CN104560947A - Application of DP (diglycerol phosphate) serving as additive in PCR (polymerase chain reaction) - Google Patents
Application of DP (diglycerol phosphate) serving as additive in PCR (polymerase chain reaction) Download PDFInfo
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- CN104560947A CN104560947A CN201310483541.XA CN201310483541A CN104560947A CN 104560947 A CN104560947 A CN 104560947A CN 201310483541 A CN201310483541 A CN 201310483541A CN 104560947 A CN104560947 A CN 104560947A
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- CN
- China
- Prior art keywords
- pcr
- ethylhexyl
- glyceryl ester
- phosphate
- phosphate glyceryl
- Prior art date
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- 239000000654 additive Substances 0.000 title claims abstract description 18
- 230000000996 additive effect Effects 0.000 title claims abstract description 16
- 150000002319 glycerophosphoglycerols Chemical class 0.000 title abstract description 6
- 238000003752 polymerase chain reaction Methods 0.000 title abstract description 6
- 230000000694 effects Effects 0.000 claims abstract description 10
- 238000012408 PCR amplification Methods 0.000 claims abstract description 7
- 238000000034 method Methods 0.000 claims abstract description 5
- 238000006243 chemical reaction Methods 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 9
- 239000007864 aqueous solution Substances 0.000 claims description 5
- 239000012634 fragment Substances 0.000 claims description 3
- 239000012747 synergistic agent Substances 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 230000002195 synergetic effect Effects 0.000 claims 1
- 108020004414 DNA Proteins 0.000 abstract description 8
- 102000053602 DNA Human genes 0.000 abstract 2
- 241000894006 Bacteria Species 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 238000013467 fragmentation Methods 0.000 description 3
- 238000006062 fragmentation reaction Methods 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 150000007523 nucleic acids Chemical group 0.000 description 2
- -1 polyoxyethylene Polymers 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000010359 gene isolation Methods 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention aims to provide an application of DP (diglycerol phosphate) serving as an additive in a PCR (polymerase chain reaction). The high-temperature tolerance degree of DNA (deoxyribonucleic acid) polymerase is increased with a DP adding method, so that the PCR can be performed more stably at high temperature, and the PCR amplification effect is improved.
Description
Technical field
The present invention relates to a kind of raising PCR additive, especially di(2-ethylhexyl)phosphate glyceryl ester is used for improving PCR effect as additive.
Background technology
Polymerase chain reaction (being called for short PCR) is a kind of Protocols in Molecular Biology, for in vitro enzyme' s catalysis DNA, reacted by a few steps such as high-temperature denatured, low-temperature annealing and appropriateness extend and form one-period, circulation is carried out, and target DNA can be made at short notice to increase rapidly.There is high specificity, highly sensitive, easy and simple to handle, the feature such as save time.It not only can be used for the fundamental researchs such as gene isolation, clone and nucleic acid sequence analysis, also can be used for medical diagnosis on disease or any place having DNA, RNA.Invented by American scientist Dr.Mullis, due to round pcr on Theory and applications across significance of times, therefore Mullis obtains Nobel chemistry Prize in 1993.
In recent years, it is found that, in PCR reaction system, add a certain amount of additive can improve pcr amplification efficiency and specificity, reduce or eliminate the generation of side reaction.Common additive has: DMSO, bovine serum albumin (BSA), glycerine, polyoxyethylene glycol, tween 20 etc.But these additives respectively have its limitation.Developing new pcr amplification additive is a very important job, to be applicable to the fragment amplification of various complicated.
Di(2-ethylhexyl)phosphate glyceryl ester (diglycerol phosphate, DP) is a kind of solute of thermophile bacteria body accumulation, and main discovery is present in thermophile bacteria and the ancient bacterium of hyperthermophilic.Its Main Function is the function with biomacromolecules such as protected protein, nucleic acid, cytolemma, enzymes under high temperature, thus reaches the infringement of assisting thermophile bacteria to resist external high temperature.
Summary of the invention
The object of this invention is to provide a kind of PCR additive, to improve pcr amplification effect, Be very effective, consumption is few, and cost is low, easy to operate, use safety.
What realize the object of the invention is a kind of di(2-ethylhexyl)phosphate glyceryl ester PCR additive, adds di(2-ethylhexyl)phosphate glyceryl ester in PCR reaction system.
The final concentration of described di(2-ethylhexyl)phosphate glyceryl ester in PCR reaction system is 3-5mg/ml.
The beneficial effect of a kind of PCR synergistic agent of the present invention is as follows:
By adding di(2-ethylhexyl)phosphate glyceryl ester in PCR reaction system, improve the tolerance degree of polysaccharase to temperature, thus make PCR react at high temperature can be more stable carrying out, thus improve the effect of PCR.
Di(2-ethylhexyl)phosphate glyceryl ester is utilized to have following functions as the synergistic agent of the raising PCR effect of additive: (1) adds di(2-ethylhexyl)phosphate glyceryl ester in PCR reaction system, really improve PCR effect, and cost is low.(2) di(2-ethylhexyl)phosphate glyceryl ester on subsequent operations without impact.(3) method of di(2-ethylhexyl)phosphate glyceryl ester raising pcr amplification effect provided by the invention is simple to operate, is easy to carry out.
Embodiment
The present invention is described in detail by following examples by reference to the accompanying drawings, but is not limited to following embodiment.
The method that the present invention relates to PCR optimization adds di(2-ethylhexyl)phosphate glyceryl ester to realize in PCR reaction system, and concrete steps are as follows:
1. configure the di(2-ethylhexyl)phosphate glyceryl ester aqueous solution: take commercially available di(2-ethylhexyl)phosphate glyceryl ester 3-6mg, be placed in sterilized 1.5ml centrifuge tube, add aqua sterilisa and make it dissolve, be finally settled to 1ml, gained solution carries out 121 DEG C, 20min sterilizing.
