CN105176974B - The zero background unwindase for eliminating primer dimer relies on system and amplification method and application - Google Patents
The zero background unwindase for eliminating primer dimer relies on system and amplification method and application Download PDFInfo
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- CN105176974B CN105176974B CN201510579700.5A CN201510579700A CN105176974B CN 105176974 B CN105176974 B CN 105176974B CN 201510579700 A CN201510579700 A CN 201510579700A CN 105176974 B CN105176974 B CN 105176974B
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Abstract
The invention discloses a kind of zero background unwindase for eliminating primer dimer to rely on system and amplification method and application, belongs to molecular biology and molecular diagnostic techniques field.The present invention is directed to false positive signal caused by primer dimer in HDA systems, the primer dimer come by using the ATP and dNTPs of appropriate low concentration in elimination system, obtains zero simple and general background HDA detection methods.The method need not increase extra heat inactivation step or extra enzyme, without the special primer of well-designed structure, only need the appropriate concentration for reducing ATP and dNTPs in substance system that false positive background caused by primer dimer is completely eliminated, with simple and convenient, it is versatile, the advantages that effect is good.
Description
Technical field
The invention belongs to molecular biology and molecular diagnostic techniques field, and in particular to a kind of to eliminate the zero of primer dimer
Background unwindase relies on system and amplification method and application.
Background technology
It is that one kind has to rely on unwindase isothermal amplification technology (helicase-dependent amplification, HDA)
The technology of the exponential amplification target nucleic acid of effect.The technical modelling biology nucleic acid in vivo reproduction process, using DNA helicase in isothermal
Under the conditions of untie double-stranded DNA, with single strand binding protein (single-stranded DNA-binding protein) maintain DNA
Single-chain state, nucleic acid amplification then is realized in the presence of archaeal dna polymerase, without similar to complicated and accurate required by PCR
Thermal cycle (denaturation-annealing-extension) process.HDA has been considered as a kind of strong PCR alternative, particularly
In the area of resource-constrained and when needing to detect on the spot, HDA illustrates the simplicity and practicality of brilliance, nowadays by
It is widely used in pathogen detection and SNP partings.
Early stage HDA is carried out at relatively low temperature (about 37 DEG C), using E. coli UvrD helicase and it is other two kinds it is auxiliary
Albumen MutL and single strand binding protein is helped to realize amplified reaction.In order to further improve HDA reaction efficiency, many research people
Member has made unremitting effort.A kind of thermostabilization UvrD unwindases are cloned and be purified into Kong Research team from thermophilic anaerobic bacillus
(thermostable UvrD helicase) come realized at higher temperature (60~65 DEG C) HDA react, without auxiliary egg
Efficient amplification is realized in lean type system.Another HDA improved method by using one kind by unwindase and archaeal dna polymerase fusion and
Into bifunctional protein, can successfully realize more long segment (2.3kb) amplification.Also Research team is proposed before amplification with interior
Enzyme cutting processing target gene group DNA can accelerate HDA speed and improve its sensitivity.In addition, also someone's research is drawn：Primer
Rich in A, either C ratios are advantageous to obtain more effective amplified reaction containing T or G at 5 ' ends.Nevertheless, primer dimer is still deposited
.The problem of this is common disturbs the specificity accumulation of target nucleic acid chain, generates the testing result of mistake.
Recently, Zhang Research team reports that the HDA detection methods of a new target conversion eliminate primer dimerization
The interference of body, realize the super sensitivity detection to transcription factor.In whole HDA amplified reactions, single primer is used only in they, this
Primer and the stem of well-designed hairpin structure template are complementary, eliminate the primer dimer that primer pair is formed in traditional HDA and make
Into non-specific amplification.However, the method additionally digests the hair fastener template not combined with object simultaneously completely using excision enzyme
Need to carry out heat inactivation to excision enzyme to prevent background signal from producing.Furthermore because the method is based on given hair fastener template,
It cannot be used for the amplification of other nucleic acid chains and the detection of various non-nucleic acid objects.
So far, background caused by primer dimer can be completely eliminated in the method for also no simple general-purpose.With HDA
The expansion of application, need badly and develop a kind of general and simple method further to improve HDA application.
The content of the invention
The defects of in order to overcome above-mentioned prior art to exist, it is an object of the invention to provide one kind to eliminate primer dimer
Zero background unwindase rely on system and amplification method and application, this method is simple and practical, versatile, effect is good；The system
The HDA of zero background can be realized.
