Mitochondrial DNA

Background

In addition to nuclear DNA, each cell contains mitochondrial DNA, suitably named for its location in the mitochondria. Mitochondrial DNA is used to create and store ATP, which is used to power the cell. It is theorized that mitochondria may have been bacteria adapted into the cell in a symbiotic relationship; the bacteria would receive nutrients from the cell in return for energy created from oxygen.

^ Mitochondria_______________________

Mitochondrial DNA is inherited only from the mother, because while the sperm and oocyte both contribute chromosomes, only the oocyte contributes cytoplasm and organelles. Mitochondria also have a high mutation rate- approximately 10 times as often as nuclear DNA. These two features make it possible to trace ancestry by comparing the mutations present in individuals to those of their mothers, grandmothers, great-grandmothers, etc., etc. Work in the 1980s by Alan Wilson and others at UC Berkeley created a family tree, hypothesizing that all humans alive today evolved from a single ancestor. This "mitochondrial Eve" probably existed in Africa about 200,000 years ago.

^ a family tree_________________________

Mitochondrial DNA is extremely valuable in science. Because it is more prolific than nuclear DNA, it can be used in cases where much time has passed and DNA has been severely degraded.

Procedure

Skin cells are obtained either by a saline mouthwash or root ends from scalp, eyebrow, or arm hairs. A region is amplified using PCR, which cycles the DNA through three stages: first, a 94°C incubation to denature the DNA, a 58°C incubation to bond the primers to their complementary sequences, and a 72°C incubation for DNA polymerase to create complementary strands. Because of the large quantity of mitochondrial DNA already present, it does not need to be cycled as long as nuclear DNA. Because of the similarity of mitochondrial DNA, the bands resulting from gel electrophoresis will appear to be the same. Samples may be submitted to the Sequencing Service  of the Dolan DNA Learning Center, which can generate sequences and post them on the internet.

Results

From our results, we saw that the band width and positions are nearly identical to the human eye. This is understandable, as all human beings on the planet today share 99.9% of their DNA. The similarity allows us to trace our evolution and understand how we compare to each other, as well as to other species.

The next step in this lab will be sending our samples in to be sequenced, so we can compare our DNA in greater detail.

"Disease" Gene Testing

Background

DNA testing has multiple purposes, and is used in the study of evolution, as well as both forensic and paternal tests. One of its most valuable applications is checking for genetic diseases. Through DNA testing, one can test whether an individual is at risk for inheriting an illness such as cystic fibrosis or sickle cell anemia. Scientists take a sample of cells, amplify them using PCR, and analyze them through gel electrophoresis. In this lab, we will use the above steps to test for a gene (which is actually an intron. This is due to the ethical problems of testing for genetic diseases in class).

Procedure

Day 1: Collecting a Sample
A sample of skin cells is taken from the subject by chewing gently on the insides of the cheeks and then rinsing with a saline solution. The sample, once collected in a test tube, is spun in a centrifuge to clump all the cells together. The test tube is then immersed in two hot water baths, one at 56°C and one at 95°C. This is done to break the cell membrane and release the DNA. Once loose in the cytoplasm, however, DNAse would quickly destroy the sample DNA. This is why Instagene Matrix beads are added to the sample before it is immersed in the hot water baths. The test tube is then stored in a refrigerator until the next day.
Day 2: Polymerase Chain Reaction (PCR) Amplification

The sample of cells taken from an individual's cheek is much too small to be visible using gel electrophoresis (discussed during Day 3). PCR is therefore necessary in order to increase the size of the DNA sample before gel electrophoresis. In PCR, a primer is added to the solution. This primer acts like a heat-seeking missile, searching for the target gene sequence. The sample is then placed in a thermal cycler, which cycles through three temperatures- one to denature, or "unzip" the DNA, one to anneal to (find) their complementary sequences of DNA, and one to make new nucleotides attach to their complements on the unzipped strands. These three steps constitute one cycle; one PCR amplification consists of 40 of these cycles.
Day 3: Gel Electrophoresis
The bands of DNA are separated by size during gel electrophoresis, with the smaller bands being further towards the bottom (having progressed faster than the larger ones). There are three possible results.

