PCR amplification system and PCR amplification method for high GC content gene

An amplification system and content technology, applied in the field of bioengineering, can solve the problems of limited scope of application, harsh requirements for primers and templates, etc., and achieve the effect of short operation time, simple equipment and few operation steps

Inactive Publication Date: 2010-02-10
陈学军
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although the above-mentioned methods and means have achieved certain effects, the requirements for primers and templates are generally too harsh, and the scope of application is relatively limited, so the improvement on these basis is extremely urgent and has great application value

Method used

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  • PCR amplification system and PCR amplification method for high GC content gene
  • PCR amplification system and PCR amplification method for high GC content gene
  • PCR amplification system and PCR amplification method for high GC content gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment one: PCR amplification of cyclodehydratase (Cyclodehydrase) gene in actinomycetes

[0063] Cyclodehydratase is a key enzyme in the metabolic pathway of actinomycetes (Kelly WL, et al. J Am Chem Soc. 2009, 131, 4327-4334). The target gene amplified in this example has a length of 993bp, a melting temperature of 98.2°C, and a GC content of over 70%. Conventional PCR amplification techniques are difficult to correctly amplify the target gene with high specificity using degenerate primers, but using the present invention Described PCR amplification technique then can successfully amplify, and the specific implementation mode is as follows:

[0064] 1. Template Preparation

[0065] 1) Mycelia culture: Streptomyces lawn is dug from the inclined surface and inoculated into the seed medium, shaken and cultivated at 250rpm at 32°C for 72 hours, replanted in 25ml fermentation medium with 4% replanting amount, shaken at 250rpm The bed was shaken at 32°C for 48 hours. ...

Embodiment 2

[0119] Example 2: PCR amplification of the hamster guanosine triphosphate-binding protein transcription termination factor (GSPT2) gene

[0120] Hamster guanosine triphosphate-binding protein transcription termination factor plays a key role in the transition from the completion of mitosis to the gap phase (G1 phase) before DNA replication to the DNA replication phase (S phase) in the cell cycle of yeast and animal cells (Le Goff C, et al.Genes Cells.2003, 7, 1043-10557), the length of the target gene amplified in this example is 415bp, the melting temperature is 92.8°C, and the GC content is 60.48%. Conventional PCR is difficult to amplify increase, and use the PCR amplification technique of the present invention to amplify successfully, the specific implementation is as follows:

[0121] 1. Template Preparation

[0122] Use the Blood Genomic DNA Extraction Kit (spin column type) purchased from Jerry Biological Company to extract, the specific steps are as follows:

[0123]...

Embodiment 3

[0168] Example 3: PCR amplification of rice Dof (DNA binding with one finger) zinc finger protein gene

[0169] Rice Dof protein has multiple functions and participates in the regulation of various life activities in rice (Tanaka M, et al. Planta. 2009). The length of the protein gene is 472bp, the melting temperature is 97.4°C, and the GC content is 71.4%. It needs to add expensive 7-Deaza-dGT and various additives to amplify the target gene. It can be effectively amplified, and the specific implementation method is as follows:

[0170] 1. Template Preparation

[0171] Rice total DNA was extracted by a modified cetyltrimethylammonium bromide (CTAB) method.

[0172] 1) Take about 1 g of tender rice leaves, wash them with ultrapure water, blot them dry with filter paper, freeze and grind them;

[0173] 2) Divide into four 7ml centrifuge tubes, add 3ml CTAB to each tube, incubate at 65°C for 30min, then add 15μl proteinase K (10mg / ml) at 55°C for 30min;

[0174] 3) Add chlor...

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Abstract

The invention discloses a PCR amplification system and a PCR amplification method for high GC content gene, belonging to the technical field of biotechnology; the PCR amplification system comprises DNA polymerase, MgCl2, dNTP, upstream primer, downstream primer, DNA template and double distilled water; and the invention is characterized in that the PCR amplification system also comprises tri(hydroxymethyl)aminomethane hydrochloride, dithiothreitol, bovine serum albumin and additive. The PCR amplification technology in the invention can amplify target genes containing over 80% of GC, can amplify high GC content target genes with a length up to 1.5Kb and can successfully amplify template genes with different degrees of complexity and different sources such as people, animals, plants and microorganisms; in addition, the PCR amplification technology has less operating steps, short operating time, simple instruments and devices, thus being generally applicable to the scientific research and medical diagnosis.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a PCR amplification system and amplification method suitable for genes with high GC content. Background technique [0002] PCR (Polymerase Chain Reaction), the full name of polymerase chain reaction, is a specific DNA in vitro amplification technology widely used in diagnostics, molecular biology, microbiology, and genetics. Although people have successfully amplified a large number of gene fragments with basic PCR procedures since 1988, non-specific results and time-consuming processes have gradually failed to meet wider needs. Especially in the face of high GC content genes with special secondary structures, including some primers, enhancers and other control elements, people are often helpless. So over the years, people have made many improvements to the conventional PCR program, and tried many methods to improve product specificity, including hot start, tw...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/34
Inventor 陈依军魏茂陈
Owner 陈学军
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