The configuration of 2.PCR reaction system: DNA fragmentation to be amplified is different, dNTP, Mg in PCR reaction system
2+, template add-on different; In above-mentioned reaction system, add appropriate di(2-ethylhexyl)phosphate glyceryl ester solution, the addition of distilled water should do corresponding reducing according to adding of di(2-ethylhexyl)phosphate glyceryl ester solution.
3.PCR reacts operation: denaturation, sex change, annealing temperature and time should be selected according to template, and the extension time is selected according to the length of the object fragment that will increase.
The detection of 4.PCR amplified production: detect with agarose gel electrophoresis.
Embodiment 1: di(2-ethylhexyl)phosphate glyceryl ester is to the optimized expansion of the DNA fragmentation of 9kb
1. configure the 4mg/ml di(2-ethylhexyl)phosphate glyceryl ester aqueous solution.
The configuration of 2.PCR reaction system:
10×PCR Buffer 2.5ul
dNTP（2.5mM） 5ul
Primer 1(2.0uM) 1ul
Primer 2 (2.0uM) 1ul
Mg
2+（25mM） 1.5ul
After configuring above-mentioned reaction system, add 14ul di(2-ethylhexyl)phosphate glyceryl ester aqueous solution additive respectively, in accompanying drawing, 1,2 represent control group respectively and add di(2-ethylhexyl)phosphate glyceryl ester solution additive group, like this, the final concentration of di(2-ethylhexyl)phosphate glyceryl ester solution in PCR reaction system is 2.8mg/ml.Control group changes di(2-ethylhexyl)phosphate glyceryl ester solution into 14ul sterilizing distilled water, as in accompanying drawing 5.
3.PCR reacts operation:
Denaturation 94 DEG C of 4min
Sex change 94 DEG C of 30s
Anneal 58 DEG C of 30s } 32
Extend 72 DEG C of 5min
Further extension 72 DEG C of 5min
Detect with agarose gel electrophoresis after reaction under 4.PCR: result as shown in the figure, Maker used is Tian Gen company kb marker, as can be seen from the figure the group adding di(2-ethylhexyl)phosphate glyceryl ester additive is obviously bright than control group band, illustrates that di(2-ethylhexyl)phosphate glyceryl ester can improve PCR effect really.
Accompanying drawing explanation
Fig. 1 utilizes the DNA fragmentation result that di(2-ethylhexyl)phosphate glyceride optimized expansion length is about 9kb in the present invention, in figure, 1,2 represent control group respectively and add di(2-ethylhexyl)phosphate glyceryl ester solution additive group.
Claims (4)
1. di(2-ethylhexyl)phosphate glyceryl ester improves a method for expanding effect as PCR Synergistic additives, it is characterized in that, first di(2-ethylhexyl)phosphate glyceryl ester is dissolved in sterilized water and prepares di(2-ethylhexyl)phosphate glyceryl ester solution.
2. PCR additive according to claim 1, is characterized in that: the concentration of described di(2-ethylhexyl)phosphate glyceryl ester solution is the di(2-ethylhexyl)phosphate glyceryl ester aqueous solution of 3-6mg/ml.
3. PCR synergistic agent according to claim 1 and 2, is characterized in that: add the di(2-ethylhexyl)phosphate glyceryl ester aqueous solution in PCR reaction system after, the final concentration of di(2-ethylhexyl)phosphate glyceryl ester is 0.8-3mg/ml.
4. di(2-ethylhexyl)phosphate glyceryl ester according to claim 1 improves the method for pcr amplification effect, and it is characterized in that, described pcr amplification comprises: Standard PCR, the PCR of high GC fragment PCR, complex sequence.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310483541.XA CN104560947A (en) | 2013-10-16 | 2013-10-16 | Application of DP (diglycerol phosphate) serving as additive in PCR (polymerase chain reaction) |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310483541.XA CN104560947A (en) | 2013-10-16 | 2013-10-16 | Application of DP (diglycerol phosphate) serving as additive in PCR (polymerase chain reaction) |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104560947A true CN104560947A (en) | 2015-04-29 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310483541.XA Pending CN104560947A (en) | 2013-10-16 | 2013-10-16 | Application of DP (diglycerol phosphate) serving as additive in PCR (polymerase chain reaction) |
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| CN (1) | CN104560947A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0354474A2 (en) * | 1988-08-04 | 1990-02-14 | Thomas Glonek | Method and composition for cryopreservation of tissue |
| EP0965268A1 (en) * | 1998-04-08 | 1999-12-22 | IBET - Instituto de Biologia Experimental e Tecnologica | Thermostabilization, osmoprotection, and protection against desiccation of enzymes, cell components and cells by di-glycerol-phosphate |
-
2013
- 2013-10-16 CN CN201310483541.XA patent/CN104560947A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0354474A2 (en) * | 1988-08-04 | 1990-02-14 | Thomas Glonek | Method and composition for cryopreservation of tissue |
| EP0965268A1 (en) * | 1998-04-08 | 1999-12-22 | IBET - Instituto de Biologia Experimental e Tecnologica | Thermostabilization, osmoprotection, and protection against desiccation of enzymes, cell components and cells by di-glycerol-phosphate |
Non-Patent Citations (2)
| Title |
|---|
| PEDRO LAMOSA ET AL.,: "Thermostabilization of Proteins by Diglycerol Phosphate, a New Compatible Solute from the Hyperthermophile Archaeoglobus fulgidus", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
| POOJA SHIVANAND ET AL.,: "Halophilic bacteria and their compatible solutes-osmoregulation and potential applications", 《CURRENT SCIENCE》 * |
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