If the gene being tested for is present in both chromosomes, a band of DNA strands 941 base pairs long will form. If it is present in neither, a band of DNA strands 641 base pairs long will form. If the gene is present in one chromosome but not the other, one band of each will form.

Results

There was a major source of human error for our result, which was that the gel was punctured by the pipette tip one two or three wells and somehow all of the samples bled through to some extent. All of our control samples were barely visible viewed in the correct light, as well as Lizzie's results (she was heterozygous, meaning she carried the 'disease' but did not herself show symptoms). Over half the class's results were homozygous negative, meaning they had the disease, so although our other test samples did not yield results, it would not be unreasonable to hypothesize that at least two of our tablemembers tested homozygous negative.

Testing for Genetically Modified Organisms Through the Use of PCR

   Background

What are GMOs?

GMOs, or genetically modified organisms, are animals or plants that have had traits added or amplified for human benefit. Genetic modification can be as simple as selective breeding of a species in order to bring out a quality. However, in recent years it has become possible to insert a gene from one species directly into another species. The most common uses for GMOs are in the agriculture business. Crops are often modified to improve features such as frost, pest, or drought resistance, to give plants longer shelf life, or to improve appearance and taste.

How are They Made?

The GOI (gene of interest) is identified in one species and isolated. This gene is placed in a plasmid, which is inserted into an agrobacteria. The agrobacteria is then inserted into a plant cell. The cell sees the inserted gene as one of its own, and the plant will develop with the specified trait.

Identification

There are two methods used to identify GMOs: ELISA and PCR. ELISA (enzyme-linked immunosorbent assay) identifies genetically modified organisms by identifying specific proteins. The disadvantages of this method are that the produce being tested must be fresh, and each test must be specific to a certain crop. However, ELISA is inexpensive and relatively simple. PCR (polymerase chain reaction), on the other hand, identifies all sequences of DNA foreign to the plant. Because a relatively small number of DNA sequences are inserted into crops, it is easy to test for many at once (a PCR test can identify up to 85% of all GM crops).

Controversy

GMOs have both advantages and disadvantages. Positive effects include the reduction of pesticides and herbicides in agriculture, and the possibility of nutritional value and reduced stress on farmland. However, opponents of GMOs warn that "superweeds" and "superpests", no longer affected by resistant crops, could develop. Other fears include reduction of plant diversity and potential allergic reactions resulting from crossing species. Although there are requirements in Europe to label genetically modified foods, there are no such laws in the United States due to the influence of agribusiness lobbies.

Procedure
1. Put 1 gram of cornflour into a mortar. Add 10 ml of distilled water, and grind into a pipetable mixture. Repeat with the broccoli. This is done to break down the cell wall.

The cornflower supplied in this lab is a non-GMO control. It verifies that the test will not give results for non-GMO foods with GMO primers.

2. Pipet 50 μL of the cornflour mixture and the broccoli into their respective tubes (labeled "non-GMO" and "test"). Add 50 μL of InstaGene to each tube.
3. Place the tubes in a 95°C water bath for five minutes. This breaks the cell and nuclear membranes. Afterwards, centrifuge the tubes.

The InstaGene added in step two prevents DNAse in the cytoplasm from destroying cell DNA that is released once the nuclear membrane is broken.

4. Prepare six test tubes. The contents should be as follows:

1. 20 μL non-GMO control (cornflour), 20 μL plant master mix
2. 20 μL non-GMO control, 20 μL GMO master mix
3. 20 μL test food (broccoli), 20 μL plant MM
4. 20 μL test food, 20 μL GMO MM
5. 20 μL GMO positive control, 20 μL plant MM
6. 20 μL GMO positive control, 20 μL GMO MM

The master mixes are primers which will search for a specific sequence of DNA. Because tubes one, three, and five contain plant primers, they should not show up as bands after the PCR testing. Tube two contains GMO primers, but the food is guaranteed to be non-GMO. If a band shows up from the test, this indicates contamination or some other failure in the experiment. On the other hand, tube six must produce a band, because it contains the GMO-positive control with GMO primers. A lack of a band would also mean a failure in the experiment. Tube four is the only one with completely unpredictable results.

