The animal immune system is a sophisticated weapon in the fight against foreign invaders, which include harmful bacteria, viruses and fungi. The first part of the immune system is the non-specific defense that attempts to prevent all unknown substances from entering the body. In a human, this includes skin, earwax and mucous membranes. If this defense is compromised, usually by a cut on the skin, an inflammatory response occurs, adding heat to the area and clotting the blood to block the opening.
Any foreign particle that triggers an immune response is called an antigen. If an antigen is able to make it past the first-line, non-specific response, the body targets the invader with a specific attack. First, the body rapidly makes copies of an antibody that can bind to the antigen. There are countless variations of antibodies in human blood, each with a shape that matches it to one specific antigen. The binding of an antibody to an antigen flags the invader for destruction by other immune system cells.
Scientists utilize the ultra-specific nature of the immune response to create tests for certain diseases. This will be the basis for our lab, in which an infection will spread throughout the classroom through "bodily fluids." When testing for a certain disease, the matching antibodies are added to the subject's blood samples. If the patient has the infection, the antibodies will bind to the antigens. Next, secondary antibodies are added to the sample. Secondary antibodies are isolated by exposing one species to antibodies from a different species. The exposed animal treats the antibodies as a foreign invader, creating new secondary antibodies to bind to the original ones. These secondary antibodies, attached to enzyme, are then added to the blood sample, binding to the original antibody-antigen complex if disease is present.
The method is called Enzyme-linked Immunosorbant Assay (ELISA) and can be useful in many different applications. It can be used to test for HIV, SARS or any other type of cantagious infection (but not genetic diseases). It can also be used for pregnancy testing by targeting the hormones released by the fetus in a mother's womb.
We will record the name of the person with whom we swapped bodily fluids, as well as the time the exchange occurred. At the end, we will use ELISA to determine if and to what level we have the disease. Using the fluid-swap logs, we will hopefully be able to figure out who initially had the disease and, therefore, caused the epidemic.
Results:
Based on the chart, we have concluded that Chloe and Taylor were the original disease-carriers. Every person they came in contact with ended up with a full-blown version of the disease, so they must have started the pandemic. There are several sources of error that could potentially screw up the experiment, but we were careful to avoid them. If the wells are not completely washed after the second antibody is added, there could be a false positive. Enzyme may remain in the well even if the second antibody did not bind. The same could occur if the primary antibody is not fully washed out. The second antibody would still bind, and a false positive would also occur. Finally, it is possible for to people to analyze the color of the liquid differently. What appears blue to one person may appear light blue to another. Luckily, although the lab was probably not perfecct, we were able to find the two individuals who began the spread of the disease.
Monday, May 16, 2011
Wednesday, April 13, 2011
Sushi (or Lack Thereof): Evolutionary Relationships through Muscle Protein Profiles
The last few decades in science have centered on genomics, the study of genomes, especially the human genome. Though genomics is still an evolving field, proteomics, the study of the structures and functions of proteins, has emerged as one of the hottest fields in science. The genome, which consists of long stretches of non-coding regions, does not tell the entire story. Protein, the product of the coding sequences of DNA, can give a more straight-forward view. Proteins are also altered after translation from RNA, indicating that life cannot be entirely explained by simply referring to a sequence of DNA bases. Furthermore, it is possible that the non-coding regions of DNA have a role in protein regulation, but that cannot be determined without consulting the proteins themselves. Because of the importance of proteins in all molecular interactions, proteomics projects to be a ground-breaking science that could very well change our perception of life.
Still, proteomics is an incredibly complex field because the proteome of an organism isn't constant. Every cell type utilizes different proteins to fulfill its duties, and these proteins vary as an individual grows and matures- and that is only within a single organism. Proteomics becomes vastly more complex when comparing species
In the past, scientists had to examine anatomical features or track development and behavior to determine evolutionary relationships. Though the technique was often correct, it was extremely prone to error. Genomics and proteomics are much more accurate, as they illustrate relationships at the molecular level. The more similar the genomes or proteomes of two species, the more closely related they are (the more recently they diverged from a common ancestor).
