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PCR amplification additive composition used for high-GC gene, and PCR amplification method of high-GC gene

An additive and composition technology, applied in the field of bioengineering, can solve the problems of organic solvents harmful to human body and inconvenient use, and achieve the effect of high specificity, single band, and low requirement for DNA polymerase

Inactive Publication Date: 2014-08-13
江苏佰龄全基因生物医学技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In the prior art, organic solvents such as ethylene glycol, ethyl acetate, n-pentane, etc. are used to increase the amplification efficiency of DNA fragments with GC content. The disadvantage is that the organic solvents involved are harmful to the human body and are inconvenient to use.

Method used

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  • PCR amplification additive composition used for high-GC gene, and PCR amplification method of high-GC gene
  • PCR amplification additive composition used for high-GC gene, and PCR amplification method of high-GC gene
  • PCR amplification additive composition used for high-GC gene, and PCR amplification method of high-GC gene

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Embodiment 1

[0028] The PCR amplification of embodiment 1FMR-1 gene

[0029] Fragile X syndrome is a common inherited mental retardation syndrome. The molecular genetic basis of its pathogenesis is the fragile X syndrome mental retardation gene FMR-1 (CGG) n Structural mutation. The length of the target gene amplified in this example is 558bp, and the GC content is 78%. The specific implementation method is as follows:

[0030] 1. Template preparation

[0031] Using a commercially available genomic DNA extraction kit, the concentration of extracted DNA is 100 ng / μl, the 260 / 230 value is greater than 2.0, and the 260 / 280 value is greater than 1.8.

[0032] 2. Primer design

[0033] According to the sequence of the causative gene FMR1 of fragile X syndrome, the primers were designed by using Oligo6.0 software as follows:

[0034] Upstream primer: 5'-CGACCTGTCACCGCCCTTCAG-3'

[0035] Downstream primer: 5'-AGCCCCGCACTTCCACCACC-3'

[0036] 3. PCR reaction

[0037] PCR amplification sys...

Embodiment 2

[0051]The PCR amplification of embodiment 2FMR-1 gene

[0052] Fragile X syndrome is a common inherited mental retardation syndrome. The molecular genetic basis of its pathogenesis is the fragile X syndrome mental retardation gene FMR-1 (CGG) n Structural mutation. The length of the target gene amplified in this example is 558bp, and the GC content is 80%. The specific implementation method is as follows:

[0053] 1. Template preparation

[0054] Using a commercially available genomic DNA extraction kit, the concentration of extracted DNA is 100 ng / μl, the 260 / 230 value is greater than 2.0, and the 260 / 280 value is greater than 1.8.

[0055] 2. Primer design

[0056] According to the sequence of the causative gene FMR-1 of fragile X syndrome, the primers were designed by Oligo6.0 software as follows:

[0057] Upstream primer: 5'-GCGCTCAGCTCCGTTTCGGTT-3'

[0058] Downstream primer: 5'-AGCCCCGCACTTCCACCACC-3'

[0059] 3. PCR reaction

[0060] In this embodiment, there a...

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Abstract

The invention provides a PCR amplification additive composition used for high-GC gene. The amplification additives are selected from two or more of 7-deaza-dGTP, betaine or betaine analog, DMSO, glycerin, formamide and polyethylene glycol. Compared with additives and the prior art, the PCR additive and an amplification method can realize the amplification of target gene with the template GC content of above 80%, and can realize the successful amplification of complex gene to be used in clinic detection; a PCR amplification obtained in the invention has the advantages of high specificity, single brand and no purification, and can be directly used in subsequent operations; and the PCR method has a low requirement on DNA polymerase, and can match with various commercial PCR kits.

Description

technical field [0001] The invention relates to a PCR amplification additive for genes with high GC content and a PCR amplification method for genes with high GC content, belonging to the technical field of bioengineering. Background technique [0002] Polymerase Chain Reaction (PCR) is an in vitro nucleic acid amplification technique developed in the mid-1980s. The PCR process is generally divided into three stages: denaturation, annealing, and extension. First, the DNA is denatured and melted under high temperature conditions. When the temperature drops to about 55°C, the primer binds to the template DNA single strand, and then proceeds under the action of DNA polymerase. DNA synthesis. Compared with the previously commonly used bacteria to amplify target genes, it has the advantages of strong specificity, high sensitivity, simplicity, good repeatability, and easy automation. PCR technology has been developed for nearly 30 years. With the continuous advancement of techno...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 潘海波邢楠楠谢阳华琴
Owner 江苏佰龄全基因生物医学技术有限公司
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