Course: SAT 170 Introductory Biotechnology

Unit 6 Learning Module

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- Unit 6
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- Unit 6 Glossary (Selected)

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DNA Technology Part 1

The advancement of biotechnology into what is commonly referred to as modern biotechnology is largely reliant on advances in our understanding of, and ability to manipulate DNA. Thus, before we further investigate the techniques and topics of modern biotechnology, we must familiarize ourselves with DNA technology.

Recombinant DNA
The production of recombinant DNA has perhaps been one of the most significant contributions of DNA technology. A recombinant DNA molecule is one which is formed as two pieces of DNA from different sources are combined. The original sources of these DNA fragments are often from very distantly related organisms. For example, one common form of recombinant DNA combines a fragment of DNA collected from a bacterium, known as a plasmid, and a segment of human DNA.  A plasmid is a small, circular, double-stranded piece of DNA that is found outside of the bacterial chromosome free-floating in the cytoplasm. Plasmid DNA is replicated independently of the chromosomal DNA and can be transferred between bacterial cells. Plasmid DNA can be removed from a bacterium, cut, fused with other DNA, and then re-inserted into the bacterium. This plasmid, now with a combination of DNA nucleotides from two sources, is an example of recombinant DNA.

See Figure 1: EcoR1 is a restriction enzyme that was isolated from the bacterium Escherichia coli. In this illustration it is being used to cut a plasmid (shown in yellow) as well as to remove a fragment of DNA from a linear chromosome (shown in blue). Since these two pieces of DNA now have complementary sticky ends they can combine to form a recombinant DNA molecule.

Figure 1

- DNA is cut using special enzymes with nuclease activity, meaning these enzymes are able to cut the phosphodiester bonds of a chain of nucleic acids such as DNA and RNA. There are exonucleases which makes cuts to the ends of a nucleic acid chain, often removing only a single nucleotide at a time. These enzymes are mostly used to regulate the expression of DNA by removing mRNA molecules once a gene no longer needs to be expressed. Recall from Unit 4 that mRNA serves as a copy of the DNA of a particular gene for the purpose of translating that gene into a functional protein. The poly A tail helps to protect the mRNA for a certain period of time (depending on its length) by giving something for these exonucleases to remove.
- There are also nuclease enzymes that are able to cut in the middle of a chain of nucleic acids. Such enzymes are referred to as endonucleases. The function of endonucleases is to protect an organism from a foreign piece of DNA, such as that which is introduced when a host cell becomes infected with certain viruses. As we discussed in unit 3, when a virus infects its host cell, it will inject its genetic material and take over that host cell’s replication, transcription and translation machinery. This is because viruses are parasites that rely on a host cell in order to replicate.

Figure 2

- There are special types of endonuclease which make cuts at a very specific site in the DNA. These endonucleases are known as restriction enzymes. These restriction enzymes will make a cut in the DNA upon recognizing that specific sequence. These specific orders of nucleotide bases are known as restriction sites. There are many different restriction enzymes that have been isolated from a variety of organisms. Restriction enzymes can cut DNA in such a way that they cut through both strands of the DNA molecule. The resulting DNA fragments will have what are referred to as “blunt ends”. However, when DNA is cut with other types of restriction enzymes, it leaves “sticky ends”. These “sticky ends” are so named because these restriction enzymes make a zig-zag cut in the DNA molecule leaving regions on each end that are single stranded. These single stranded regions will readily reattach to a complimentary sequence. Thus, a piece of DNA that is cut from one source with a specific restriction enzyme (e.g. a particular gene of the human genome, like insulin) can be inserted into a plasmid that has been cut with the same restriction enzyme as they will have complementary “sticky ends”.
- DNA ligase can then be used to “glue” the two DNA fragments together at these “sticky ends” by forming a new phosphodiester bond between the nucleotides of those fragments.

See Figure 2: Notice how the restriction enzyme cuts between the G and the AATTC of the top strand and the CTTAA and G of the bottom strand. This will create the “sticky ends” needed for creating recombinant DNA molecules.
Separation of DNA fragments: Gel Electrophoresis

Figure 3

Gel Electrophoresis is one of the staple tools in molecular biology and is of critical value in many aspects of genetic manipulation and study. It is a method that separates nucleic acid and protein molecules. Recall that we discussed the use of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS Page) for the separation of proteins in unit 5. We will describe the process of electrophoresis in a bit more detail here, focusing on the use of this technique for the separation of DNA fragments. The term electrophoresis describes the movement of charged particles under the influence of an electric force. In this technique molecules are forced across a span of gel, driven by an electrical current. Activated electrodes at either end of the gel provide the driving force, while the buffer in which the gel is immersed within the electrophoresis chamber helps to conduct the current. The electrical current from one electrode repels molecules of the same charge while the other electrode has an opposite charge and can simultaneously attract the molecules. The gel matrix functions as a "molecular sieve," separating the molecules by size. The sample of proteins or DNA is applied to the gel, and due to the electrical force, the molecules of that sample will travel a certain distance at a certain speed. The distance and speed the molecules travel primarily depends on the size of the molecule. Thus molecules of different sizes can be separated. Another factor that can impact how quickly a molecule will travel within the gel is the overall shape of the molecule; bulkier molecules tend to get stuck in the gel matrix and travel more slowly. Once the molecules have been separated, the gel can be stained in order to visualize the separated molecules. These molecules will create a banding pattern (a series of bands spread from one end of the gel to the other) in each lane of the gel.

See Figure 3: Notice the placement of the gel within the electrophoresis chamber. Once the DNA samples are loaded using a micropipette (which is the action being shown here), the buffer will be added and a current will be applied to the chamber.

Figure 4

There are two basic types of materials used to make gels: agarose and polyacrylamide. We discussed the use of polyacrylamide gel in the SDS Page procedure used for analyzing proteins. Agarose gel analysis is the most commonly used method for analyzing DNA fragments. Agarose is a type of sugar that forms really long chains. It is isolated from agar, which is a material found in the cell walls of certain algae. A powder form of this agarose material is mixed with water and brought to a boil to dissolve the powder. This mixture will then be placed into a mold in order to create the specific shape needed for electrophoresis (think of how you make Jello by boiling water with a powder mix, once the Jello mixture cools it takes a semi-solid form). The concentration of the agarose can be adjusted to give you better separating power of DNA fragments. For example, smaller DNA fragments can be easily separated by using a high concentration of agarose and thus creating a molecular sieve with very small openings or passageways. Unlike proteins, DNA does not need to be pre-treated with sodium dodecyl sulfate, as DNA has an inherent negative charge because of the negatively charged phosphate groups of the DNA nucleotides. Thus, the positively charged electrode will attract the DNA molecules while the negative electrode will repel the DNA molecules. These DNA molecules will be pushed and pulled toward the positive electrode at different rates depending on their size. Since most DNA molecules are relatively large, the DNA samples that will be used for electrophoresis will be digested with restriction enzymes prior to running the gel. This ensures that the fragments will be small enough to make their way through the gel matrix to be separated. Since the restriction enzymes cut at very specific sequences, different DNA samples will be cut in different ways by the same enzymes resulting in different sized fragments. This is the basis for DNA fingerprinting which we will discuss further in Unit 14.

Figure 4: Once electrophoresis is complete, the DNA banding pattern can be observed. The DNA bands can be stained with a colored dye or can be seen by exposing them to ultraviolet light as is shown here.