Changed gradient gel electrophoresis

Principle

Double-stranded DNA molecules When a general polyacrylamide gel electrophoresis, its migration behavior is determined to its molecular size and charge. Different length DNA fragments can be distinguished, but the same length DNA fragment is the same as the migration behavior in the glue, so it cannot be distinguished. DGE / TGGE technology is added to the general polyacrylamide gel, which is added to the denaturing agent (urea and the formamide) gradient, thereby distinguishing the same length but the sequence different DNA fragment is separated. A particular DNA fragment has its unique sequence, its sequence composition determines its unlockingdomain, MD) and Melting Behavior. A few hundred base pairs of DNA fragments typically have several unlocated regions, each of which has a continuous base pair (Fig. 1). When the degrees concentration gradually increases its lowest solution region concentration, the segment of this region is subjected to a resumed base pair. These regions are sequentially restriction when the concentration of each other decay region is sequentially reached. Until the degenerative concentration reached the highest solution to the highest solution region concentration, the highest restroom area also resolves the chain, thereby completely solving the double-stranded DNA.

Different double-stranded DNA fragment

Different double-stranded DNA fragments are different because of their sequence composition, so the unlocking region of the unconventional zone and each of each of the decomposition areas are different. of. When they perform DGGE, the degree of density agent is relatively small, and the minimum decimal region of the double stranded DNA fragment cannot be made, and the migration behavior of the DNA fragment is in the same manner as in the general polyacrylamide gel. However, once the denaturing concentration reaches the highest solution region temperature of the DNA fragment, the DNA fragment is completely solved to become a single-stranded DNA molecule, and they can continue to migrate in the glue. Therefore, if the sequence difference between the different DNA fragments occurs at the highest solution area, these segments cannot be distinguished. This problem can be solved by adding a GC-rich DNA fragment (GC clip, general 30-50 base pairs) in one end of the DNA fragment. The highest solution area containing the DNA fragment of the GC clip is in the sequence of the GC clip, its solution concentration is high, and the DNA fragment can be prevented from completely resolving in DGGE rubber. When the GC clip is added, the sequence differences in the DNA fragment can be discarded. However, once the DNA fragment migrates to a specific position, the degenerator concentration is just the decline in the decrease of the minimum decay region of the double-stranded DNA fragment, and the minimum solution region of the double-stranded DNA fragment immediately occurs. The migration rate of the DNA fragment in part of the partially decomposed DNA fragment will decrease sharply. Therefore, the same length, DNA fragments of the sequence, will reach the solution concentration of the respective minimum unlocking regions at different locations in the glue, so they will have a partially decreasing chain at different locations in the glue. The glue is distinguished.

Features

DGGE / TGGE has been widely used to analyze the biodiversity of bacterial, blue bacteria, bacteria, micro eukaryotic, eukaryotic and viral community in natural environments [ 8]. This technology can provide advantageous type information in communities and simultaneously analyze multiple samples, which have the characteristics of repeatable and easy operation, suitable for survey of spatial changes in populations, and can hybridize to specific probes by sequence analysis or with specific probes Analysis identification community composition. DGGE and TGGE respectively have the same length of the same length, but only one base fragment of the DNA fragment is separated by the gradually increased chemical degenerative gradient and the linear temperature gradient. DNA molecules are separated at a particular temperature, depending on the hydrogen bond content of the complementary chain (rich in regional melt temperature of GC) and the gravity of the adjacent base.

DGGE method

Advantages

1, almost all mutations

2 can be detected, and the mutant molecules can be intact Earth-bearing molecules are separately analyzed for further analysis

3, no need to mark

4, only one step in the electrophoresis

5, can be used without Amplified genomic DNA

6 can detect DNA modifications

disadvantage of methylation

2, it is necessary to perform pre-experiments require expensive "GC splint"

3, which cannot determine the mutation in the DNA fragment position

4, you need to use Gradient gel containing toxic substance formamide

5, DNA fragment size limit is limited to 100-500 bp