Testing for Genetic Diseases via DNA extraction, PCR, and gel electrophoresis

Introduction:

a) DNA testing is the extraction and analysis of the entire genome or, more commonly, a specific gene of interest. DNA testing has multiple applications. For example, it can be used for forensics, paternity testing, or, in this case, genetic disease diagnosis. Of course, we're not actually testing for a real genetic disease in class, but the principle is the same. DNA testing involves three main steps - DNA extraction, PCR, and gel electrophoresis - which will be discussed later in part c (techniques).
b) The purpose of this lab is to test ourselves for a "genetic disease". We will be utilizing all the techniques below in order to achieve this goal.
c) First, we start with DNA extraction - cheek cells will be swabbed and placed into a test tube containing a lysis buffer to help break open the cell/nuclear membranes. We will then put the cheek cells into a hot water bath of 95 degrees Celsius to further lyse open the membranes. Instagene matrix beads are used to kill DNAse, which would otherwise kill the exposed DNA. Second is PCR (polymerase chain reaction), which is used to make many copies of the DNA so that the DNA will be easier to test. The more copies, the more likely it will show up on the gel (part 3). A main ingredient of PCR is the primer, which targets the specific gene of interest. That way, we make copies of just the gene of interest (in this case, the "disease" gene), rather than the entire genome, which would be unnecessary. Third, we run the mass-produced genes on a gel. This process is called gel electrophoresis. The electric current run through the gel pulls the slightly negatively charged DNA toward the positive end - the smaller segments of DNA (in this case, the "diseased" gene is smaller than the "healthy" gene) will be able to work their way through the matrix faster and will thus travel farther toward the positive end. The larger pieces (the "healthy" gene) will be bogged down in the matrix and will not make it as far. Thus, gel electrophoresis has three potential outcomes:
1. Two bands that did not travel far --> indicates two "healthy" genes
2. Two bands that did travel far --> indicates two "diseased" genes
3. One band that did not and one band that did travel far --> indicates one "healthy" gene and one "diseased" gene.
Only outcome #2 will result in the expression of the disease, since the disease is recessive (both alleles must be recessive in order for the recessive genes to be expressed in the phenotype).

Results:

I do not have a picture of the gel results to post on this blab, but I can tell you that 3 of the people at my lab table tested positive for the "disease". In other words, each of us had 2 bands that travelled far and matched the bands of the "diseased" control gene. The fourth person's bands did not show up, so we cannot determine whether she was positive or negative. Luckily this "disease" is an intron, or else we'd all be in a bad spot!

Testing for GM foods via DNA extraction, PCR, and gel electrophoresis

Introduction:

a) GMO stands for Genetically Modified Organism. Many GMOs are agricultural plants, which have been enhanced to be cold or drought resitant, pest resistant, bigger, stronger, healthier, etc. Genetic modification can be used to create the "perfect apple" that can withstand forces of nature and be produced in vast amounts, increasing the farmer's yield and profit.
GMOs are made by inserting a plasmid with a gene of interest (say, a gene for a redder color) into agrobacteria. Usually the plasmid is a Ti plasmid, meaning Tumor-inducing. The plant is then infected with the agrobacteria containing the Ti plasmid and the plant accepts the bacteria and allows the protein to be made from the red-color gene. The plant's fruit then becomes a beautiful red color!



GMOs can be identified by a certain method called PCR. PCR stands for Polymerase Chain Reaction and it involves making many copies of DNA so the DNA can be easier to test. DNA polymerase, a primer specific to the Ti plasmid, nucleotides, and plasmid DNA are needed for PCR to occur.
The controversy surrounding GMOs involves a conflict between technology, natural selection, and morals. Some people are greatly in favor of GMOs, saying that humans are only speeding up the process of natural selection and we have the technology so why not use it? Others say that GMOs go against nature and humans have no right to "play God". GMOs also result in a decrease in genetic variation in a population, which can have terrible consequences if a certain strain of virus were to emerge - the entire population of crops would be rapidly wiped out. An example from history - the potato famine in Ireland. Too much dependence on one type of crop is bound to result in catastrophe. But whether humans will be able to resist the possibility of creating and shaping "perfect" crops, animals, and maybe even humans is doubtful.



