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Introduction
Introduction
Gel electrophoresis separates large molecules of DNA according to their size. Electrophoresis involves running a current through a gel containing negatively charged DNA molecules. Gel electrophoresis of DNA separates them based on their sizes.
Using electrophoresis, we can see how many different DNA fragments are present in a sample and how large they are relative to one another.
The set up
The set up
Gels for DNA separation are often made out of a polysaccharide called agarose, which comes as dry, powdered flakes. When the agarose is heated in a buffer (water with some salts in it) and allowed to cool, it will form a solid, slightly squishy gel. At the molecular level, the gel is a matrix of agarose molecules that are held together by hydrogen bonds and form tiny pores.
At one end, the gel has pocket-like indentations called wells, which are where the DNA samples will be placed:
Before the DNA samples are added, the gel is placed in a gel tank. One end of the tank is connected to the positive electrode, while the other end is connected to the negative electrode. The tank is filled with a salt-containing buffer solution that can conduct current. The buffer fills the gel box to a level where it just barely covers the gel. The end of the gel with the wells is positioned towards the negative electrode. The end without wells is positioned towards the positive electrode.
Running the Gel
Running the Gel
DNA fragment often too small in amount for to be visualised in gel electrophoresis. For this reason, DNA sample is amplified by PCR before running.
After the gel is casted into the box, each DNA samples to be examine is carefully transferred into the wells. One well is reserved for the DNA ladder, a standard reference that contains DNA fragments of known lengths. Commercial DNA ladders come in different size ranges, so we would want to pick one with good "coverage" of the size range of our expected fragments.
Next, turn on the power of the gel box. Current begins to flow through the gel. The DNA molecules have a negative charge because of the phosphate groups in their sugar-phosphate backbone, so they start moving through the matrix of the gel towards the positive pole. When the power is turned on and current is passing through the gel, the gel is said to be running.
As the gel runs, shorter pieces of DNA will travel through the pores of the gel matrix faster than longer ones. After the gel has run for a while, the shortest pieces of DNA will be close to the positive end of the gel, while the longest pieces of DNA will remain near the wells. Very short pieces of DNA may have run right off the end of the gel if we left it on for too long. The phenomenon is known as 跳海. Therefore, carefully observe if the DNA is travelling too fast. Alter the running voltage and time to control the speed of DNA.
Interpreting the results
Interpreting the results
Once the fragments have been separated, we can examine the gel and see what sizes of bands are found on it. When a gel is stained with a DNA-binding dye and placed under UV light, the DNA fragments will glow, allowing us to see the DNA present at different locations along the length of the gel. A well-defined “line” of DNA on a gel is called a band. Each band contains a large number of DNA fragments of the same size that have all traveled as a group to the same position. A single DNA fragment would not be visible by itself on a gel. By comparing the bands in a sample to the DNA ladder, we can determine their approximate sizes.
Application
Application
- Check clone
- After cloning, we would like to check if we have done it correctly. Aside from sequencing, we can also use gel electrophoresis to conduct a indefinite (less certain) review of our cloning process. If the length of the DNA sample matches with the expected length of cloning products, it may be assumed that the clone is correct. However, gel electrophoresis is much less accurate and definite than DNA sequencing.
- Forensic science, victim identification
- Variable number tandem repeats (VNTRs) are continuous repetitive sequence. The number of repeats varies greatly from people to people. Therefore different people have different lengths of VNTRs. After PCR then running gel electrophoresis, we can compare the bands of different people, hence identifying the owner of the tissue samples.
- Parentage tests
- Compare the bands (from same segment of DNA) of the child and the adult. Have of the bands of the child is identical with the parent.
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