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Induction design plan for PCR-DGGE research on environment microorgan population

An environmental sample, chain reaction technology, applied in biochemical equipment and methods, determination/inspection of microorganisms, electrochemical variables of materials, etc., can solve problems such as inseparability

Inactive Publication Date: 2003-11-19
RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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Problems solved by technology

[0003] Denaturing gradient gel electrophoresis (DGGE) has been used in the field of environmental microorganisms to study the community structure of microorganisms in environmental samples since 1993. It is based on the PCR reaction of 16SrRNA (prokaryotes) and 18S rRNA of all microorganisms in environmental samples. (eukaryote) and other conserved gene intervals based on the amplification of DNA fragments, these amplified DNA fragments can represent the information of all different microorganisms, and these DNA fragments of different microorganisms amplified using the same primer have The same number of bases, so general electrophoresis cannot separate them, which raises new questions for subsequent further research

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  • Induction design plan for PCR-DGGE research on environment microorgan population
  • Induction design plan for PCR-DGGE research on environment microorgan population

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[0009] Genomic DNA of various microorganisms in 7 soil samples were extracted at the same time, and the DNA gel recovery kit (Shanghai Sangong, product number: SK111) was used to purify the crude DNA extract according to the operation instructions. Genomic DNA was used as a template for polymerase chain reaction (PCR), using Applied Biosystem's GeneAmp PCR system 2700 gene amplification instrument, using a primer pair specific to the 16S rRNA gene fragments of most bacteria and archaea: F 357 GC and R 518 , and their sequences are: F 357 GC: (5'-CGC CCGCCG CGC CCC GCG CCC GGC CCG CCG CCC CCG CCC CCC TAC GGG AGG CAG CAG-3'), R 518 : (5'-ATT ACC GCG GCT GCT GG-3'), the length of the amplified fragment is about 230bp, and at the same time, set the primer pair F without GC hairpin structure 357 and R 518 The same sample was amplified as a control. The 100 μL PCR reaction system was composed as follows: 100 ng template, 30 pmol each primer, 200 μmol / L dNTPs: (each 10 mmol / L), 1...

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Abstract

A prime design scheme for using PCR-DGGE to research the microbial community structure in environmental specimen includes such steps as choosing environmental specimen (agricultural field soil), extracting the genomic DNAs of all microbes, purifying for using them as template of PCR amplification, choosing universal prime pair for prokaryotic 16 SrRNA gene amplification, adding GC hairpin structure to terminal 5', PCR amplifying, electrophoretic separating in DGGE, and observing if the PCR resultant is fully separated.

Description

Technical field: [0001] Polymerase chain reaction (PCR) technology is a basic technology in the field of molecular biology at present, and it is mainly used to amplify a DNA segment located between two known sequences. It has a wide range of applications in genetics, forensic science and other fields. In addition, PCR amplification technology has the following applications in molecular cloning and DNA analysis: (1) nucleic acid probe production, (2) DNA library establishment, (3) DNA sequence determination (4) mutation analysis, etc. Background technique: [0002] Denaturing gradient gel electrophoresis (DGGE) was originally used for gene mutation detection. DNA fragments with single-base mutations at any site can be separated from the original DNA fragments, and the mutation sites can be found after sequence determination and comparison . The principle of separation of different DNA fragments by denaturing gradient gel electrophoresis is that DNA fragments of the same leng...

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Application Information

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IPC IPC(8): C12Q1/25C12Q1/68G01N27/26
Inventor 齐鸿雁罗海峰张洪勋薛凯王晓谊
Owner RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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