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Optimized method for long-fragment nucleic acid polymerase chain reaction expanding based on nano metallic particles

A nano metal particle, nucleic acid polymerase technology, applied in the field of molecular biology, can solve the problems of limitation, high price, unknown amplification effectiveness, etc., and achieve the needs of convenient amplification of long fragments, low cost, and wide application range. Effect

Inactive Publication Date: 2008-03-05
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although in the process of product competition, the world-renowned major biological reagent companies have developed long-fragment amplification kits suitable for different lengths and different templates, which greatly facilitates our needs for amplifying long fragments, but these kits are also It has inevitable disadvantages: high price and unknown amplification effectiveness, which is undoubtedly a great limitation for the further expansion and application of Long-PCR method

Method used

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  • Optimized method for long-fragment nucleic acid polymerase chain reaction expanding based on nano metallic particles
  • Optimized method for long-fragment nucleic acid polymerase chain reaction expanding based on nano metallic particles
  • Optimized method for long-fragment nucleic acid polymerase chain reaction expanding based on nano metallic particles

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preparation example Construction

[0031] 1. Preparation of elemental nano-metal colloid solution:

[0032] The elemental nanometer metal colloid solution can be self-made or a commercially available product, which belongs to the prior art and will not be described in detail.

[0033] 2. Pretreatment of elemental nano-metal colloid solution:

[0034] In order to meet the special needs of PCR reactions, the prepared or commercially available elemental nano-metal colloid solutions need to use certain methods to kill DNase and RNase. Specifically: Dispense into 1ml small centrifuge tubes after inactivating the enzyme, and put it under the ultraviolet light for 30 minutes; or use a micro air filter membrane to inactivate the enzyme.

[0035] 3. Configuration of PCR system

[0036] The Long-PCR system is based on the common reaction system of Taq enzyme and Pfu enzyme, and the ratio of the two enzymes in a system with a volume of 25 μL can be about 2.5U: 1.25U; other DNA polymerases can be based on General PCR re...

Embodiment 1

[0056] High-concentration nano-gold colloidal solution and nano-platinum colloidal solution optimize the amplified DNA fragment with a fragment length of 12kb

[0057] 1. Configuration of PCR reaction system:

[0058] Configure the system in a thin-walled PCR tube in the following order and dosage.

[0059] 10×PCR buffer 2.5μL

[0060] dNTP substrate (2.5mM) 5μL

[0061] Primer 1 (2.0 μM) 3.75 μL

[0062] Primer 2 (2.0μM) 3.75μL

[0063] Template (0.1μg / μL) 0.5μL

[0064] Mg 2+ (25mM) 2.8μL

[0065] Taq enzyme (5U / μL) 0.5μL

[0066] Pfu enzyme (2.5U / μL) 0.5μL

[0067] Among them, the template is λDNA, which was purchased from Canada BBI Company, and Taq enzyme and Pfu enzyme were purchased from Beijing Sanbo Yuanzhi Bioengineering Company.

[0068] The system adding gold colloidal solution and platinum colloidal solution is equipped with 8 tubes respectively, and is numbered from 0 to 7.

[0069] 2. Add 0 μL, 1.0 μL, 2.0 μL, 2.5 μL, 3.0 μL, 3.5 μL, 4.0 μL, 4.5 μL of ...

Embodiment 2

[0078] High-concentration nano-gold colloidal solution and nano-platinum colloidal solution optimize the amplified DNA fragment with a fragment length of 15kb

[0079] 1. Configuration of PCR reaction system:

[0080] Configure the system in a thin-walled PCR tube in the following order and dosage.

[0081] 10×PCR buffer 2.5μL

[0082] dNTP substrate (2.5mM) 5μL

[0083] Primer 1 (2.0 μM) 3.75 μL

[0084] Primer 2 (2.0μM) 3.75μL

[0085] Template (0.1μg / μL) 0.5μL

[0086] Mg 2+ (25mM) 2.8μL

[0087] Taq enzyme (5U / μL) 0.5μL

[0088] Pfu enzyme (2.5U / μL) 0.5μL

[0089] Among them, the template is λDNA, which was purchased from Canada BBI Company, and Taq enzyme and Pfu enzyme were purchased from Beijing Sanbo Yuanzhi Bioengineering Company.

[0090] The system adding gold colloidal solution and platinum colloidal solution is equipped with 7 tubes respectively, and is numbered from 0 to 6.

[0091] 2. Add 0 μL, 2.5 μL, 3.0 μL, 3.5 μL, 4.0 μL, 4.5 μL, 5.0 μL from tube 0...

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Abstract

The present invention is one optimized long segment nucleic acid PCR amplification system based on nanometer metal particle, and belongs to the field of molecular biology technology. One Long-PCR system is configured on the basis of available PCR amplification process, and the Long-PCR system has at least 10 vol% of 0.01 wt% concentration nanometer metal colloid solution or nanometer metal particles in the ultimate concentration not lower than 0.001 wt% added into the PCR system. The present invention has simple operation, obvious optimizing effect, low cost and wide application.

Description

technical field [0001] The invention relates to a method for polymerase chain reaction (PCR) amplification in the technical field of molecular biology, in particular to an optimization method for long-segment polymerase chain reaction (Long-PCR) amplification of high-concentration nanometer metal reagents . Background technique [0002] Polymerase chain reaction (PCR), also known as in vitro enzymatic gene amplification, is a gene amplification technology that simulates DNA replication in vitro. This technology was invented by K.Mullis in 1985. Under the action of enzymes and the guidance of specific primers, a large number of gene duplications that only occur when organisms cell division and proliferation can be realized in a test tube in a very short period of time. Generally speaking, within a few hours. Amplify the gene of interest to a million-fold. [0003] Over the past 20 years, PCR technology has continued to improve and develop. During this process, a series of P...

Claims

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Application Information

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IPC IPC(8): C12P19/34C12Q1/68
Inventor 张治洲汪名春王群曹小红
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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