1. Read the background essay below.
2. Watch the video linked below.
3. Submit answers to the following questions.Discussion Questions
- 3.1 Why does DNA move through the gel matrix when electrical current is applied to the gel?
- 3.2 What factors affect the rate at which DNA fragments move through the gel?
- 3.3 Would you expect DNA pieces of a particular size to move faster or slower in a gel with a higher percent of agarose? Explain why.
- 3.4 Describe the steps used to analyze a gel once the electrophoresis is completed.
- 3.5 The DNA ladder provides a reference for the size of the DNA. Why do you think control samples are also usually run?
Background Reading
Gel electrophoresis is a technique used in a wide variety of scientific, criminal, and legal investigations. Although all organisms have a high percentage of genes in common, there are significant genetic differences among members of the same species, even among members of the same family. Scientists use gel electrophoresis to help tease out these differences. As shown in this animation, the technique can also be used in genetic engineering to determine if a sample of bacterial plasmids has been successfully altered to contain a newly inserted gene. But what makes this comparison possible? What makes one molecule of DNA distinguishable from the next?
Gel electrophoresis sorts DNA molecules according to their size and shape. For example, a sample might contain fragments of varying lengths that result from cutting DNA molecules with restriction enzymes, it might contain uncut DNA plasmids, or it might contain a combination of molecules of various sizes and shapes.
To sort these molecules, DNA samples are loaded into wells at one end of the gel, which sits in a bath of buffer solution. The gel is then placed in a special box with electrodes leading from a power source. A negative electrode is attached to one end of the box while a positive electrode is attached to the opposite end. This is important because the DNA molecule itself is negatively charged — a result of the negatively charged phosphate groups that form part of the DNA backbone. When an electrical current is applied to the gel, the DNA molecules move with the current toward the positive end of the gel box. The size and shape of a DNA segment determine the rate at which it moves through the gel.
The gel presents a porous barrier — a matrix — through which DNA segments must pass en route from one end of the gel to the other. The speed with which linear segments of DNA move through the gel when the electrical current is applied is determined by size — the longer the segment, the slower it moves. However, uncut plasmids of identical size can take on different shapes, and this too affects the speed with which they move through the gel. Supercoiled plasmids move much more quickly through the gel than relaxed plasmids because the former are more compact. Finally, when the electrical current is turned off, the molecules in the sample stop moving and are “trapped” at the point in the gel they’ve reached by that time.
Staining the gel with ethidium bromide after it has run and then exposing it to UV light reveals the location of groups of DNA molecules of similar size and shape. Some lanes of the gel serve as reference points for the test sample. The control sample and the DNA ladder both contain DNA fragments of known size and are used for this purpose. By comparing the bands from the test sample to bands in the ladder and the controls, scientists can assess the relative size and shape of the DNA molecules they contain. In the example shown, a relatively large, slow-moving band in the test lane indicates that the plasmids in the sample do indeed carry a newly inserted gene.
Ass2
. Read the background essay below.
2. Watch the video linked below.
3. Submit answers to the following questions.Discussion Questions
- 3.1 List two important safety measures you should follow when making an agarose gel.
- 3.2 Why is it important to properly dissolve the agarose before pouring the gel into the tray?
- 3.3 List three factors that can influence the rate of movement of DNA molecules during gel electrophoresis.
- 3.4 Why do you think smaller DNA fragments are better separated, or resolved, in higher concentrations of agarose, and larger fragments in lower concentrations?
Background Reading
We hear a lot about forensic DNA analysis and genetic engineering. These procedures require the ability to isolate DNA fragments of interest from the rest of an organism’s genome. However, if you’ve ever extracted DNA from plant or animal cells, or seen the sticky clump that often results from this process, you know that the final product isn’t much to look at. So, how can a scientist or lab technician isolate a DNA fragment of interest?
Scientists use gel electrophoresis to separate nucleic acids according to their size. A key component of this process is the gel itself—which is usually made of agarose, a complex carbohydrate found in the cell walls of certain types of algae and seaweed.
For use in gel electrophoresis, purified, powdered agarose must first be fully dissolved in a heated buffer solution. This agarose solution is allowed to cool a bit and then poured into a gel tray whose ends have been sealed with tape or with removable dams that are provided with some electrophoresis equipment. Here, the gel will solidify and take the shape of the gel tray as it cools to room temperature. Once cooled, the agarose gel can serve as a solid, yet porous medium through which relatively large fragments of DNA can travel.
An electrical field generated by a power supply is what pushes the DNA molecules through the gel. However, the speed with which the molecules move, and the ultimate separation among their groups, depends on a number of factors, including the size of the molecules, the concentration of agarose in the gel, and the voltage applied.
Acting as a filter, or sieve, the gel allows relatively small molecules to pass through, while it also slows the molecules’ movement. Relatively small molecules pass through the gel matrix quickly and easily—and therefore move farther in a given period of time—compared to large molecules. Also, the shape of the DNA molecule will influence its movement through the gel. Small, compact DNA molecules, like super-coiled plasmids, will move faster than open, circular plasmids of the same molecular weight and number of base pairs.
