CN102876659A - Improved high-specificity polymerase chain reaction (PCR) method - Google Patents
Improved high-specificity polymerase chain reaction (PCR) method Download PDFInfo
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- CN102876659A CN102876659A CN2011102003852A CN201110200385A CN102876659A CN 102876659 A CN102876659 A CN 102876659A CN 2011102003852 A CN2011102003852 A CN 2011102003852A CN 201110200385 A CN201110200385 A CN 201110200385A CN 102876659 A CN102876659 A CN 102876659A
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- high specific
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- interlayer
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Abstract
The invention belongs to the technical field of biology and novel medicines. The overall technical conception is that: the specificity of polymerase chain reaction (PCR) can be improved to some extent by hot-start PCR and touchdown PCR, and the two methods are combined and improved to some extent, so that non-specific products and backgrounds are reduced to the maximum, and a high-specificity PCR method is obtained.
Description
Technical field
Be applicable to the various experiments relevant with genetic expression and research field.
Technical background
(Polymerase Chain Reaction) is called for short in the polymerase chain reaction, is called for short PCR.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 the diagnosis of disease or the place of any DNA of having, RNA, have high specificity, highly sensitive, easy and simple to handle, the characteristics such as save time.Even so, in many PCR experiments false positive and non-specific amplification often appear still, for many experiments have brought huge puzzlement.Therefore, a kind of PCR method of high specific seems particularly important for scientific experiment and research.
Summary of the invention
The object of the present invention is to provide a kind of PCR method of high specific.
Overall technology design of the present invention is: heat start PCR and touchdown PCR can improve the specificity of PCR to a certain extent, this paper combines these two kinds of methods, and improve to a certain extent, reduce to the full extent non-specific product and background, thereby obtain a kind of PCR method of high specific.
The improvement PCR method of high specific is as follows:
(1) wax-wrapped pill is added dNTP, MgCl simultaneously
2, primer and part 10 * damping fluid heat 80 wax-wrapped pills and melt, and the room temperature cooling forms a kind of wax interlayer on the surface.
(2) add DNA Taq enzyme at the wax interlayer, dna profiling, and part 10 * damping fluid enter the PCR circulation.
(3) PCR programdesign: renaturation temperature initial value is set to 70 ℃, descends 1 ℃ until the suitableeest annealing temperature, each temperature cycle 2-4 time at every turn.
After (4) the 3rd steps finished, again in sex change, renaturation was extended, and carries out about 30 circulations under the suitableeest program.
(5) last, extend 5-10min after 72 ℃.
(6) at last 4 ℃ of lower preservations.
The present invention not only can avoid because the mispairing of primer and non-target site and primer form the amplification that dimer causes, can also avoid because Mg
2+Inappropriate, the pair of primers of Tm (texturing temperature) value and Ta (annealing temperature) value apart from each other, the false-positive appearance that the complicated reasons such as template DNA cause.Compare with traditional PCR, can significantly increase the amount of special product, reduce simultaneously or eliminated non-specific product.
Below in conjunction with example the present invention is further described
The improvement PCR method of high specific is as follows:
(7) wax-wrapped pill is added dNTP, MgCl simultaneously
2, primer and part 10 * damping fluid heat 80 wax-wrapped pills and melt, and the room temperature cooling forms a kind of wax interlayer on the surface
(8) add DNA Taq enzyme at the wax interlayer, dna profiling, and part 10 * damping fluid enter the PCR circulation.
(9) PCR programdesign: renaturation temperature initial value is set to 70 ℃, descends 1 ℃ until the suitableeest annealing temperature, each temperature cycle 2-4 time at every turn.
After (10) the 3rd steps finished, again in sex change, renaturation was extended, and carries out about 30 circulations under the suitableeest program.
(11) last, extend 5-10min after 72 ℃.
At last 4 ℃ of lower preservations.
Claims (5)
1. the improvement PCR method of high specific is as follows:
(1) wax-wrapped pill is added dNTP, MgCl simultaneously
2, primer and part 10 * damping fluid heat 80 wax-wrapped pills and melt, and the room temperature cooling forms a kind of wax interlayer on the surface
(2) add DNA Taq enzyme at the wax interlayer, dna profiling, and part 10 * damping fluid enter the PCR circulation.
(3) PCR programdesign: renaturation temperature initial value is set to 70 ℃, descends 1 ℃ until the suitableeest annealing temperature, each temperature cycle 2-4 time at every turn.
After (4) the 3rd steps finished, again in sex change, renaturation was extended, and carries out about 30 circulations under the suitableeest program.
(5) last, extend 5-10min after 72 ℃.
(6) at last 4 ℃ of lower preservations.
2. the improvement PCR method of high specific according to claim 1 is characterized in that step (1) adds dNTP, MgCl simultaneously with wax-wrapped pill
2, primer and part 10 * damping fluid heat 80 wax-wrapped pills and melt, and the room temperature cooling forms a kind of wax interlayer on the surface.
3. the improvement PCR method of high specific according to claim 1 is characterized in that step (2) adds DNA Taq enzyme at the wax interlayer, and dna profiling, and part 10 * damping fluid enter the PCR circulation.
4. the improvement PCR method of high specific according to claim 1, it is characterized in that step (3) PCR programdesign: renaturation temperature initial value is set to 70 ℃, descends 1 ℃ until the suitableeest annealing temperature, each temperature cycle 2-4 time at every turn.
5. the improvement PCR method of high specific according to claim 1, it is characterized in that the 3rd step of step (4) finishes after, again in sex change, renaturation is extended, and carries out under the suitableeest program about 30 circulations.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011102003852A CN102876659A (en) | 2011-07-12 | 2011-07-12 | Improved high-specificity polymerase chain reaction (PCR) method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011102003852A CN102876659A (en) | 2011-07-12 | 2011-07-12 | Improved high-specificity polymerase chain reaction (PCR) method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102876659A true CN102876659A (en) | 2013-01-16 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011102003852A Pending CN102876659A (en) | 2011-07-12 | 2011-07-12 | Improved high-specificity polymerase chain reaction (PCR) method |
Country Status (1)
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1238387A (en) * | 1999-06-22 | 1999-12-15 | 夏庆杰 | Double-chain primer polymerase chain reaction |
| CN1900307A (en) * | 2005-07-22 | 2007-01-24 | 陕西师范大学 | Process for preparing competitive template for quick quantitatively detecting heatstable bacterium QC-PCR |
| CN1900308A (en) * | 2005-07-22 | 2007-01-24 | 陕西师范大学 | Method of quick quantitatively detecting heatstable bacterium QC-PCR |
-
2011
- 2011-07-12 CN CN2011102003852A patent/CN102876659A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1238387A (en) * | 1999-06-22 | 1999-12-15 | 夏庆杰 | Double-chain primer polymerase chain reaction |
| CN1900307A (en) * | 2005-07-22 | 2007-01-24 | 陕西师范大学 | Process for preparing competitive template for quick quantitatively detecting heatstable bacterium QC-PCR |
| CN1900308A (en) * | 2005-07-22 | 2007-01-24 | 陕西师范大学 | Method of quick quantitatively detecting heatstable bacterium QC-PCR |
Non-Patent Citations (1)
| Title |
|---|
| 李建科等: "耐热菌的竞争定量PCR 检测方法优化与建立", 《中国农业科学》, vol. 39, no. 2, 10 February 2006 (2006-02-10), pages 375 - 380 * |
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Application publication date: 20130116 |