Use method of DGGE (Denatured-Gradient Gel Electrophoresis) technique

Abstract

The invention discloses a use method of a DGGE (Denatured-Gradient Gel Electrophoresis) technique, which is characterized by comprising the following steps of: (1) collecting algal cells; (2) extracting total DNA (DeoxyriboNucleic Acid); (3) designing and optimizing PCR (Polymerase Chain Reaction) primers; (4) detecting by using agarose electrophoresis; (5) preparing denaturing gradient gel; (6) optimizing the electrophoresis conditions; and (7) analyzing microalgae by utilizing the DGGE technique. 18S rDNA, 28S RDNA and ITS (Intertek Testing Service) region sequences of common microalgae are downloaded from a GenBank, MegAlign analysis is carried out by utilizing DNA Star software to select a sequence high-mutation region, Primerprimer 5.0 is used for the primer design of conserved regions at both sides of the high-mutation region, and then, various parameters of the designed primers are estimated by using Oligo software to select the optimal primer for synthesis. The invention can accurately detect the structural composition of a phytoplankton community and is beneficial to the outbreak warning of a phytoplankton red tide.

Description

Technical field: [0001] The invention relates to the technical field of application of denaturing gradient gel electrophoresis technology in the detection of species and / or quantity of marine phytoplankton, in particular to a method for using denaturing gradient gel electrophoresis technology. Background technique: [0002] Plankton is a unique group that lives with the current in water bodies, and is easy to move passively under the action of wind and current, with no or only weak swimming ability; plankton includes two categories: phytoplankton and zooplankton. Plankton are small in size, short in life cycle, fast in reproduction, very sensitive to changes in the environment, and can respond quickly to changes in the nutritional status of water bodies. In waters with good water quality, the diversity index and uniformity index of plankton were larger; on the contrary, the species diversity of plankton decreased, and the distribution was uneven. Therefore, changes in the s...

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Problems solved by technology

However, at the same time, this technology also has defects: (1) only the main microorganisms in the environment can be detected, and the microorganisms accounting for more than 1% of the total can be detected; (2) the size of the fragments that can be separated by DGGE is between 200-700bp (3) DNA with different sequences may migrate to the same position of the gel, and sometimes the bands cannot be separated well even if the bands are recovered, re-amplified, and re-electrophoresis ; (4) There is a problem of heterogeneity between different copies of genes in some species. Even if they have only slight differences, DGGE can separate them, so a species does not only show one band on the DGGE map, which is overestimated (5) In addition, there are certain limitations in each step of the whole analysis process: some microbial species may be lost during sample collection and storage; due to the different cell wall compositions of different species, the same Different extraction methods have different extraction efficiencies for different species; PCR itself has the problem of amplification preference, and chimeras and heteroduplexes may be generated during the amplification process, which have a certain impact on the experimental results

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