The present invention is to be achieved through the following technical solutions：
The invention discloses a kind of zero background unwindase for eliminating primer dimer to rely on system, and the system includes：2.5μL
10 × annealing buffer II, 1 μ L 100mM MgSO4, 2 μ L 500mM NaCl, 1 μ L 5 μM of reverse primers, 5 μM of 1 μ L
Forward primer, 1 μ L 52.5mM ATP solution, 1 μ L 3.5mM dNTPs, 0.75 μ LEnzymatic mixture and 1 μ L
5 × SYBR Green I.
Preferably, 40bp chain is chosen as template strand, then the 18 base identical 18bp held with template strand 5 ' chain
As forward primer, to hold the 24bp chain of 24 base complementrities to be used as reverse primer with template strand 3 '.
The invention also discloses a kind of zero background unwindase for eliminating primer dimer to rely on amplification method, including following step
Suddenly：
1) according to purpose fragment template design primer to be amplified, including forward primer and reverse primer；
2) HDA reaction systems are built, including：Forward primer, reverse primer, template, buffer solution, reaction needed for enzyme and
ATP and dNTPs；
3) each reactant in system is well mixed, carries out HDA isothermal duplications, amplification knot is detected according to fluorescence signal
Fruit, judgement realize that zero background rotation enzyme relies on amplified reaction.
The HDA reaction systems of HDA reaction system reference standards described in step 2), place is turned down to ATP and dNTPs concentration
Reason.
Fluorescence signal detection amplification described in step 3) is to use96 System carry out glimmering in real time
Light detects, and per 60s, once, design temperature is 60 DEG C to measure.
Described template is single-stranded for the single-stranded of synthesis or after cellular telomerase amplification.
Preferably, the zero background unwindase for eliminating primer dimer relies on amplification method, comprises the following steps：
1) according to template sequence, forward primer and reverse primer are designed：
Template sequence is：5'-AAT CCG TCG AGC AGA GTT AGG GTT AGG GTT AGG GTT AGG G-
3'；
Designing forward primer is：5'-AAT CCG TCG AGC AGA GTT-3'；
Designing reverse primer is：5'-CCC TAA CCC TAA CCC TAA CCC TAA-3'；
2) HDA reaction systems are built, including：2.5 μ L 10 × annealing buffer II, 1 μ L 100mM MgSO4, 2 μ L
500mM NaCl, 1 μ L 5 μM of reverse primers, 1 μ L 5 μM of forward primers, 1 μ L 52.5mM ATP solution, 1 μ L 3.5mM
DNTPs, 0.75 μ L5 × SYBR Green I of enzymatic mixture and 1 μ L；
3) each reactant in system is well mixed, carries out HDA isothermal duplications, amplification knot is detected according to fluorescence signal
Fruit, judgement realize that zero background rotation enzyme relies on amplified reaction.
The invention also discloses zero background unwindase of above-mentioned elimination primer dimer to rely on system in detection cell telomere
Application in enzymatic activity.
Compared with prior art, the present invention has technique effect beneficial below：
The present invention is directed to false positive signal caused by primer dimer in HDA systems, by using the ATP of appropriate low concentration
The primer dimer come with dNTPs in elimination system, obtain zero simple and general background HDA detection methods.The method need not
Increase extra heat inactivation step or extra enzyme, without the special primer of well-designed structure, it is only necessary to appropriate
False positive background caused by primer dimer is completely eliminated in the concentration for reducing ATP and dNTPs in substance system, has simple side
Just, it is versatile, the advantages that effect is good.Specific advantage is as follows：
1st, the present invention is solved in traditional HDA technologies caused by the common non-specific amplification triggered as primer dimer
False positive issue, eliminate the amplification background of primer dimer；
2nd, the present invention simply reduces the technology of primer dimer amplification background compared to some, completely eliminates primer dimerization
The amplification background of body, thoroughly solves the problems, such as primer dimer initiation；
3rd, the present invention is compared to needing to add specific enzymes, need the primer of special construction or need to increase extra enzyme-deactivating
For other HDA improving technologies of step, it is only necessary to ATP and dNTPs concentration is suitably reduced in former HDA systems, you can realize
The HDA of zero background, without additional step, extra cost, and there is versatility, effect is more preferable.