In this lab, we will attempt to infer evolutionary relationships by studying the muscle protein profiles of various sea creatures, isolated from sushi. First, we will add a buffer to begin the denaturation process of the muscle proteins. The buffer will also give the proteins a negative charge, so that they will run on the gel. We will then run our proteins on a gel, just as if they were DNA. The resulting gel will give us a general profile of the muscle proteins in each species, so we can create a cladogram.
Results:
Visible lanes from left to right: Kaleidoscope Marker, salmon, scallop, shrimp, actin/myosin marker
Still, proteomics is an incredibly complex field because the proteome of an organism isn't constant. Every cell type utilizes different proteins to fulfill its duties, and these proteins vary as an individual grows and matures- and that is only within a single organism. Proteomics becomes vastly more complex when comparing species
In the past, scientists had to examine anatomical features or track development and behavior to determine evolutionary relationships. Though the technique was often correct, it was extremely prone to error. Genomics and proteomics are much more accurate, as they illustrate relationships at the molecular level. The more similar the genomes or proteomes of two species, the more closely related they are (the more recently they diverged from a common ancestor).
In this lab, we will attempt to infer evolutionary relationships by studying the muscle protein profiles of various sea creatures, isolated from sushi. First, we will add a buffer to begin the denaturation process of the muscle proteins. The buffer will also give the proteins a negative charge, so that they will run on the gel. We will then run our proteins on a gel, just as if they were DNA. The resulting gel will give us a general profile of the muscle proteins in each species, so we can create a cladogram.
Results:
Monday, March 28, 2011
Extraction, Amplification and Electrophoresis of Human Mitochondrial DNA
Imagine that we could trace our family tree back to the first human ever to set foot on earth. Well actually, it doesn't take much of an imagination, as the study of mitochondrial DNA may offer us just that possibility. From an early age, everyone learns what DNA is. Mom and dad pass some stuff to their children that makes them look and act alike. But a lesser known fact is that there is a different type of DNA that we obtain solely from our mothers.
In all eukaryotic organisms are organelles called mitochondria. It is theorized that mitochondria were once their own organism but were engulfed by a more sophisticated eukaryotic cell. Mitochondria utilize oxygen from the atmosphere to create energy that fuels the functioning of the cells, making it one of the most vital organelles in our bodies.
Mitochondria actually carries its own set of DNA that codes for the machinery that allows it to synthesize energy (ATP). This DNA comes directly from our mothers, creating a lineage based on nearly-identical mitochondrial DNA, differing only due to mutation. But why do we procure this DNA just from our mothers? Sperm, hosting the male genome, must be small and elusive to make its way to the female egg, so it carries little more than the necessary DNA, with just enough mitochondria to power its journey. The egg, on the other hand, contains all the typical organelles. Therefore, the mother is the one to contribute its cytoplasm and organelles, including the mitochondria, to the zygote.
The mitochondrial (mt) genome was sequenced far before the human genome because it only contains 16,569 nucleotides and 37 genes. The mt genome has few introns, but it does have one long non-coding stretch that is highly mutative. The region's supervariable quality creates SNPs that help establish familial relationships. Based on rates of mutation, scientists determined that the "mitochodrial Eve" first appeared 200,000 years ago in Africa- the origin of modern humans. The non-coding control region is especially useful because each cell contains hundreds of thousands of copies of each mt gene.
For our experiment, we will use a template DNA instead of our own. First, we will add Chelex beads to the sample to break open the membrane and destroy ions that block PCR, and then we will separate the cells by vortexing. Next, we will amplify the extracted DNA through the process of PCR that I have described thousands of times. As a refresher, PCR requires DNA polymerase, primers, DNA nucleotides, and DNA ligase. It is essentially the rapid, controlled process of DNA replication. We will use specific primers that will target a segment of the non-coding region of mt DNA.
Finally, we will run our mt DNA samples through gel electrophoresis, in which the DNA molecules will travel a certain distance, depending on their length (number of nucleotides).
In all eukaryotic organisms are organelles called mitochondria. It is theorized that mitochondria were once their own organism but were engulfed by a more sophisticated eukaryotic cell. Mitochondria utilize oxygen from the atmosphere to create energy that fuels the functioning of the cells, making it one of the most vital organelles in our bodies.
