；10μ
The 0.4 μ L of the second outer primer SEQ ID NO:4 of mol/L；The 0.4 μ L of the first inner primer SEQ ID NO:7 of 10 μm of ol/L；10μ
The 0.4 μ L of the second inner primer SEQ ID NO:8 of mol/L；7.4 μ L deionized waters；It mixes and is centrifuged, be then placed in centrifuge tube
In PCR instrument.
Reaction system seven includes long Taq mixed liquor (Shenzhen HYK Gene Technology Co., Ltd.'s production) 10 μ L；One
Expand product dilution (100 times of dilution) 1.0 μ L of reaction；The 0.4 μ L of the first outer primer SEQ ID NO:3 of 1 μm of ol/L；1μ
The 0.4 μ L of the second outer primer SEQ ID NO:4 of mol/L；The 0.4 μ L of the first inner primer SEQ ID NO:7 of 10 μm of ol/L；10μ
The 0.4 μ L of the second inner primer SEQ ID NO:8 of mol/L；7.4 μ L deionized waters；It mixes and is centrifuged, be then placed in centrifuge tube
In PCR instrument.
Reaction system eight includes long Taq mixed liquor (Shenzhen HYK Gene Technology Co., Ltd.'s production) 10 μ L；One
Expand product dilution (100 times of dilution) 1.0 μ L of reaction；The 0.4 μ L of the first outer primer SEQ ID NO:3 of 0.1 μm of ol/L；0.1
The 0.4 μ L of the second outer primer SEQ ID NO:4 of μm ol/L；The 0.4 μ L of the first inner primer SEQ ID NO:7 of 10 μm of ol/L；10μ
The 0.4 μ L of the second inner primer SEQ ID NO:8 of mol/L；7.4 μ L deionized waters；It mixes and is centrifuged, be then placed in centrifuge tube
In PCR instrument.
Reaction system ten includes long Taq mixed liquor (Shenzhen HYK Gene Technology Co., Ltd.'s production) 10 μ L；One
Expand product dilution (100 times of dilution) 1.0 μ L of reaction；The 0.4 μ L of the first outer primer SEQ ID NO:5 of 10 μm of ol/L；10μ
The 0.4 μ L of the second outer primer SEQ ID NO:6 of mol/L；The 0.4 μ L of the first inner primer SEQ ID NO:7 of 10 μm of ol/L；10μ
The 0.4 μ L of the second inner primer SEQ ID NO:8 of mol/L；7.4 μ L deionized waters；It mixes and is centrifuged, be then placed in centrifuge tube
In PCR instrument.
Reaction system 11 includes long Taq mixed liquor (Shenzhen HYK Gene Technology Co., Ltd.'s production) 10 μ L；
One expands product dilution (100 times of dilution) 1.0 μ L of reaction；The 0.4 μ L of the first outer primer SEQ ID NO:5 of 1 μm of ol/L；1μ
The 0.4 μ L of the second outer primer SEQ ID NO:6 of mol/L；The 0.4 μ L of the first inner primer SEQ ID NO:7 of 10 μm of ol/L；10μ
The 0.4 μ L of the second inner primer SEQ ID NO:8 of mol/L；7.4 μ L deionized waters；It mixes and is centrifuged, be then placed in centrifuge tube
In PCR instrument.
Reaction system 12 includes long Taq mixed liquor (Shenzhen HYK Gene Technology Co., Ltd.'s production) 10 μ L；
One expands product dilution (100 times of dilution) 1.0 μ L of reaction；The 0.4 μ L of the first outer primer SEQ ID NO:5 of 0.1 μm of ol/L；
The 0.4 μ L of the second outer primer SEQ ID NO:6 of 0.1 μm of ol/L；The 0.4 μ L of the first inner primer SEQ ID NO:7 of 10 μm of ol/L；
The 0.4 μ L of the second inner primer SEQ ID NO:8 of 10 μm of ol/L；7.4 μ L deionized waters；It mixes and is centrifuged, then by centrifuge tube
It is placed in PCR instrument.