Brief description of the drawings
Fig. 1 is the method flow schematic diagram of the present invention；
Fig. 2 is the real-time fluorescence curves figure of HDA under different ATP concentration；
Fig. 3 is the real-time fluorescence curves figure of the HDA under different dNTPs concentration；
Fig. 4 is detection principle diagrams of the zero background HDA to telomerase activation；
Fig. 5 is the fluorescence real-time curve chart for adding or not adding HeLa extracts in zero background HDA；
Fig. 6 is the real-time fluorescence curves figure that varying number cellular telomerase amplified production is added in zero background HDA；
Fig. 7 is the logarithm of cell quantity and the linear relationship of fluorescence intensity；
Fig. 8 is testing results of the zero background HDA to the telomerase activation of different cell lines.
Embodiment
With reference to specific embodiment, the present invention is described in further detail, it is described be explanation of the invention and
It is not to limit.
Referring to Fig. 1, zero background unwindase of elimination primer dimer of the invention relies on amplification method, including following step
Suddenly：
1) according to purpose fragment template design primer to be amplified, including forward primer and reverse primer；
2) HDA reaction systems are built, including：Forward primer, reverse primer, template, buffer solution, reaction needed for enzyme and
ATP and dNTPs；
3) each reactant in system is well mixed, carries out HDA isothermal duplications, amplification knot is detected according to fluorescence signal
Fruit, judgement realize that zero background rotation enzyme relies on amplified reaction.
Embodiment 1
Template, forward primer and reverse primer are used as using the chain of three synthesis respectively
1) design of template, forward primer and reverse primer
Template sequence is：5'-AAT CCG TCG AGC AGA GTT AGG GTT AGG GTT AGG GTT AGG G-
3'；
Forward primer is：5'-AAT CCG TCG AGC AGA GTT-3'；
Reverse primer is：5'-CCC TAA CCC TAA CCC TAA CCC TAA-3'；
Using the 40bp chains of synthesis as masterplate, drawn using the chain of 18 base identical 18bp before being held with template strand 5 ' as forward direction
Thing, using the chain of the 24bp with 24 base complementrities in template strand 3' ends as reverse primer, at this moment this pair of primers has two alkali
Base is complementary, and primer dimer non-specific amplification most probably occurs, and is experimentally confirmed the non-specificity of primer dimer initiation
Amplification generates really.
2) zero background HDA implementation
The zero background HDA systems (25 μ L) of one standard include 2.5 μ 10 × annealing buffers of L II, 1 μ L100mM
MgSO4,2 μ L 500mM NaCl, 15 μM of μ L reverse primers, 15 μM of μ L forward primers, 1 μ L 52.5mM ATP solution, 1 μ L
3.5mM dNTPs,0.75μL Enzymatic mixture and 1 μ L 5 × SYBR Green I.
Whole reaction existsReal-time fluorescence detection is carried out in 96 System (Roche, Germany), per 60s
Once, design temperature is 60 DEG C to measure.
3) detection of product
Proved according to real-time fluorescence curves Fig. 2 as ATP concentration declines (from 3mM to 1.8mM), specific amplification and primer
The signal that dimer triggers all declines, but the speed that background declines is faster, and when ATP is 2.1mM, signal to noise ratio reaches maximum.
Fig. 3 prove with dNTPs concentration decline (from 0.20mM to 0.08mM), it is similar with ATP trend, the signal of specific amplification and
The background that primer dimer triggers all declines, and when dNTPs is 0.14mM, the background that primer dimer triggers is completely eliminated,
Now, signal to noise ratio reaches maximum.When adding 1 μ L 52.5mM ATP solution to HDA systems, during 1 μ L 3.5mM dNTPs, i.e. body
ATP concentration is 2.1mM in system, and dNTPs concentration is 0.08mM, and background is completely eliminated, and now HDA reaction systems acquired results
Signal to noise ratio it is maximum.The HDA amplification systems show that the present invention eliminates the notable effect of the background that primer dimer triggers in HDA
Fruit.