GloFish - the first genetically modified pet

b) The purpose of this lab is to test grocery store produce to see if it is genetically modified. This can be done in the real world as well - scientists must varify that a farmer's crop is organic by performing similar tests as the one we will do in class. A fruit is not organic if it has been genetically modified.
c) We will be using many techniques for DNA extraction in this lab. First, we will use a mortar and pestle to grind up the produce, breaking open the cell walls. Next, a hot water bath will be used to lyse open the cell and nuclear membranes. This leaves the DNA vulnerable, so Instagene matrix beads will be added to kill the DNAse in the cells. DNAse is present in all cells to kill foreign DNA - in this case, we do not want DNAse to kill the cell's own DNA, so the DNAse must be killed first. During PCR, we will use two primers - one to target the plant DNA (green primer) and the other to target GM DNA (red primer). The green primer serves as a control - all plant cells contain plant DNA, so if the plant DNA does not show up during gel electrophoresis, then we know the lab was not successful. If the plant DNA does show up, then we know PCR worked and if there is no GM DNA that shows up, then we know that the plant is not a GM product. Gel electrophoresis is used to separate and analyze the DNA found in the cells.
d) We are testing for GM DNA (the Ti plasmid) in the plant cells. That is the variable. Plant DNA is the control (as explained above). My hypothesis is that the non-organic produce (the corn, apple, etc.) will be genetically modified. Some 85% of non-organic foods are genetically modified. But the organic foods will be all natural.

Results:

According to the results, the bands of our test foods match the bands of the GM+ food - thus, all our test foods (the corn powder and the lettuce) are genetically modified. 

Glowing Bacteria: Transformation of pGLO plasmid from sea jelly to bacteria

Introduction:
a) Genetic transformation is the process in which a gene of interest is inserted into or taken up by another organism's cell (usually a bacterium), changing the organism's trait(s). Transformation can be used to transfer a insect resistant gene into crop plants or to enable bacteria to digest oil spills. In this lab, we will be transferring a sea jelly's GFP gene (Green Fluorescent Protein) contained in a pGLO plasmid into bacteria to make the bacteria glow! The pGLO plasmid also contains a gene for ampicillin resistance and a special gene regulation system which can be used to control expression of the fluorescent protein in transformed cells. The gene for GFP can be switched on by adding sugar arabinose to the bacteria's nutrient medium. The bacteria will also be grown on antibiotic plates, killing all the bacteria which did not take up the pGLO and thus do not have antibiotic resistance. This will allow us to determine which bacteria contain the pGLO plasmid.
b) The purpose of this experiment is to transfer the pGLO plasmid (which is derived professionally from a sea jelly gene) into the bacteria. Hopefully we will be able to see the bacteria glow, like a sea jelly! It is a simple way of seeing how genetically modified organisms are made in the real world of biotechnology.
c) There are many techniques we will be utilizing in this lab. Among them are proper pipetting techniques, to fill the test tubes with the Calcium Chloride solution and to put the pGLO plasmid into the tube.. We will use sterile loops to collect bacteria from a dish and put the bacteria in the solution. Heat shock is a technique used to induce transformation - the solution of CaCl2, plasmids, and bacteria is iced for about 10 minutes before put into a hot water bath and then iced again. This causes a sort of current, allowing plasmids to go in through the bacteria's pores. After heat shock, we will use pipetting techniques to transfer the solution onto a petri dish. Incubation of the dish will allow bacteria to grow faster so we can see the results!
d) My hypothesis is that the experiment will work! I think we will be successful and will be able to create glowing bacteria.

Comparing Gene Expression in Lung Cancer Cells and Normal Lung Cells Using Microarrays

Introduction:

a) All cells (except for gametes) contain the same DNA. What makes cells unique is how they express this DNA - sometimes the difference between a healthy cell and a diseased cell is a simple change in gene expression. Microarray analysis allows us to compare gene expression by first creating a gene chip, in which specific DNA sequences are put into individual spots on the chip. Meanwhile, cDNA (complementary DNA) is created from mRNA found in the healthy and diseased cells. The cDNA is then added to the chip and the fragments bind to their complementary strands of DNA that were placed there originally. By analyzing the chip, we can see what sequences are being expressed based on where the cDNA binds. For instance, let's say that the cDNA from the diseased tissue is labeled red and the cDNA from the healthy tissue is labeled green. When we analyze the results, spots that are green indicate that those particular sequences are only expressed in healthy tissue. Spots that are red indicate sequences that are only expressed in diseased tissue. Spots that are yellow (red light + green light = yellow light) indicate sequences which are expressed in both and spots that don't turn any color are not expressed in either.
b) The purpose of this experiment is to analyze gene expression in lung cancer cells versus healthy lung cells by using microarrays. Potentially, by gaining more knowledge about gene expression in this way, we could develop ways to alter cancer cells or cure them somehow. The more we understand about what makes a cancer cell different, the closer we get to solving the problem.
c) Most of the techniques or materials were explained in part "a". The microarray chip serves to isolate known DNA sequences, so that when we apply the cDNA to the chip, we can determine which sequences are active. The red and green labels show us which sequences are specifically expressed in either, both, or neither of the two cell types. We will use pipetting techniques to transfer the cDNA to the miniature microarray.
d) For my hypothesis, I think genes which are involved in typical cell functions (such as protein synthesis or cell respiration) will be expressed in both the healthy and the cancer cells. I think genes which are involved in replication will likely be turned off in the cancer cells, since they are known to divide uncontrollably. After that,  I will have to wait and see how the experiment turns out. The controls of this experiment are the DNA sequences on the microarray. We already know where specific sequences are located on the chip. The variables are the cDNA sequences, which are unknown (until they bond to the DNA on the chip of course).

