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Optimizing method of amplifying nucleic acid polymerase chain reaction

A nucleic acid polymerase and chain reaction technology, applied in the biological field, can solve the problems of complicated extraction and purification of SSB protein, expensive commercial kits and high preparation cost, and achieves the effects of simple operation, low cost and easy storage.

Inactive Publication Date: 2006-12-13
苏州市长三角系统生物交叉科学研究院有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the complex technology of extracting and purifying SSB protein and the high requirement of reagent purity, the preparation cost is very high, and the commercial kit is very expensive, and the price is 6-7 times that of conventional PCR reagents; at the same time, in order to maintain the biological activity of the single-chain binding protein, Requires strict storage at -20°C, and its biological activity has a short period of time

Method used

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  • Optimizing method of amplifying nucleic acid polymerase chain reaction
  • Optimizing method of amplifying nucleic acid polymerase chain reaction
  • Optimizing method of amplifying nucleic acid polymerase chain reaction

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Experimental program
Comparison scheme
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Embodiment 1

[0030] The optimization of embodiment 1 titanium powder to PCR reaction

[0031] For the optimization and improvement of some PCR amplifications that form non-specific amplification, the following steps are included:

[0032] Takara Ex Taq Enzyme (5U / μL)

0.25 μL

10×PCR buffer

2.5 μL

dNTP substrate (10mM)

0.75 μL

Mg 2+ (250mM)

0.35μL

Primer 1 (1 μM)

2.5 μL

Primer 2 (1 μM)

2.5 μL

template

1μL

h 2 o

Add water to make up to a total volume of 25 μL

[0033] Among them, the template is λDNA, purchased from Huamei Bioengineering Company, Ex Taq enzyme is purchased from Takara Company, and the length of the amplified fragment is 283bp.

[0034] 2. Add treated optimized material titanium powder to the above PCR system, add no less than 100 μg to each 25 μL reaction system, and do corresponding control experiments without optimization medium; and control experiments with colloidal gold o...

Embodiment 2

[0038] Example 2 Optimization of Titanium Wire for Multiplex PCR Amplification

[0039] Optimization and improvement for multiplex PCR amplification, including the following steps:

[0040] 1. Prepare a PCR system, the composition of which is as in Example 1, except that 8 pairs of primers are used for multiplex PCR amplification to amplify DNA molecules with lengths of 159bp, 283bp, 485bp, 573bp, 660bp, 785bp, 942bp and 1240bp respectively.

[0041] 2. Add the treated titanium wire, the optimized medium material of this patent, with a diameter of 0.5mm and a length of 3mm. At the same time, do the corresponding control experiment without the optimization medium; and the control experiment of adding colloidal gold optimization.

[0042] 3. The template molecule is amplified by PCR technology, and the amplification conditions are the same as in Example 1.

[0043] 4. Perform agarose gel electrophoresis detection on the amplified DNA sample.

[0044]The results are shown in Fi...

Embodiment 3

[0045] Embodiment 3 The optimization of other different optimization materials to PCR reaction

[0046] For the optimization and improvement of some PCR amplifications that form non-specific amplification, the system and steps are exactly the same as in Example 1, except that the optimized medium is other materials.

[0047] attached Figure 4 Description: The top row is 1, 2 - adding nickel wire, adding nickel wire with a diameter of 0.5 mm and a length of 3 mm to each system; 3, 4 - adding bismuth particles, adding spherical bismuth with a diameter of 2 mm to each system 1 particle; 5, 6- add antimony particles, add 1 spherical particle with a diameter of 2mm to each system; 7, 8- add silica particles, add 4 spherical particles with a diameter of 1mm to each system; 9, 10 - add selenium powder, the mass added to each system is 400μg; the lower row is 11 - molecular weight markers, 12, 13 - add tin foil, each system is added with a thickness of 0.2mm, a length of 2mm, and a ...

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Abstract

An optimizational method by way of nucleic acid polymerase chain augmenting reaction. First, prepare optimizational medium material. Second, disinfect the material. Third, optimizate PCR system and detect PCR product. Finally, optimizational material is separated from the reaction system. Add the material to PCR system to realize PCR augmenting optimization.

Description

technical field [0001] The invention relates to a method in the field of biotechnology, in particular to an optimization method for nucleic acid polymerase chain reaction amplification. Background technique [0002] Nucleic acid polymerase chain reaction (Polymerase Chain Reaction, PCR) is a nucleic acid amplification technology that simulates natural DNA replication in vitro. The detailed mechanism is very complicated, and a certain degree of interfering side reactions will always occur during the chain reaction. For example, primer templates may be mismatched, primers combine to form dimers, etc. These side reactions can cause non-specific amplification (smeared tailing bands and non-specific bands in subsequent electrophoresis detection), leading to low amplification efficiency, and severe ones may lead to failure of normal amplification reactions. Improving the specificity of PCR amplification not only depends on the optimized design of primer sequences, but also largel...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/34
Inventor 李海阔黄洁欢张晓东胡钧樊春海张治洲
Owner 苏州市长三角系统生物交叉科学研究院有限公司