Reaction condition: continuing 2 minutes under the conditions of 94 DEG C, a circulation；It is for 10 seconds under the conditions of 94 DEG C, it is held under the conditions of 52 DEG C
For 10 seconds under the conditions of 20 seconds, 72 DEG C continuous, 30 recycle altogether；Continue 3 minutes circulations under the conditions of 72 DEG C, obtains target and produce
Object, target product is stored in 10 DEG C of environment, with specific reference to table 7.
Electrophoresis result: as shown in figure 3, swimming lane 1,5,9: the blank of each outer primer pair；Swimming lane 2,6,10: outer primer concentration
For the amplified production of 10uM；Swimming lane 3,7,11: outer primer concentration is the amplified production of 1uM；Swimming lane 4,8,12: outer primer concentration is
The amplified production of 0.1 uM.Swimming lane 1-4 is general primer to SEQ ID NO:1 and SEQ ID NO:2's as a result, master when high concentration
To be 439bp product, reduce other products with concentration and gradually increase, purpose product occupies the minority；Swimming lane 5-8 is primer pair SEQ ID
NO:3 and SEQ ID NO:4's as a result, slightly there are other products in when high concentration, principal product is purpose product；Swimming lane 9-12 is to draw
For object to SEQ ID NO:5 and SEQ ID NO:6's as a result, slightly there are other products in when high concentration, principal product is purpose product.Figure
Result shown in 3 illustrates the second outer primer to SEQ ID NO:3, SEQ ID NO:4 and third outer primer to SEQ ID NO:5, SEQ
ID NO:6 can form primer duplex structure under low-temperature annealing and significantly reduce the interference for expanding reaction to two.
Experiment three
Experiment purpose: the expanding effect of a tubular type nest-type PRC of the invention is verified.
System configurations: with above-mentioned three kinds of outer primers to three reaction systems of configuration, each reaction in three test tubes respectively
System includes: long Taq mixed liquor (Shenzhen HYK Gene Technology Co., Ltd.'s production) 10 μ L；The mankind of 25ng/ μ L are complete
1.0 μ L of blood DNA molecular；The 0.4 μ L of the first outer primer of 10 μm of ol/L；The 0.4 μ L of the second outer primer of 10 μm of ol/L；10μmol/L
0.4 μ L of the first inner primer SEQ ID NO:7；The 0.4 μ L of the second inner primer SEQ ID NO:8 of 10 μm of ol/L；7.4 μ L are gone
Ionized water；It mixes and is centrifuged, then centrifuge tube is placed in PCR instrument, each reaction system configuration is referring to the following table 8.
Reaction condition: continuing 4 minutes under the conditions of 94 DEG C, a circulation；It is for 20 seconds under the conditions of 94 DEG C, it is held under the conditions of 65 DEG C
Continue 30 seconds under the conditions of 20 seconds, 72 DEG C continuous, altogether 15 circulations；Continue 3 minutes circulations under the conditions of 72 DEG C, obtains intermediate produce
Object, one, which expands reaction, terminates.Then continue 2 minutes under the conditions of 94 DEG C, a circulation；It is for 10 seconds under the conditions of 94 DEG C, 52 DEG C of conditions
Under it is for 20 seconds, it is for 10 seconds under the conditions of 72 DEG C, altogether 25 circulation；Continue 3 minutes circulations under the conditions of 72 DEG C, two expand instead
It should terminate, obtain target product and be stored in 10 DEG C of environment, with specific reference to table 9.
Electrophoresis result: swimming lane 1,3,5 as shown in Figure 4: the blank of each primer pair, swimming lane 2,4,6: the gene of each primer pair
Group amplified production.SEQ ID NO:1 and SEQ ID NO:2: four bands of display；SEQ ID NO:3 and SEQ ID NO:4: display
For the purpose of band；SEQ ID NO:5 and SEQ ID NO:6: band for the purpose of display, Fig. 4 electrophoresis result illustrate there is complementary series
Outer primer can preferably realize the amplification of a tubular type nest-type PRC to more common outer primer.
