Many of the procedures used in recombinant DNA technology rely on a researcher's ability to purify a DNA fragment of interest. In an important procedure called agarose gel electrophoresis, DNA fragments are separated by size as they move through a gel matrix. In this animation, we will examine how gel electrophoresis is performed and then describe a method called blotting, which allows researchers to identify the DNA fragments of interest along the length of a gel.
These tubes contain identical DNA fragments, but they will be cleaved with different restriction enzymes to yield fragments of different sizes. Enzyme 1 cuts the DNA into fragments A and B, which we color for tracking purposes. Enzyme 2 cleaves the DNA at a different recognition sequence, yielding fragments C and D. Adding both enzymes yields fragments A, E, and D.
Gel electrophoresis is one of the most useful means of separating and purifying DNA fragments for further analysis. In this technique, a jello-like slab of material called agarose is molded with wells, placed in a buffer solution, and hooked up to positive and negative electrodes. The DNA solutions, to which blue dye is added, are then pipetted into the wells. A well is also reserved for the placement of DNA of known sizes, and then the power supply is turned on.
The blue tracking dye is negatively charged and migrates toward the positive electrode, as does the DNA. The DNA backbones contain negatively charged phosphate groups, which are attracted to the positive electrode. The smallest fragments move the fastest, being entangled less in the agarose matrix of the gel.
When the blue dye reaches the bottom of the gel, the power is turned off. A fluorescent dye is then used to stain the DNA fragments. A DNA fragment in the size standards lane is the smallest—in this case about 500 base pairs (bp)—followed by DNA fragment D. The largest fragment is 23,000 bp long. Each band of DNA may contain millions of fragments of the same size.
In many cases, a researcher may want to determine which DNA fragment contains a DNA sequence of interest. To do this, the researcher prepares the DNA in the gel to make a copy, known as a blot. First, the gel is soaked in a basic solution so that the double-stranded DNA denatures into single strands.
The gel is then transferred to a salt solution and a nylon filter is placed on top of the gel. Absorbent towels are placed on top of the filter. The salt solution draws the DNA through the gel toward the nylon filter, where the DNA adheres.
The filter is treated so that the DNA adheres to it permanently, and then the filter is placed in a solution with a radioactive probe. The probe consists of single-stranded DNA that is complementary and will hybridize to the band of interest.
The filter is washed to remove any unhybridized probe. A piece of x-ray film is placed over the filter. The radioactive probe exposes the film, revealing the locations of hybridization.
Knowing which bands contain the sequence of interest, an identical agarose gel can be created, and a band of interest can be cut out of the gel. The DNA in this band can then be manipulated for further analysis.
In gel electrophoresis, charged molecules migrate through a matrix in an electric field. DNA molecules are negatively charged along their length, so they migrate toward the positive electrode. Within an agarose matrix, the DNA molecules become separated according to size during their travel—the pores in the agarose gel allow short DNA molecules to snake through easily, but cause more friction for the longer DNA molecules, slowing their progress. Therefore, the shorter the DNA fragment, the quicker it can travel through an agarose gel.
The electrophoresis procedure can be a starting point for additional identification and isolation of the DNA fragments. For example, the DNA fragments within an agarose gel can be transferred to a solid support, such as a nylon filter—making a blot of the DNA fragments in the gel—for further analysis. DNA fragments of interest can also be sliced out of a gel and used in recombinant DNA applications.