Mitochondria actually carries its own set of DNA that codes for the machinery that allows it to synthesize energy (ATP). This DNA comes directly from our mothers, creating a lineage based on nearly-identical mitochondrial DNA, differing only due to mutation. But why do we procure this DNA just from our mothers? Sperm, hosting the male genome, must be small and elusive to make its way to the female egg, so it carries little more than the necessary DNA, with just enough mitochondria to power its journey. The egg, on the other hand, contains all the typical organelles. Therefore, the mother is the one to contribute its cytoplasm and organelles, including the mitochondria, to the zygote.
For our experiment, we will use a template DNA instead of our own. First, we will add Chelex beads to the sample to break open the membrane and destroy ions that block PCR, and then we will separate the cells by vortexing. Next, we will amplify the extracted DNA through the process of PCR that I have described thousands of times. As a refresher, PCR requires DNA polymerase, primers, DNA nucleotides, and DNA ligase. It is essentially the rapid, controlled process of DNA replication. We will use specific primers that will target a segment of the non-coding region of mt DNA.
Finally, we will run our mt DNA samples through gel electrophoresis, in which the DNA molecules will travel a certain distance, depending on their length (number of nucleotides).
MOMS
GGel Electrophoresis
Our lab was a success, with everyone's mitochondrial DNA showing up on the gel. All our mt DNA strands were the same size, so they traveled the same distance on the gel. Because of this, we cannot make any differentiations between our mt DNA. For a small fee, however, we could send it off to be sequenced by a lab. We just might do that...
Friday, March 18, 2011
Teenage Mutant Electrophoresis
DNA testing has opened a completely new realm of scientific possibilities. The process has evolved over the years from a tedious task in which a lone scientist charted nucleotides by hand to a routine procedure often rapidly performed by robots. The technique has numerous applications, including testing for evolutionary relationships, crime scene investigation, testing familial relationships, and individual testing for genetic predispositions for certain diseases.
In our lab, we will be testing for a hypothetical disease, as disease testing is unethical in a high school environment. The gene for the "disease" we will be testing for comes in two separate forms: a long, sterile segment and a short, disease-causing segment. The short form is a recessive disorder, so we will need two copies (homozygous recessive) to have the predisposition. With Polymerase Chain Reaction (PCR) we will be able to target and amplify this specific gene, although in reality it is an intron that does not code for protein.
There are several techniques for DNA testing, but we will be using gel electrophoresis. First, we need to extract our DNA, which we will take from cheek cells. After rinsing with a saline solution and collecting the cells, we will put the cells in a 95-degree Celsius water bath to break through the cellular and nuclear membrane. Hiding in the cytoplasm, however, are enzymes called DNAse that kill any DNA they find. This is a protection against foreign DNA, like from a virus, but it cannot tell the difference between viral DNA and its own genetic material. Therefore, we will add Instagene Matrix Beads that dismantle DNAse and allow us to extract our DNA, unharmed.
This will be a tiny amount of DNA that won't be visible on a gel, so we must amplify the genetic material using PCR. PCR requires primer for our specific sequence, ample DNA nucleotides, DNA polymerase, and DNA ligase. During PCR, our DNA samples will rapidly replicate by a power of 2, giving us more than enough material to be seen in gel electrophoresis.
Lane 1 represents an individual with the genetic disorder (homozygous recessive). The recessive gene is smaller, so it travels farther in the gel. Lane 1, therefore, must have two copies of the recessive gene because there is only one band.
Lane 2 is homozygous dominant and does not have the disease. There is one band that did not travel very far, so it must represent two copies of the larger, dominant gene.
Lane 3 is heterozygous and doesn't have the disease. There are two bands, so it must have had one copy of each type of the gene.
I'm not diseased!!! At first I believed I was diseased because I only saw one solid band designating the recessive gene, but there was a faint semblance of a band below it, meaning that I am heterozygous- a carrier. David and Taylor are diseased because they just have one far-traveling band. Schuyler is homozygous dominant, having zero copies of the disease gene.
There were some potential sources of error, but we avoided them. We loaded our gel properly, not puncturing the well or contaminating lanes with incorrect sample. Overall, this lab went perfectly.
In our lab, we will be testing for a hypothetical disease, as disease testing is unethical in a high school environment. The gene for the "disease" we will be testing for comes in two separate forms: a long, sterile segment and a short, disease-causing segment. The short form is a recessive disorder, so we will need two copies (homozygous recessive) to have the predisposition. With Polymerase Chain Reaction (PCR) we will be able to target and amplify this specific gene, although in reality it is an intron that does not code for protein.