Compared with the existing technology, in the reaction system of single tube nested PCR amplification method building of the present invention, 5 ' ends of outer primer
Including complementary series, when two expand the stage of reaction compared with such as 52 DEG C of low temperature thermal oxidation, primer double-strand mutually can be formed from connection
Structure, the primer duplex structure can be the dimer formed between primer hairpin structure, such as the first outer primer or second
The dimer formed between outer primer is also possible between dimer between primer, such as the first outer primer and the second outer primer
The dimer of formation.Therefore two expansion the stages of reaction only have inner primer to can just work normally to intermediate product carry out amplification form mesh
Mark product.Single tube nested PCR amplification method reaction system building of the invention is simple, and reaction process is not necessarily to intermediate product again
Packing, the technical issues of not only reducing prior art nested PCR amplification method, it is ensured that target product after Single tube amplification
Purity is higher.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
SEQUENCE LISTING
<110>Guangzhou Kang Xin Rui Jiyin health Science and Technology Ltd.
<120>single tube nested PCR reaction system and amplification method
<130>
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 25
<212> DNA
<213>artificial sequence
<400> 1
gtgctgtgct gttggaaggt gcaag 25
<210> 2
<211> 27
<212> DNA
<213>artificial sequence
<400> 2
gggaagaact cagcgaactc agcactc 27
<210> 3
<211> 35
<212> DNA
<213>artificial sequence
<400> 3
gagtgctgag gtgctgtgct gttggaaggt gcaag 35
<210> 4
<211> 35
<212> DNA
<213>artificial sequence
<400> 4
cttgcaccgg gaagaactca gcgaactcag cactc 35
<210> 5
<211> 32
<212> DNA
<213>artificial sequence
<400> 5
cttgcacgtg ctgtgctgtt ggaaggtgca ag 32
<210> 6
<211> 33
<212> DNA
<213>artificial sequence
<400> 6
gagtgcggga agaactcagc gaactcagca ctc 33
Claims (10)
1. a kind of single tube nested PCR reaction system, it is characterised in that: in reaction system include double-stranded DNA template, outer primer to
Inner primer pair, the outer primer include complementary series and target area binding sequence from 5 ' ends to 3 ' ends, and the target area binding sequence is remote
Base complementrity from the end of complementary series and the complementary series, the outer primer are used under the first annealing temperature to denaturation
The DNA profiling of unwinding expands and obtains intermediate product, and the inner primer is used under the second annealing temperature in denaturation unwinding
Between product amplification and obtain target product, the outer primer forms primer duplex structure under the second annealing temperature.
2. single tube nested PCR reaction system according to claim 1, it is characterised in that: the primer duplex structure includes
Primer hairpin structure or primer dimer structure.
3. single tube nested PCR reaction system according to claim 2, it is characterised in that: the outer primer is to including first
Outer primer and the second outer primer, the double-strand knot that the primer duplex structure is formed between the first outer primer and the second outer primer
Structure.
4. single tube nested PCR reaction system according to claim 2, it is characterised in that: the outer primer is to including first
Outer primer and the second outer primer, the primer hairpin structure include the double-strand knot that the first outer primer or the second outer primer itself are formed
Structure.
5. single tube nested PCR reaction system according to claim 1, it is characterised in that: the model of first annealing temperature
Enclosing is 65-72 DEG C, and the range of second annealing temperature is 50-62 DEG C.
6. single tube nested PCR reaction system according to claim 5, it is characterised in that: first annealing temperature is 65
Degree, second annealing temperature are 52 degree.
