DGGE/TGGE (Denaturing Gradient Gel Electrophoresis/Temperature Gradient Gel Electrophoresis) analysis method of specific 18S rDNA (Deoxyribose Nucleic Acid) fragment without being based on GC clamp strategy

An analysis method and fragment technology, applied in microorganism-based methods, microorganism determination/inspection, biochemical equipment and methods, etc., can solve the problems of high cost, low GC long primer amplification efficiency, etc., and reduce experimental operation steps. , the effect of saving experimental costs

Active Publication Date: 2013-03-20
FUZHOU UNIV
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Problems solved by technology

However, when studying the structure of the flora, it is necessary to simultaneously synthesize two sets of primers including long primers containing GC splints and common primers witho

Method used

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  • DGGE/TGGE (Denaturing Gradient Gel Electrophoresis/Temperature Gradient Gel Electrophoresis) analysis method of specific 18S rDNA (Deoxyribose Nucleic Acid) fragment without being based on GC clamp strategy
  • DGGE/TGGE (Denaturing Gradient Gel Electrophoresis/Temperature Gradient Gel Electrophoresis) analysis method of specific 18S rDNA (Deoxyribose Nucleic Acid) fragment without being based on GC clamp strategy
  • DGGE/TGGE (Denaturing Gradient Gel Electrophoresis/Temperature Gradient Gel Electrophoresis) analysis method of specific 18S rDNA (Deoxyribose Nucleic Acid) fragment without being based on GC clamp strategy

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Embodiment 1

[0047] Example 1: In this example, red yeast rice purchased in Yongchun area and Youxi area was taken as the research object, and the amplified fragments of the primer pair NS1-fung and NS1-GCfung (the sequences of primers NS1, fung, and GCfung are detailed in the sequence list) were used for DGGE Used to analyze the fungal flora in distiller's yeast.

[0048] The specific steps of this embodiment are as follows:

[0049] 1. Genomic DNA extraction

[0050] Refer to literature (Heng Zhu, Feng Qu and Li-Huang Zhu. Isolation of genomic DNAs from plants, fungi and bacteria using benzyl chloride. Nucleic Acids Research. 1993,21(2):5279-5280.) Extraction by benzyl chloride method Total genomic DNA of microorganisms in koji.

[0051] 2. Amplification of specific 18S rDNA fragments

[0052] Using the genomic DNA extracted in step 1 as a template, the primer pair NS1-fung and the primer pair NS1-GCfung were used to amplify specific 18S rDNA fragments respectively. The PCR conditio...

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Abstract

The invention provides a DGGE/TGGE (Denaturing Gradient Gel Electrophoresis/Temperature Gradient Gel Electrophoresis) analysis method of specific 18S rDNA (Deoxyribose Nucleic Acid) fragment without being based on a GC clamp strategy. The specific 18S rDNA fragment is a fragment containing a terminal region of 18S rDNA, which is far away from an ITS (Internal Transcript Space) sequence; and with a 18S rDNA sequence of a Saccharomyces cerevisiae standard strain as a reference, the terminal region of the 18S rDNA, which is far away from the ITS sequence, is equivalent to a fragment which starts from any nucleotide within a range of the first to the 30th and ends with the 291th-locus nucleotide. According to the invention, a result consistent with that obtained by the use of the GC clamp can be obtained under the same condition by adopting the DGGE/TGGE analysis of the specific 18S rDNA without adopting the GC clamp, therefore, the experiment cost can be reduced and the experiment operation steps are reduced.

Description

technical field [0001] The present invention relates to a DGGE / TGGE analysis method for a specific 18S rDNA fragment, in particular to a DGGE / TGGE analysis method for a specific 18S rDNA fragment not based on the GC splinting strategy. Background technique [0002] DGGE (Denaturing Gradient Gel Electrophoresis) and TGGE (Temperature Gradient Gel Electrophoresis) are two electrophoresis techniques based on the sequence difference of DNA fragments. They were commonly used in the effective detection of gene mutations in the early days and have become an important means for the study of microbial diversity. . These two techniques can separate DNA fragments of the same length but different sequences. The separation of the fragments depends on the different solutions of double-stranded DNA when swimming in a gel containing a chemical denaturant gradient (DGGE) or a temperature gradient (TGGE). chain behavior to achieve. In order to detect all the mutations in the sequence as eff...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12R1/865
Inventor 倪莉黄志清黄山靳爽
Owner FUZHOU UNIV
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