There are several techniques for DNA testing, but we will be using gel electrophoresis. First, we need to extract our DNA, which we will take from cheek cells. After rinsing with a saline solution and collecting the cells, we will put the cells in a 95-degree Celsius water bath to break through the cellular and nuclear membrane. Hiding in the cytoplasm, however, are enzymes called DNAse that kill any DNA they find. This is a protection against foreign DNA, like from a virus, but it cannot tell the difference between viral DNA and its own genetic material. Therefore, we will add Instagene Matrix Beads that dismantle DNAse and allow us to extract our DNA, unharmed.
This will be a tiny amount of DNA that won't be visible on a gel, so we must amplify the genetic material using PCR. PCR requires primer for our specific sequence, ample DNA nucleotides, DNA polymerase, and DNA ligase. During PCR, our DNA samples will rapidly replicate by a power of 2, giving us more than enough material to be seen in gel electrophoresis.
Lane 1 represents an individual with the genetic disorder (homozygous recessive). The recessive gene is smaller, so it travels farther in the gel. Lane 1, therefore, must have two copies of the recessive gene because there is only one band.
Lane 2 is homozygous dominant and does not have the disease. There is one band that did not travel very far, so it must represent two copies of the larger, dominant gene.
Lane 3 is heterozygous and doesn't have the disease. There are two bands, so it must have had one copy of each type of the gene.
There were some potential sources of error, but we avoided them. We loaded our gel properly, not puncturing the well or contaminating lanes with incorrect sample. Overall, this lab went perfectly.
Tuesday, February 1, 2011
Robot Plants
Genetically Modified Organisms (GMOs), usually plants or bacteria, have been genetically engineered to increase efficiency or to fit a certain purpose. Scientists can take a desirable gene from the same species- or even from a completely different species- and transfer it to another organism, which will then express the gene as if it was its own. Plants, including the food we eat, are often genetically modified to improve yield by giving them characteristics such as disease or frost resistance, or to increase size or improve taste. Bacteria can be engineered to create human proteins such as insulin, or even to clean up oil spills.
This cutting-edge science, like all new discoveries, has garnered plenty of criticism and controversy. Besides the ethical concerns of playing God, people are worried that tinkering with the genetic harmony of the world could have disastrous effects that we are unable to predict. Although scientists have proved that GM crops are much safer than crops sprayed with pesticides, skeptics worry that GM crops could create multi-resistance superbugs or, through cross-pollination, superweeds.
With a growing population and static supply of land, GM proponents believe this technology is absolutely necessary to make sure we can produce enough food for the world. By creating plants that can persevere in harsh conditions, the amount of land available for crops is vastly increased. Therefore, the benefits outweigh the potential setbacks.
For this lab, we will be testing whether or not certain plants have been genetically modified. Because GM products in the U.S. do not need to be labeled, this is an important technique. The easiest and most effective way to determine whether or not a food has been genetically altered is through a process called Polymerase Chain Reaction (PCR). PCR is a way to, through replication, amplify a small amount of DNA very rapidly. The process has many applications, including crime scene analysis, where DNA samples are often too tiny to test, and in the identification of fossils containing very little intact DNA.
First, we will need to extract the DNA from our food source. To do this, we must break down the cell wall by grinding the material with a mortar and pestle, and then break open the cell membrane and nuclear membrane by putting the sample in an extremely hot water bath. DNA is supposed to stay in the nucleus, however, and an enzyme called DNAse (kills foreign DNA), will destroy our sample. To prevent this, we will use Instagene Matrix Beads, which kill enzymes, keeping our DNA safe from DNAse.
The second day we will use PCR to mass produce our DNA sample for testing. The ingredients necessary for PCR are DNA polymerase, lab-created RNA primer, which targets specific genes, nucleotides, and the DNA we want to amplify. Almost all GM plants contain a Ti (tumor-inducing) plasmid. Because plants do not have vital organs, a tumor is not devastating to its health. Ti plasmids are used because they are the easiest way to transfer genetic material from a bacterium to a plant cell. We will have two separate primers for our PCR: one to test the GM DNA and another to test a control plant's DNA. This control is necessary to make sure the lab actually worked. If our results for the GM food turn out negative, it may simply be because our experiment failed. The control prevents this potentially incorrect conclusion.