Results:

The microarray results are shown above. Pink = expressed in lung cancer cells only (#1,5). Blue = expressed in healthy lung cells only (#3,6). Purple = expressed in both (#2). Clear = expressed in neither (#4).

Solving the Mystery using Restriction Enzymes and Gel Electrophoresis

Introduction:
a) Restriction enzymes are a specific type of protein which can be found in bacteria. They act as a defense against viruses by cutting up viral DNA at specific locations called palindromes (these specific sites can also be called restriction sites). Gel electrophoresis is a process used to analyze DNA - the DNA is placed in a gel and an electric current is sent through. DNA is negatively charged, thus it will be pulled toward the positive node. However, the smaller, lighter pieces of DNA can move faster and travel farther along the gel. If this cut-up DNA is run on a gel, we can determine the length of each fragment of DNA based on how far they traveled along the gel. The different fragments can be seen as dark bands along the gel. This is called Restriction Fragment Length Polymorphism (RFLP).
b) The purpose of this experiment is to analyze different suspects' DNA via gel electrophoresis and compare their DNA to the crime scene's. The suspect whose DNA matches the crime scene's (meaning the bands are the same) is the culprit.
c) The purpose of the restriction enzymes is to cut up the suspects' DNA - each person has a unique genetic code, thus each person's DNA will be cut at different locations and will result in unique DNA fragment lengths. A loading buffer containing blue dyes is added to the DNA samples before injection into the gel - this allows us to monitor the process of gel electrophoresis. The gel electrophoresis and RFLP will then show a unique banding pattern of each person's DNA fragment lengths on a gel.  We can then analyze these bands to determine which DNA matches the DNA of the crime scene.
d) I cannot predict which person will be the culprit since I have not seen the results yet. However, the control is the crime scene DNA and the variables are the suspects' DNA.

Procedure:
Basically explained above...

Results:
It turns out Suspect #3 was the criminal! The DNA bands from Suspect #3 clearly matched the DNA bands of the Crime Scene - the evidence is incriminating.

Discussion:
a) Via gel electrophoresis, the fragments were separated by length along the gel - the shorter fragments traveled farthest toward the positive node while the longer fragments remained further behind. By comparing the bands, it was clear that the DNA bands of Suspect #3 exactly matched the bands of the Crime Scene.
b) Possible sources of error:

• Improper pipetting
• Contamination of DNA
• Not run on the gel long enough to accurately disperse DNA fragments

Creating biofuels from cellulose using enzyme cellobiase

Introduction:
a) Enzymes speed up the rate of chemical reactions without being used up by first binding to the reactants (substrate). The location of the binding is called the enzyme's active site. This bond lowers the activation energy (the energy needed to start the reaction), making the reaction occur faster. Enzymes' effectiveness can be affected by pH, temperature, and salinity. The reaction tends to occur faster if the concentration of enzymes or substrates is increased, but there is a point when the solution becomes saturated with either enzymes or substrates and cannot work any faster. The enzyme we will be using on this lab is cellobiase, which breaks down cellobiose - a sugar derivative of cellulose, a polysaccharide found in plant cell walls. The biofuel agency uses enzymes such as cellobiase to break down cellulose in plant matter and convert it to glucose. Glucose can then be converted to ethanol by microbial fermentation. Ethanol can then be used as an energy source to power certain motors and engines.
b) The purpose of this lab is to experience first hand in the classroom what biofuel companies do on a massive scale. Biofuel companies are constantly experimenting to find the most efficient way to make biofuels - and the method we will use in this experiment is only one method out of many.
c) We will use pipetting techniques to carefully and accurately transfer enzymes, buffers, and substrates to the solution. On Day #1, we will transfer a little bit of the solution at various time points to another test tube with a strong base to stop the reaction. The base will also turn p-nitrophenol (a product of the reaction) yellow, so we can judge how much product (including the glucose) has been created. On Day #2, we will have to carefully grind up mushrooms and add the mushroom extract to the reaction and measure how this affects the speed of the reaction.
d) I predict that, as time goes on, more product will be produced from the reaction. Also, I think the mushroom, which contains more enzymes, including cellobiase, will speed up the reaction rate. The variable of this experiment is the mushroom. The control is the amount of product produced under normal circumstances (Day #1).