5. Place tubes in the PCR machine (thermal cycler). The PCR machine uses fluctuating temperatures to replicate DNA.

6. Load 20 μL of each sample into the gel electrophoresis apparatus and run the gel for 3 minutes at 200 V.

Results

Bands appeared in lanes 2, 4, and 6. This means that the test food, broccoli, is genetically modified. There is an extra band in lane 2, the cornflour, which is likely due to contamination during the procedure.

pGLO Transformation

Background

Genetic transformation is a process which entails taking a gene from one organism, and inserting it into another so as to alter or add a trait. Uses of genetic transformation include modifying plants to resist frost or pests, strengthening agriculture; altering bacteria to help in environmental disasters by giving them the capability to digest oil; and gene therapy, in which sick cells are transformed with the use of healthy genes.

To move genes from one organism to the other, the gene must be isolated and added to a plasmid. Plasmids are a small circular pice of DNA in addition to the chromosome. They allow bacteria to share genes and help them adapt to their environment more rapidly. To force the bacteria to accept the DNA, the plasmids and bacteria are subjected to heat shock, or a rapid raising and lowering of temperature. This triggers the bacteria's' instincts to absorb new DNA, a survival skill.
This lab involves taking the Green Fluorescent Protein gene from jellyfish and inserting it into bacteria, which will cause them to become bioluminescent.

DNA Chips

Background

A DNA chip (also known as a gene chip, genome chip, or microarray) is used in the study of genes and how they affect cell functions. It is a solid glass slide containing a pattern of spots, each of which is a copy of a certain gene. To compare, for example, cancerous and noncancerous tissue, scientists label each with a different colored dye and apply them to the DNA chip. Each labeled gene attaches to its compliment on the chip; the brighter the resulting color, the stronger the gene is expressed in that specific tissue. A computer is used to see which genes are expressed in cancerous tissue (marked in one color), which in healthy tissue (marked in another), and both (a mix of the two colors). Genes expressed in the cancerous tissue but not in the healthy tissue could be important and require further examination.

Procedure

1. Obtain miniature "microarray"
2. Add 20µl of each sample of cDNA, incubated at 70°, to each spot of the microarray
3. Add 20µl of hybridization solution to each sample
4. Observe results

Results

Samples 3 and 6 were blue. Those genes are expressed only in healthy cells.

Sample 2 was purple, meaning it is expressed in both healthy and cancerous cells.

Sample 4 was purple. It is not expressed at all in lung cells.

Samples 1 and 5 were pink, meaning they are expressed only in cancer cells. Sample 1 was more vivid, meaning it is more likely to be an important factor than sample 5.

CSI : AHS

Introduction

          DNA is essentially the blueprint to create a person, and as we are all unique, so our DNA is unique as well. This has a great number of uses in proving relatedness of individual organisms, identifying species, and studying ancient peoples. DNA is also particularly important in the field of forensic science. Although human beings are 99.9% the same, that 0.1% can be crucial in telling one individual from another. DNA evidence can be used either to incarcerate a man, or to set him free.

 Restriction Enzymes and DNA Analysis

          DNA analysis is carried out through Restriction Fragment Length Polymorphism, or RFLP. Restriction enzymes are normally used within bacteria to protect them from invading viruses, cutting a virus's DNA at the recognition sequence, or one specific point in DNA that the restriction enzyme is made to cut. If there are multiple recognition sequences, the restriction enzyme will cut at multiple points. However, restriction enzymes are not limited to the bacteria in which they originate. In the RFLP process, these "scissors" snip DNA into fragments, the resulting sequence unique to each individual because of his or her specific DNA.When compared to DNA evidence obtained from a victim or crime scene, the cut DNA patterns can be compared to determine from which suspect the DNA originated.