Finally, on the third day, we will use Gel Electrophoresis to test our DNA samples. Compared to the marker and our plant control, we will be able to determine whether or not our sample food has been genetically modified.
This cutting-edge science, like all new discoveries, has garnered plenty of criticism and controversy. Besides the ethical concerns of playing God, people are worried that tinkering with the genetic harmony of the world could have disastrous effects that we are unable to predict. Although scientists have proved that GM crops are much safer than crops sprayed with pesticides, skeptics worry that GM crops could create multi-resistance superbugs or, through cross-pollination, superweeds.
With a growing population and static supply of land, GM proponents believe this technology is absolutely necessary to make sure we can produce enough food for the world. By creating plants that can persevere in harsh conditions, the amount of land available for crops is vastly increased. Therefore, the benefits outweigh the potential setbacks.
For this lab, we will be testing whether or not certain plants have been genetically modified. Because GM products in the U.S. do not need to be labeled, this is an important technique. The easiest and most effective way to determine whether or not a food has been genetically altered is through a process called Polymerase Chain Reaction (PCR). PCR is a way to, through replication, amplify a small amount of DNA very rapidly. The process has many applications, including crime scene analysis, where DNA samples are often too tiny to test, and in the identification of fossils containing very little intact DNA.
First, we will need to extract the DNA from our food source. To do this, we must break down the cell wall by grinding the material with a mortar and pestle, and then break open the cell membrane and nuclear membrane by putting the sample in an extremely hot water bath. DNA is supposed to stay in the nucleus, however, and an enzyme called DNAse (kills foreign DNA), will destroy our sample. To prevent this, we will use Instagene Matrix Beads, which kill enzymes, keeping our DNA safe from DNAse.
The second day we will use PCR to mass produce our DNA sample for testing. The ingredients necessary for PCR are DNA polymerase, lab-created RNA primer, which targets specific genes, nucleotides, and the DNA we want to amplify. Almost all GM plants contain a Ti (tumor-inducing) plasmid. Because plants do not have vital organs, a tumor is not devastating to its health. Ti plasmids are used because they are the easiest way to transfer genetic material from a bacterium to a plant cell. We will have two separate primers for our PCR: one to test the GM DNA and another to test a control plant's DNA. This control is necessary to make sure the lab actually worked. If our results for the GM food turn out negative, it may simply be because our experiment failed. The control prevents this potentially incorrect conclusion.
Finally, on the third day, we will use Gel Electrophoresis to test our DNA samples. Compared to the marker and our plant control, we will be able to determine whether or not our sample food has been genetically modified.
Thursday, January 27, 2011
Playing God: Transforming Bacteria with Sea Jelly Fluorescence Gene
Genetic transformation, with diverse applications like bioremediation, gene therapy and genetically modified foods, is one of the most important techniques in the biotech field. In this experiment, we will transform bacteria with a gene from a sea jelly that codes for Green Fluorescent Protein. If everything goes as planned, our bacteria should glow under UV light.
A bacterium is a prokaryotic single-celled organism with one long, linear chromosome and sometimes a circular plasmid containing specialized DNA. Genetic transformation occurs when a bacterium ingests a foreign DNA strand and expresses it by incorporating the strand into its own DNA. For our lab, we will transform bacteria with a plasmid containing the GFP gene and a gene for ampicillin resistance. The plasmid also contains a gene regulation system, so that the gene for GFP is only expressed in the presence of arabinose.
Bacteria lack a nucleus and contain all their vital genes in the linear chromosome. Because they multiply rapidly, bacteria colonies can become visible on agar overnight. To make the bacteria competent for transformation, we must alther their cell walls to allow the passage of DNA. We will do this by bathing the cells in calcium chloride and shocking them with heat. The CaCl neutralizes the cell wall and prevents it from repelling the negatively charged DNA. The heat shock therapy stresses the cell and causes it to take in foreign DNA, in this case the GFP plasmid.
