The main separation principle of denaturing gradient gel electrophoresis is to use the melting properties of DNA strands to achieve the purpose of separation. The low melting point and high melting point DNA have different degrees of local melting, and the migration rate of molecules does not pass, and then separation.
1. Experimental principle
Denaturing gradient gel electrophoresis (DGGE) is a gel system that separates DNA fragments based on their melting properties. The double helix structure of the nucleic acid can be melted under certain conditions, which is called denaturation. The temperature at which 50% of nucleic acid denatures is called the melting temperature (Tm). The Tm value mainly depends on the amount of GC in the DNA molecule. DGGE sets the gel under double denaturation conditions: temperature 50 ~ 60 ℃, denaturant 0 ~ 100%. When a double-stranded DNA fragment passes through a gel with a gradient of 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 this segment of DNA. The zone is still double-stranded. This partial melting DNA molecule mobility changes to achieve the effect of separation. The change of Tm depends on the DNA sequence, even a base substitution can cause the increase and decrease of Tm value. Therefore, DGGE can detect any single base substitution, frameshift mutation, and deletion mutation of less than 10 bases in the DNA molecule.
In order to improve the mutation detection rate of DGGE, a high melting point region-GC clip can be added artificially. GC clamp is to add a 30-40bp GC structure to the 5 ′ end of one side primer, so that a high melting point region can be generated on the side of the PCR product, so that the corresponding sequence of interest is in the low melting point region And easy to analyze. Therefore, the mutation detection rate of DGGE can be increased to nearly 100%.
As a mutation detection technology, DGGE has the following advantages: (1) The mutation detection rate is high. The mutation detection rate of DGGE is over 99%. (2) The length of detection fragments can reach 1kb, especially suitable for fragments of 100 ~ 500bp. (3) Non-isotopic. DGGE does not require isotope incorporation, which can avoid isotope pollution and damage to human body. (4) The operation is simple and fast. DGGE generally obtains results within 24 hours. (5) Good repeatability. However, this method requires special equipment, and the synthesis of primers with GC clips is relatively expensive.
2. Experimental supplies
1. PCR amplification instrument; denaturing gradient gel electrophoresis instrument; gel imaging and analysis system; ultraviolet transmission instrument; high speed centrifuge; electrophoresis instrument; electrophoresis tank.
2. Urea, deionized formamide, acrylamide, methylene bisacrylamide, agarose
3. Micro sampler (200μl, 20μl); Tip head (200μl, 20μl). Tip head box (200μl, 20μl); Eppendorf tube (0.5ml, 0.2ml), Eppendorf tube rack.
4. PCR amplification related reagents
3. Experimental procedure
1. PCR reaction (same as above)
Among them, the upstream primer plus 40bp [GC] Clamp.
2. The PCR product was confirmed by agarose gel electrophoresis.
3. Vertical denaturing gradient gel electrophoresis
The direction of increasing denaturation gradient is perpendicular to the direction of electrophoresis. The glue used is a 6% polyacrylamide gel with a denaturing concentration of 0-100%. Among them, the glue containing 7 M urea and 40% deionized formamide is 100% denatured, and the glue without urea and deionized formamide is 0% denatured. Vertical denaturing gradient gel electrophoresis is mainly used to detect the optimal melting conditions (ie, denaturation concentration) of primers.
The PCR product is loaded with the sample buffer and loaded, 300-400 μl / well, voltage 150V, temperature 60 ° C, 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 denaturation concentration of the melting region detected by vertical denaturing gradient gel electrophoresis, parallel denaturing gradient gels with corresponding denaturing concentrations are prepared to detect whether there is mutation in each specimen.
After adding the sample buffer to the PCR product, 25μl ～ 30μl / well, voltage 150V, temperature 60 ℃, time 3 ～ 6 hours.
5. After staining for 5 minutes, the gel imager analyzes the results.
Note: Many of the experimental drugs and reagents used in the operation of the denaturing gradient gel electrophoresis experiment are toxic and have toxic effects such as carcinogenesis and denaturation. It is necessary to strictly follow the steps to protect yourself from experimental harm.
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