Embodiment 2
The detection of cellular telomerase, schematic diagram such as Fig. 4, comprises the following steps：
1) Telomerase extracts
1 × 10 will collected in logarithmic phase6Individual culture cell loads in 1.5ml Eppendorf centrifuge tubes, with frost PBS
Buffer solution (pH=7.4) cleans, and centrifuges (2000 revs/min, 10 minutes, 4 DEG C), is repeated twice.Obtained cell is resuspended in 200 μ L
Ice-cold 1 × CHAPS lysis buffers, freeze on ice 30 minutes, then with 12000 revs/min, 4 DEG C centrifuge 20 minutes.Will be upper
In the clean centrifuge tube of the careful immigration of clear liquid, -80 DEG C of preservations.
The cell extract of inactivation is incubated 15 minutes to be heated to 85 DEG C.
2) extraction of cellular telomerase
Forward primer is：5'-AAT CCG TCG AGC AGA GTT-3'；
Reverse primer is：5'-CCC TAA CCC TAA CCC TAA CCC TAA-3'；
All solution are diluted by RNase-free water.Telomere enzyme extract is diluted with 1 × CHAPS lysis buffers.Telomere
Enzymatic amplification system is that 6 μ L telomere enzyme extracts, 1 10 μM of μ L forward primers, 1 μ L 2mM dNTPs, and 1 μ 10 × Telomerases of L prolong
Stretch reaction buffer (20mM Tris-HCl, 1.5mM MgCl2, 1mM EGTA, 63mM KCl, 0.05%Tween 20).Entirely
System is in 37 DEG C of reactions, insulation 30 minutes.
3) zero background HDA implementation
By the above-mentioned Telomerase amplified productions of 2 μ L and 2.5 μ 10 × annealing buffers of L II, 1 μ L 100mM MgSO4,2 μ L
500mM NaCl, 15 μM of μ L reverse primers, 15 μM of μ L forward primers, 1 μ L52.5mM ATP solution, 1 μ L 3.5mM dNTPs,
0.75μL Enzymatic mixture and 1 μ L 5 × SYBR Green I.Whole reaction system is 25 μ L.
Whole reaction existsImplementation fluoroscopic examination is carried out in 96System (Roche, Germany), is surveyed per 60s
Determine once, design temperature is 60 DEG C.
4) detection of product
It can be seen that by real-time fluorescence curves Fig. 5, when telomerase product is added in system, specific amplification occurs, signal
Produce；When in system without Telomerase amplified production is added, signal 0, the background that primer dimer triggers is eliminated.Explanation
In zero background HDA reaction systems, primer dimer reasons for its use is eliminated completely, and specific amplification signal normally produces
It is raw.Fluorescence intensity is different as caused by Fig. 6 can draw the cellular telomerase amplified production of varying number, by fluorescence intensity and carefully
Born of the same parents' number log-linear graph of a relation 7, it can be seen that fluorescence intensity and cell number logarithm are linear within the specific limits.Linear regression
Equation is：F=0.273lg (N) -0.441 (R2=0.986, N are cell number).
The result (Fig. 8) drawn by detecting the different cell of telomerase activation can be seen that the application of the method
Extensively, and the degree of accuracy is high.In normal cell MRC-5 (people's normal chick embryo lung fibroblast) and heat-inactivated HeLa cells telomerase
Activity is very low, and in the HeLa not inactivated, the tumour such as MDA-MB-231 (human breast cancer cell) and A549 (human lung adenocarcinoma cell)
Telomerase activity in cells is high.In fig. 8, normal cell MRC-5 (people's normal chick embryo lung fibroblast) and heat-inactivated HeLa
The signal of the Telomerase amplified production of cell almost as blank control, illustrates Telomerase almost without activity.And do not inactivate
The Telomerase amplified production of tumour cell such as HeLa, MDA-MB-231 and A549 signal it is very high, illustrate the activity of Telomerase
It is high.The method of the opposite end telomerase activity detection illustrates that the application of the present invention is wide, and reliability is high, and obtained effect is fine.
In summary, the present invention reduces its background, this Research team has been probed into several heavy to improve HDA efficiency
Influence of the factor wanted to HDA, including enzyme concentration, reaction temperature, ATP concentration and dNTPs concentration.We have found that under enzyme concentration
Drop causes amplification rate to decline, but primer dimer still has.The increase of reaction temperature can not only reduce primer dimerization
The influence of body, also accelerates non-specific amplification, reduces the difference with template triggering specific amplification.Unexpected effect
It is that when adjusting ATP and dNTPs to a relatively low level at the same time, primer dimer is eliminated.By the present invention in that with
Appropriate ATP and dNTPs concentration eliminates the influence of primer dimer, obtains a kind of simple and general zero background HDA detections
Method.Zero background unwindase of the elimination primer dimer relies on system and extensive use be present.