Transformation will not be successful in all of the bacterial cells. Therefore, we will spread the bacteria on an agar plate containing ampicillin, and only the bacteria that took up the plasmid containing GNP and ampicillin resistance will survive; the untransformed bacteria will die. Finally, the moment of truth. When we turn the lights off and apply the UV light, we hope to see a colony of glowing bacteria.
Last year when I did this lab, we thought we had done everything correctly, but the bacteria did not end up glowing. It was the ultimate disappointment in the coolest lab of the year, so I am seeking redemption.
A bacterium is a prokaryotic single-celled organism with one long, linear chromosome and sometimes a circular plasmid containing specialized DNA. Genetic transformation occurs when a bacterium ingests a foreign DNA strand and expresses it by incorporating the strand into its own DNA. For our lab, we will transform bacteria with a plasmid containing the GFP gene and a gene for ampicillin resistance. The plasmid also contains a gene regulation system, so that the gene for GFP is only expressed in the presence of arabinose.
Bacteria lack a nucleus and contain all their vital genes in the linear chromosome. Because they multiply rapidly, bacteria colonies can become visible on agar overnight. To make the bacteria competent for transformation, we must alther their cell walls to allow the passage of DNA. We will do this by bathing the cells in calcium chloride and shocking them with heat. The CaCl neutralizes the cell wall and prevents it from repelling the negatively charged DNA. The heat shock therapy stresses the cell and causes it to take in foreign DNA, in this case the GFP plasmid.
Transformation will not be successful in all of the bacterial cells. Therefore, we will spread the bacteria on an agar plate containing ampicillin, and only the bacteria that took up the plasmid containing GNP and ampicillin resistance will survive; the untransformed bacteria will die. Finally, the moment of truth. When we turn the lights off and apply the UV light, we hope to see a colony of glowing bacteria.
Last year when I did this lab, we thought we had done everything correctly, but the bacteria did not end up glowing. It was the ultimate disappointment in the coolest lab of the year, so I am seeking redemption.
Monday, November 15, 2010
DNA Chips (The Newest Product From Lays): Studying Varying Gene Expression in Lung Cancer Cells
In the nucleus, the process of transcription creates an mRNA strand using DNA as a template. This mRNA is then used as a template for translation at the ribosome, which builds protein strands based on the information in the mRNA. Each gene codes for a specific protein that affects a cells function in a variety of ways. Not all genes are expressed in every cell, however. The variable of gene expression is what allows an organism to have different cell types, such as blood cells, skin cells, lung cells, etc.
Scientists determine and study differences in gene expression using a microarray. Using cDNA made from mRNA, scientists are able to see only the genes that are expressed in each cell, rather than the entire genome, which is filled with many non-coding regions that are useless for this research. This type of experiment is often done to compare cancerous cells of a certain type to healthy cells of that type.
There is no single cause of cancer in human beings. As is evident on a microarray, there are many different genes that change in expression level when a cell becomes cancerous. In this lab, we will look at six genes and compare them between healthy lung cells and cancerous lung cells. The microarray simulation will tell us which genes are active in each cell, and how active they are. Genes that have the same level of expression in both cells would not be of interest to us, but genes that are off in one cell and on in another (or vice versa) could help us determine a cause for the cancer and, potentially, a cure. The six genes we will look at are C4BPA, ODC1, FGG, HBG1, SIAT9, and CYP24. These are genes with wildly different functions, any of which could be a reason for the cancer.
Scientists determine and study differences in gene expression using a microarray. Using cDNA made from mRNA, scientists are able to see only the genes that are expressed in each cell, rather than the entire genome, which is filled with many non-coding regions that are useless for this research. This type of experiment is often done to compare cancerous cells of a certain type to healthy cells of that type.
There is no single cause of cancer in human beings. As is evident on a microarray, there are many different genes that change in expression level when a cell becomes cancerous. In this lab, we will look at six genes and compare them between healthy lung cells and cancerous lung cells. The microarray simulation will tell us which genes are active in each cell, and how active they are. Genes that have the same level of expression in both cells would not be of interest to us, but genes that are off in one cell and on in another (or vice versa) could help us determine a cause for the cancer and, potentially, a cure. The six genes we will look at are C4BPA, ODC1, FGG, HBG1, SIAT9, and CYP24. These are genes with wildly different functions, any of which could be a reason for the cancer.
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