Agarose concentration can also affect the speed with which molecules move through a gel. Gels with high concentrations have a finer meshlike structure than gels with low concentrations. This results in more contact between the molecules and the gel matrix, and slower movement through the gel. Increasing the voltage will speed up the molecules, but if the voltage is too high, it can melt the gel.
For these reasons, scientists must carefully adjust agarose concentrations to the size range of molecules in their samples. A gel that has too high a concentration will cause molecules to pass through very slowly, or not at all, while a gel concentration that is too low will allow molecules to pass too quickly and will fail to separate them effectively. Charts like the one seen in this video are widely available and can be helpful when choosing an agarose concentration. For example, according to the chart, a concentration of 2.5 percent will effectively separate DNA molecules between about 200 bases and 5,000 bases in length. Other agarose concentrations work better for larger or smaller size ranges.
Additionally, it is important that the agarose be fully dissolved in the buffer solution prior to pouring the gel, as undissolved agarose can interfere with the movement of molecules through the gel matrix. Finally, the gel tray must be placed on a flat, level surface prior to pouring to ensure a consistent depth throughout the gel.
Ass3
1. Read the background essay below.
2. Watch the video linked below.
3. Submit answers to the following questions.Discussion Questions
- 3.1 What is the role of electric current during gel electrophoresis?
- 3.2 Describe the function of the dye and glycerol components of the sample loading buffer.
- 3.3 Explain why it is important to change the pipette tip before loading a new sample.
- 3.4 List four things that one should never do when running a gel.
Students may benefit from reading the background essay before discussing the following question:
- 3.5 Explain why the DNA molecule is negatively charged.
Background Reading
Gel electrophoresis is a technique used to detect variations in the size and shape of DNA molecules. The technique is commonly used in forensic DNA analysis and paternity testing to match tissue samples to their sources. It is also used to map genes found on chromosomes to detect mutations that can cause disease. Or, following genetic engineering procedures, gel electrophoresis can be used to determine if DNA molecules have been successfully altered.
There are two main components in a gel electrophoresis procedure. The first is the gel itself, a material made of a complex carbohydrate called agarose. This material, although solid, is also porous and, therefore, allows relatively large molecules such as DNA and RNA to pass through it.
The second main component is the electrophoresis chamber. When electrical current is applied to the chamber, it generates an electrical field, which drives DNA molecules through the gel—this is what is meant by “running the gel.” The DNA molecules move through the gel because the negative charges on the molecules’ phosphate backbone move away from the negative electrode at one end of the chamber and towards the positive electrode when electrical current is applied.
The speed with which DNA molecules move through the gel, and the distance they travel in a given period of time, depend on the size of the DNA molecules and the agarose concentration of the gel. Small molecules move more quickly and farther than large molecules. All molecules of a given size move more quickly through a gel with a low agarose concentration than through a gel with a high concentration because the molecules experience less resistance. Also, increasing the voltage can increase the rate of movement of the DNA, but up to a limit, since high voltage can also melt the agarose gel.
As the DNA molecules move through the gel while the electric current is applied, there is no way to visibly detect them. For this reason, a dye is included in the sample wells prior to running the gel so that the progress of electrophoresis can be followed even though the movement of the molecules can’t be seen.
After electrophoresis is complete, scientists can use a number of chemicals and techniques to make the DNA molecules in the gel visible. One of the most common methods is to stain the gel with a chemical called ethidium bromide (EtBr). EtBr binds directly to the DNA molecule, or intercalates with it, in between the nucleotide bases that make up the rungs of the DNA double helix. More importantly, EtBr fluoresces, or glows, under ultraviolet (UV) light. By viewing the EtBr-stained gel over a UV light source, it is possible to see the DNA bands in the gel.
The DNA bands from unknown or test samples can be compared to DNA bands from known or control samples, including a control sample known as a DNA ladder. DNA ladders are commercially available and are made up of a variety of molecules of known length. When run alongside samples with molecules of unknown length, ladder samples provide a valuable quantitative measure against which the unknowns can be compared in order to assess their size
Ass4
1. Read the background essay below.
2. Watch the video by clicking on the link.
3. Submit answers to the following questions.
- 3.1 What is the evolutionary function of RNAi?
- 3.2 What is macular degeneration? How can RNAi be used to treat macular degeneration?
- 3.3 How can scientists use RNAi to learn about the human genome and specific human gene functions?
Background Reading
In their pursuit to cure infectious as well as inherited diseases, scientists are testing several cutting-edge therapies. These include gene therapy, antisense therapy, and an approach called RNAi therapy.
As this video segment demonstrates, in an early clinical trial, RNAi seemed like a promising treatment for macular degeneration, an eye condition in which too many blood vessels grow underneath the retina, which leads to sight loss. Other potential disease targets include Huntington’s disease, hepatitis, and breast cancer. RNAi can theoretically be used to treat any infection or disease that is caused by the overproduction of a normal protein, the production of an abnormal protein, or the production of a harmful foreign protein.