7. a kind of single tube nested PCR amplification method, characterized by comprising:
Establish reaction system, include in the reaction system double-stranded DNA template, outer primer to and inner primer pair, the outer primer
It include complementary series and target area binding sequence, end and institute of the target area binding sequence far from complementary series from 5 ' ends to 3 ' ends
State the base complementrity of complementary series；
It carries out one and expands reaction, DNA profiling of the realization to denaturation unwinding is expanded and obtained by outer primer under the first annealing temperature
Obtain intermediate product；
It carries out two and expands reaction, target product to intermediate product amplification and is obtained by inner primer under the second annealing temperature, it is described
Outer primer forms primer duplex structure in the second annealing temperature.
8. single tube nested PCR amplification method according to claim 7, it is characterised in that: the primer duplex structure includes
Primer hairpin structure or primer dimer structure.
9. single tube nested PCR amplification method according to claim 8, it is characterised in that: the outer primer is to including first
Outer primer and the second outer primer, the double-strand knot that the primer duplex structure is formed between the first outer primer and the second outer primer
Structure.
10. single tube nested PCR amplification method according to claim 8, it is characterised in that: the outer primer is to including first
Outer primer and the second outer primer, the primer hairpin structure include the double-strand knot that the first outer primer or the second outer primer itself are formed
Structure.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2022537284A (en) * | 2019-06-26 | 2022-08-25 | 深▲セン▼華大智造科技股▲ふん▼有限公司 | Nested multiplex PCR high-throughput sequencing library preparation method and kit |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1536087A (en) * | 2003-04-11 | 2004-10-13 | 徐定邦 | Nested polymerase chain-type reaction method and its application |
| WO2004090150A2 (en) * | 2003-04-11 | 2004-10-21 | Dingbang Xu | Pcr method using multiple primer pairs and the solution used in it and the use of the method in the detection reagents |
| CN102459626A (en) * | 2009-06-02 | 2012-05-16 | 莫诺夸特有限公司 | A method of nucleic acid amplification |
| CN102985560A (en) * | 2010-03-30 | 2013-03-20 | 莫诺匡特私人有限公司 | A method of regulating oligonucleotide functionality |
| CN104611434A (en) * | 2015-01-29 | 2015-05-13 | 黄天谊 | Folding primer and PCR amplification method thereof |
| CN104911254A (en) * | 2015-02-13 | 2015-09-16 | 贵州省疾病预防控制中心 | Folding primer multiple PCR malaria molecule diagnostic kit and detection method thereof |
-
2017
- 2017-07-31 CN CN201710640979.2A patent/CN109321642A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1536087A (en) * | 2003-04-11 | 2004-10-13 | 徐定邦 | Nested polymerase chain-type reaction method and its application |
| WO2004090150A2 (en) * | 2003-04-11 | 2004-10-21 | Dingbang Xu | Pcr method using multiple primer pairs and the solution used in it and the use of the method in the detection reagents |
| CN102459626A (en) * | 2009-06-02 | 2012-05-16 | 莫诺夸特有限公司 | A method of nucleic acid amplification |
| CN102985560A (en) * | 2010-03-30 | 2013-03-20 | 莫诺匡特私人有限公司 | A method of regulating oligonucleotide functionality |
| CN104611434A (en) * | 2015-01-29 | 2015-05-13 | 黄天谊 | Folding primer and PCR amplification method thereof |
| CN104911254A (en) * | 2015-02-13 | 2015-09-16 | 贵州省疾病预防控制中心 | Folding primer multiple PCR malaria molecule diagnostic kit and detection method thereof |
Non-Patent Citations (1)
| Title |
|---|
| YU-TING HUANG等: "Single-tube Single-run Multiplex PCR Detects Mixed Samples with Four Species of Plasmodium", 《ZHONGGUO JI SHENG CHONG XUE YU JI SHENG CHONG BING ZA ZHI》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2022537284A (en) * | 2019-06-26 | 2022-08-25 | 深▲セン▼華大智造科技股▲ふん▼有限公司 | Nested multiplex PCR high-throughput sequencing library preparation method and kit |
| JP7281565B2 (en) | 2019-06-26 | 2023-05-25 | 深▲セン▼華大智造科技股▲ふん▼有限公司 | Nested multiplex PCR high-throughput sequencing library preparation method and kit |
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