Claims (7)
1. a kind of zero background unwindase for eliminating primer dimer relies on system, it is characterised in that
The system includes：2.5 μ L 10 × annealing buffer II, 1 μ L 100mM MgSO4, 2 μ L 500mM NaCl, the 5 of 1 μ L
μM reverse primer, 1 μ L 5 μM of forward primers, 1 μ L 52.5mM ATP solution, 1 μ L 3.5mM dNTPs, 0.75 μ L5 × SYBR Green I of enzymatic mixture and 1 μ L.
2. the zero background unwindase according to claim 1 for eliminating primer dimer relies on system, it is characterised in that chooses
40bp chain as template strand, then the 18 base identical 18bp held using template strand 5 ' chain as forward primer, with mould
Plate chain 3 ' holds the 24bp of 24 base complementrities chain as reverse primer.
3. a kind of zero background unwindase for eliminating primer dimer relies on amplification method, it is characterised in that comprises the following steps：
1) according to purpose fragment template design primer to be amplified, including forward primer and reverse primer：
Template sequence is：5'-AAT CCG TCG AGC AGA GTT AGG GTT AGG GTT AGG GTT AGG G-3'；
Designing forward primer is：5'-AAT CCG TCG AGC AGA GTT-3'；
Designing reverse primer is：5'-CCC TAA CCC TAA CCC TAA CCC TAA-3'；
2) HDA reaction systems are built, including：
2.5 μ L 10 × annealing buffer II, 1 μ L 100mM MgSO4, 2 μ L 500mM NaCl, 1 μ L 5 μM of reverse primers,
1 μ L 5 μM of forward primers, 1 μ L 52.5mM ATP solution, 1 μ L 3.5mM dNTPs, 0.75 μ LEnzyme mixes
5 × SYBR Green I of thing and 1 μ L；
3) each reactant in system is well mixed, carries out HDA isothermal duplications, amplification is detected according to fluorescence signal, sentenced
It is disconnected to realize that zero background rotation enzyme relies on amplified reaction.
4. the zero background unwindase according to claim 3 for eliminating primer dimer relies on amplification method, it is characterised in that
The HDA reaction systems of HDA reaction system reference standards described in step 2), processing is turned down to ATP and dNTPs concentration.
5. the zero background unwindase according to claim 3 for eliminating primer dimer relies on amplification method, it is characterised in that
Fluorescence signal detection amplification described in step 3) is to use96 System carry out real-time fluorescence detection, often
60s is determined once, and design temperature is 60 DEG C.
6. the zero background unwindase according to claim 3 for eliminating primer dimer relies on amplification method, it is characterised in that
Described template is single-stranded for the single-stranded of synthesis or after cellular telomerase amplification.
7. zero background unwindase of the elimination primer dimer described in claim 1 relies on system in detection Cell Telomerase Activity
In application.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1688709A (en) * | 2002-09-20 | 2005-10-26 | 新英格兰生物实验室公司 | Helicase dependent amplification of nucleic acids |
| CN102876813A (en) * | 2012-10-25 | 2013-01-16 | 中华人民共和国北京出入境检验检疫局 | Real-time fluorescence RT-HDA (Reverse Transcriptase-Helicase-Dependent Isothermal Amplification) kit and primer for detecting avian influenza virus |
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- 2015-09-11 CN CN201510579700.5A patent/CN105176974B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1688709A (en) * | 2002-09-20 | 2005-10-26 | 新英格兰生物实验室公司 | Helicase dependent amplification of nucleic acids |
| CN102876813A (en) * | 2012-10-25 | 2013-01-16 | 中华人民共和国北京出入境检验检疫局 | Real-time fluorescence RT-HDA (Reverse Transcriptase-Helicase-Dependent Isothermal Amplification) kit and primer for detecting avian influenza virus |
Non-Patent Citations (2)
| Title |
|---|
| Real-Time Detection of Transcription Factors Using Target-Concerted Helicase-Dependent Amplification Assay with Zero-Bcakground Signal;Anping Cao等;《Analytical chemistry》;20130115;第85卷;第2543-2547页 * |
| 赖解旋酶恒温基因扩增技术的研究进展;陈璐等;《医学综述》;20101231;第16卷(第13期);第1932-1934页 * |
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