RNAi therapy is based on cells’ natural response when they detect infection or genetic abnormality. Double-stranded RNA molecules in the cytoplasm signal abnormality in a cell, as RNA normally produced by the cell is single stranded. Double-stranded RNA molecules may come from viruses or may be part of the cell’s own mechanism to inhibit the production of certain proteins. When the RNAi molecules detect either of these abnormal molecules, a protein complex, which scientists call “Dicer,” cuts the double-stranded RNA into fragments. Next, a molecule called “RISC” binds to one fragment of the offending RNA and uses this fragment to detect single-stranded mRNA with the corresponding sequence. Whenever RISC encounters corresponding mRNA molecules, it cuts and degrades those molecules so that they can no longer be used to synthesize proteins. By inhibiting the production of the protein it codes for, RNAi can effectively block, or “silence,” a specific gene’s activity.
Besides its ability to selectively target genes, another advantage of the RNAi approach is that it is comparatively easy for scientists to make RNAi drugs. These drugs consist of small strands of RNA—each about 22 bases long—that can be synthesized by a machine. Further, because the RNAi mechanism passes from cell to cell, it would therefore maintain or even increase its effectiveness over time. That said, the power of the RNAi mechanism also carries potential risk. To use it effectively, scientists need to better understand how to inhibit the synthesis of a target protein without influencing the synthesis of any other pro⁷teins. The challenge, then, is to deliver the synthetic RNA molecules only to the cells that matter.
Ass5
1. Watch the following video
2. Answer the two questions below.
- 2.1 What are the two main ingredients needed for life to arise on Earth? Describe their functions.
- 2.2 What were the different steps that the scientists took to create the two RNA bases in their lab?
Ass6
1.Watch the following video.
2.Answer the following questions.Discussion Questions
- 2.1 What roles do proteins play in our bodies?
- 2.2 Describe some functions of mRNA, ribosomes and proteins within the cell.
- 2.3 Explain the process of transcription.
- 2.4 Explain the process of translation.
Ass7
1. Read the background essay below.
2. Watch the interesting video by clicking on the link.
https://www.youtube.com/watch?v=H5udFjWDM3E (Links to an external site.)
3. Submit answers to the following questions.
- 3.1 What role did the petunia play in the discovery of RNAi?
- 3.2 How does RNAi work?
- 3.3 Why did cells evolve this mechanism? Explain in detail.
Background Reading
In 1998, scientists discovered that petunias, nematodes, fruit flies, mice, and even humans use a built-in cellular mechanism to protect their genome (set of genes) from attacks by viruses. Normally, when a gene is turned on, it makes an RNA copy of itself that leaves the nucleus and serves as a message to direct the production of a specific protein. The RNA that is part of this process exists as a single strand. When a virus attacks the cell’s genome, it carries a double-stranded form of RNA that enters the cell. A cellular mechanism called RNA interference, or RNAi, recognizes this double-stranded RNA as a dangerous invader. It sets out to destroy not only all double-stranded RNA but also any single-stranded RNA messages in the cell that have a sequence like that found in the double-stranded RNA.
A few years later, scientists discovered that many, if not most, organisms use RNAi not just to prevent viral infections, but to regulate the expression of their own genes. Scientists have recently discovered a new class of RNA molecules called microRNAs. These microRNAs are produced during the early stages of an organism’s development. They bind with matching single-stranded RNA molecules that are involved with protein synthesis. When a microRNA binds to its matching messenger RNA, it forms a double-stranded RNA that is recognized by the RNAi system, which then destroys it. This RNAi mechanism silences the expression of that particular gene.
Today, scientists are using the RNAi mechanism to learn more about what particular genes do and how to alter their function. Determining gene function is a relatively simple matter of inserting double-stranded RNA molecules that have a particular sequence into cells and observing the effects after RNAi silences the corresponding gene.
Conceivably, this method may one day be used to silence gene mutations that cause human diseases such as Huntington’s disease, rheumatoid arthritis, cancer, and many others. By using either the body’s own mutations or viral invaders, scientists may develop a new type of drug—for example, one that switches off the genes of a cancer cell and leaves healthy cells unaffected. However, because RNAi’s potential effects are so powerful, scientists must first determine that they can control the mechanism so that only the target gene is silenced, and not others.
Ass8
1. Read 17.4 in the text book and pay close attention to Figure 17.15 on page 347.
2. Submit answers to the following..
- 2.1 What is the role of the ribosome in translation?
- 2.2 With a minimum of 250 words, describe the role of tRNA in protein synthesis and how mRNA works with tRNA to produce the correct sequence of amino acids in a protein? (Use these key words in your answer: ribosome, mRNA, codon, tRNA, anitcodon, complementary sequence, P site, A site, and E site.)
- 2.3 If you are given the following DNA sequence, complete the mRNA transcript and the amino acid sequence in the polypeptide. Remember to use URACIL in place of thymine for RNA.
- 2.4 DNA sequence: TAC CCT AAG GAC AAT ATA GCG CAA
- 2.5 mRNA codon sequence:
- 2.6 tRNA anticodon sequence:
- 2.7Amino acid sequence: (use the codon table in Figure 17.6 to help determine this sequence