denaturing gradient gel electrophoresis
denaturing gradient gel electrophoresis
Denaturing gradient gel electrophoresis (DGGE) is a gel system that enables the separation of DNA fragments according to their melting properties. (1) By setting the gel under double denaturation conditions, it can detect any kind of single base substitutions, code-shifting mutations, and deletion mutations of less than 10 bases in a DNA molecule, and it is widely used in gene mutation detection and related research. (2) It is used for screening and diagnosis of cancer and genetic diseases, and is also used to detect a few mutations in quantitative mutation experiments in cancer (residual lesions, mutation mapping studies, etc.). (3) Widely used to analyze the biodiversity of bacteria, cyanobacteria, archaea, microbial eukaryotes, eukaryotes and viral communities in the natural environment.
Operation method
Denaturing Gradient Gel Electrophoresis (DGGE)
Principle
1. Denaturing gradient gel electrophoresis (DGGE) is a gel system that separates DNA fragments based on their melting properties. The double helix structure of nucleic acids can be unstranded under certain conditions called denaturation. The temperature at which 50% of the nucleic acid undergoes denaturation is known as the melting temperature (Tm). the value of Tm depends mainly on the amount of GC in the DNA molecule. dGGE sets the gel under double denaturation conditions: temperature 50-60 °C and 0-100% denaturant. When a double-stranded DNA fragment passes through a gel with a gradient of increasing denaturant concentration, the fragment migrates to a point where the denaturant concentration is exactly equal to the Tm value of the low-melting-point region of the DNA, and this region starts to melt, while the high-melting-point region remains double-stranded. This localized unwinding of the DNA molecules changes the mobility to achieve the effect of separation.The change in Tm depends on the DNA sequence, even a single base substitution can cause the Tm value to rise and fall. Therefore, DGGE can detect any kind of single base substitutions, code-shifting mutations, and deletion mutations of less than 10 bases in the DNA molecule. 2. In order to increase the mutation detection rate of DGGE, a high melting point region GC clamp can be artificially added. GC clamp is to add a 30-40bp GC structure to the 5′ end of one primer, so that a high melting point region can be generated on one side of the PCR product, so that the corresponding sequence of interest is in the low melting point region, which is convenient for analysis. Therefore, the mutation detection rate of DGGE can be increased to nearly 100%. 3. As a mutation detection technology, DGGE has the following advantages: (1) high mutation detection rate, the mutation detection rate of DGGE is more than 99%. (2) The length of detected fragments can be up to 1kb, especially suitable for fragments of 100-500bp. (3) Non-isotopic: DGGE does not require isotope doping, which can avoid isotope contamination and harm to human body. (4) Simple and fast operation: DGGE can generally obtain results within 24 hours. (5) Good reproducibility. However, the method requires special instruments and it is expensive to synthesize primers with GC clips.
Materials and Instruments
DNA samples Move 1.PCR reaction In which the upstream primer was added 40bp [GC] Clamp. 2.PCR products were confirmed by agarose gel electrophoresis. 3. Vertical denaturing gradient gel electrophoresis The direction of the increasing denaturation gradient is perpendicular to the direction of electrophoresis. The gel used was a 6% polyacrylamide gel with a denaturing concentration of 0-100%. In this case, the gel containing 7 M urea and 40% deionized formamide was 100% denaturing and the gel without urea and deionized formamide was 0% denaturing. Vertical denaturing gradient gel electrophoresis was mainly used to detect the optimal unchaining conditions (i.e. denaturing concentration) of the primers. PCR product plus sample buffer after sampling, 300-400μl/well, voltage 150V, temperature 60℃, time 2-4 hours. 4. Parallel denaturing gradient gel electrophoresis The direction of increasing denaturation gradient is parallel to the direction of electrophoresis. According to the denaturing concentration of the unchained region detected by vertical denaturing gradient gel electrophoresis, parallel denaturing gradient gel with corresponding denaturing concentration is prepared to detect whether there is mutation in each specimen. PCR product plus sample buffer, 25μl-30μl/well, voltage 150V, temperature 60℃, time 3-6 hours. 5. Staining was performed for 5 minutes, and the results were analyzed by a gel imager. Caveat 1. Air bubbles should be avoided during mixing and gluing. 2. the available solutions should be stored in brown bottles at 4°C and are generally effective for several months to a year. 3. the gel temperature must be kept constant during electrophoresis. this can be achieved by submerging the gel plate in a well-stirred temperature-controlled buffer tank. For DNA, which is more easily denatured in the absence of denaturants, the temperature in the bath is chosen to be 60°C slightly above the melting point, and most of the work is done at 60°C (although slightly higher or lower temperatures can be used). Keeping the temperature constant can be done by heating the electrophoresis buffer to 60°C and just dipping the gel into it for electrophoresis. 4. The extraction of DNA in this experiment, as well as the DGGE operation in contact with a lot of drugs are poisonous, as well as carcinogenic, denaturing and other toxicity, must be strict operation, good protection to protect themselves. Common Problems I. Operational details during the experiment: 1. It is better to use a micro-sampler when spotting samples to avoid contamination and sample floating. 2. In order to avoid the edge effect, both sides can be sure to point some samples. 3. Gluing is the key. The glass plate must be washed, and when filling the gel, turn the pulley at an even speed to fill the gel liquid into the glass plate at an even speed, and the liquid level should be kept horizontal. Evaluation of experimental results The DGGE pattern is very blurred with only a few bands, which may be due to the inappropriate concentration of denaturing gel that the product results can not be completely presented on the pattern. In addition, the blurring of the graph may also be due to the low amount of sample. During the experiment, the operation process, the uniformity of the gel, and the cleanliness of the glass plate will affect the electrophoresis results. For more product details, please visit Aladdin Scientific website.
Urea Deionized formamide Acrylamide Methylene bisacrylamide Agarose
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