Results/Observations:
The solution from Day #1 turned more yellow over time (the first cuvette was the lightest and the last cuvette, the darkest). The solution with the mushroom (Day #2) also turned darker over time, only faster.

Discussion:
a) The solution from Day #1 turned more yellow over time (the first cuvette was the lightest and the last cuvette, the darkest). This indicates that more product was produced over time. On Day #2, the mushroom turned darker faster, indicating that the reaction was occurring faster. This is probably because the mushrooms contain more enzymes, such as celliobiase, which can speed up the reaction to an extent. There is a limit to how many enzymes are effective, but in the case of Day #2, the extra enzymes did help speed up the reaction.
b) Possible sources of error...

• Incorrect measuring with the pipettes
• Contamination of the solution(s)
• Timing errors

Extracting and precipitating DNA from cheek cells

Introduction:
a)  All living things contains DNA in each of their cells. Eukaryotes contain a nucleus inside their cells, which houses the DNA. Prokaryotes do not have a nucleus, but their DNA moves freely throughout the cell. DNA is a double-stranded helix composed of sugar (deoxyribose), phosphates, and bases (thymine, adenine, guanine, and cytosine). Thymine pairs with adenine and guanine pairs with cytosine. Genes are particular segments of DNA which code for a specific protein. Proteins are what give your eyes color, carry out cell communication, and build muscles.
b)  The purpose of this lab is to precipitate (or anti-dissolve) DNA, so that we can see and observe it with the naked eye. In a professional setting, such as in a biotech lab, one can use the extracted DNA to map the genome, clone genes, compare DNA, test for genetic diseases, or do forensics. However, in the classroom, we will simply be admiring our DNA.
c)  We will use a "chewing on the insides of our cheeks" technique in order to loosen cheek cells. The saline solution (which contains 0.9% salt water) will be used to give the cells an isotonic solution to keep them from bursting or shriveling. The lysis buffer (which is a detergent) will serve to dissolve the cell membranes (which are made of phospholipids), releasing the DNA out into the open. DNase is an enzyme that lives in the cell in order to kill foreign DNA; however, we do not want the DNase to kill our own DNA now that it's been released, so we add the enzyme protease to destroy it. DNA is negatively charged, due to the negative phosphates. A negative charge makes DNA polar and hydrophillic, meaning it likes water and will dissolve in it. We do not want our DNA to dissolve - in fact, we want to anti-dissolve it or precipitate it - so, we add Na+ ions to give DNA a neutral charge. This makes DNA nonpolar and hydrophobic so it will not dissolve. The hot water bath serves to speed up the enzyme reactions and helps break open the cell membranes (lysis buffer works better with hot water). Finally, the cold ethanol (rubbing alcohol) helps with precipitation by forming a very cold layer on top of the hot water solution.

Procedure:
*Our video was accidentally deleted by another group - however I will briefly go over the procedure here.

See Introduction part "c" for details on each step of the procedure. However, I will note that after the DNA had precipitated entirely, we extracted it with a pipette and put it into a necklace and wore it around school!

Results/Observations:
I observed that almost instantly after we added the cold ethanol to the hot water solution, the DNA began precipitating between the two layers. It formed a stringy, white substance that was very easy to see and very cool! It definitely looked like DNA, with the long strands all tangled together. It was very interesting.

Discussion:
a)  I did not get very much DNA, however, it was definitely there and very easy to see. The procedure largely explains how the DNA was extracted and there is not anything to analyze or explain really about the results. All I can say is that it worked!
b)  Possible sources of error could be...

• Food or other contents from the mouth could have gotten into the solution
• Insufficient chewing on the cheeks may not have extracted enough cheek cells
• Some DNA may have been destroyed in the process by a hypo/hypertonic solution (if the saline solution did not work) or DNase (if the protease did not kill it in time)