Agarose Gel Electrophoresis

          However, if the restriction enzyme is not aided in any way, the cut DNA will sit in an invisible tangle, undecipherable by forensic scientists. To give the DNA color and place it in the aforementioned patterns, the process of agarose gel electrophoresis is necessary. In electrophoresis, cut DNA fragments are loaded into an agarose gel slab, which is placed in a chamber containing a conductive buffer solution and two dyes, bromophenol blue and xylene cyanol. Electrodes are attached to the chamber, and direct current is passed through these electrodes, polarizing the it. Because DNA is negative, it will be drawn towards the positive pole in the field.The gel is filled with holes, which filter the DNA fragments according to the size of their base pairs. Smaller pairs will slip easily down through the gel, while larger ones will become stuck and take more time. Fragments of the same size ultimately stay together, forming the bands seen on a completed DNA test. While the DNA is filtering down through the agarose gel maze, so are the bromophenol blue and xylene cyanol dyes. These group around the bands of DNA, making them visible to the naked eye.

Procedure

Restriction Enzyme

1. Pipet 10 µl of restriction enzyme into each of the 6 micro test tubes containing 10 µl DNA from a suspect. Flick/tap the tubes to mix the contents.
2. Incubate for 45 minutes at 37°C. This is the temperature of the human body, and will help the restriction enzyme thrive.

Agarose Gel Electrophoresis

(Note: I was absent during this part of the lab, so I am paraphrasing the instructions in the lab packet.)

1. Place tubes in a centrifuge to bring all the liquid to the bottom
2. Add 5 µl of "loading dye" into each tube. The dye helps to make the samples denser, so they sink into the wells in the gel.
3. Place the agarose gel in the in the electrophoresis apratus, and cover it with 1x TAE buffer.
4. Load 10 µl DNA size marker and 20 µl of each of the DNA samples into separate wells, using a separate tip each time to prevent contamination.
5. Turn on the electrophoresis apparatus and let it run for 30 minutes.

Visualization of DNA Fragments

1. Add 120 ml of 100x Fast Blast DNA stain into a staining tray
2. Stain the gels for 2 minutes
3. Rinse with warm water

Results

         Because the restriction enzymes are made to cut at one specific sequence in DNA, the patterns formed by the DNA of different individuals are all unique. It is fairly simple to tell which one of the six samples is identical to the sample taken from the crime scene.

Biofuels

Introduction

Ethanol is a sustainable alternative to the oil that we use today. However, it can be difficult to break down the cellulose contained in plant cell walls, and for this reason an enzyme is needed.

Enzymes are used to speed up chemical reactions. They do this by positioning the reactant in a manner that reduces the activation energy, or the energy needed to make the reaction occur. Because they are not actually consumed in the reaction, they can be used multiple times. Reactions can also be sped up by increasing the temperature at which they occur.

Producing biofuels will become an increasingly important part of our lives in the future. Because there is no one perfect solution, multiple sources such as wind and solar power should accompany biofuels. It is crucial that we develop alternative fuel sources before our finite oil reserves are drained and we are left with no fuel whatsoever.

Procedure

Part 1
1. Measure 500 µl of stop solution into five cuvettes. The stop solution will be used to stop a sample of the reaction at a certain time, allowing us to see how much product has been created in that amount of time.

2. Pipet 2 ml of 1.5 mM substrate, or reactant, into a tube labeled "enzyme reaction". In this case the substrate is cellulose, found in plant cell walls.

3. Add 1 ml of cellobiase, an enzyme, to the tube. Cellobiase will speed up the reaction time, creating product faster.

4. At 1, 2, 4, 6, and 8 minutes, remove 500 µl from the reaction and add it to the appropriate cuvette. The stop solution in the cuvettes will halt the reaction, and dye the p-nitrophenol in the reaction yellow. The deeper the yellow, the more product has been formed.

Part 2
Repeat steps above, substituting ground mushroom for the cellulose in step 2. The mushroom is a potential natural fuel source.

Results

In each vial, the yellow was significantly more vivid. This proves that the reaction continued over the observed timeframe, creating more and more ethanol (the product). Because of the added cellubiase, the product was formed much faster than it would be if left to react without an enzyme.

The greatest chance for error is in grinding up the mushrooms with the mortar and pestle. It is possible to measure either too much or too little mushroom, and the mortar may be contaminated with chemicals from past work.