The Structure Function of GeneChip Microarrays free essay sample

Student Handout Response Sheet: Used with Lab 5 Activity #2 Question Set (The Structure Function of GeneChip Microarrays) Directions: The following questions go with Activity #2 reading on the structure and function of GeneChip microarrays. Answer each question as thoroughly and detailed as possible. Part I – Intro, and Gene Expression Microarrays (1) What is gene expression? What can affect gene expression? †¢ When a gene is used to build an mRNA copy (transcription) which is then used to guide the synthesis of a protein (translation), the gene has been â€Å"expressed†. Thus the term gene expression refers to anytime a gene is â€Å"turned on†, leading to the synthesis of the protein it codes for. †¢ Gene expression can be affected by mutations or environmental factors. Thus the expression of genes is not always consistent within an organism. (2) Are all genes expressed in all cells? Explain why or why not. How do scientists study gene expression? No, not all genes are expressed in every single cell of an organism. We will write a custom essay sample on The Structure Function of GeneChip Microarrays or any similar topic specifically for you Do Not WasteYour Time HIRE WRITER Only 13.90 / page Genes that code for vital functions needed by all cells (like getting energy from food) maybe expressed in all cells, while those needed by only specific cells will be found expressed in those cells only (such as pigments that protect skin cells). †¢ By measuring the amount of RNA copies a gene produces, scientists can study that gene’s level of expression. A highly expressed gene will produce a lot more RNA than a gene that is expressed in small amounts. A gene expression microarray can be used to detect both the presence and amount of RNA present in a cell. (3) What would researchers have to do in order to make a hypothesis about gene expression in the past, before the use of the microarrays? What can they know do with the use of microarrays? Scientists would have to refer to previous research on similar diseases or topics and extrapolate from that information. They would then have to form a hypothesis based on any links they find in this old research. †¢ With the use of microarrays, scientists are able to look at the entire genome at once, measuring the expression of every gene in the organism. They can then make comparisons of which genes are expressed in the cells of different organisms – for example: diseased versus normal cells. (3) How many genes are found in the human genome according to the latest studies? †¢ The latest scientific research points to there being approximately 30,000 genes in the human genome. However, this number is not definite. (5) Write the complementary (opposite match) DNA strand to AGGCTAGAC. TCCGATCTG (6) What is the term for the short piece of DNA bound to the glass chip? How many base pairs long is this piece of DNA? What does it represent? This piece of DNA is a probe and it is 25 base pairs long. It represents a small, unique section of the entire gene. (7) Why does the probe not have to represent the entire gene? What does the binding of the RNA to the probe show? This 25 base pair segment is unique to that specific area of the gene. It is so unique that it cannot be found anywhere else in the entire genome. †¢ So, when the RNA binds to the probe, you know that this gene was expressed since no other gene has this specific sequence. This way, you do not need to worry about building a probe that covers the entire gene. This allows the probes to be much smaller, more specific and easier to build. (8) What is hybridization? How is the process of hybridization used by microarrays? Hybridization is the basic attraction between two pieces of nucleic acids – either DNA to DNA, RNA to DNA, or RNA to RNA. It occurs due to the basic attraction between A and T as well as C and G. †¢ This process is used to determine which RNA sequences are present in a sample by â€Å"fishing† out the specific sequences from a large sample of thousands of different RNAs. Thus, if you have a piece of DNA that is ATCATG, and another piece hybridizes to it, you know the other piece (if it is RNA) must have the sequence of UAGUAC. (9) How specific and accurate is the detection of microarrays? They can detect one single specific RNA molecule in a sample of a mixture of over 100,000 different RNAs. (10) Describe the surface of the microarray chip. Be sure to include the dimensions of the entire array and a single feature. Also discuss the probes in each feature. The array is a 1. 25 cm by 1. 25 cm checkerboard, with each square (or section) known as a feature. There can be up to 6. 5 million features on one chip. †¢ Each square feature is about 11 micrometers by 11 micrometers and built on it is one specific probe. Each feature has about a million copies of a specific probe sequence. In the latest chips the features are actually as small as 5 micrometers by 5 micrometers and have up to 6. 5 million features on them. (11) What are the first few steps done when using a Gene Expression microarray? †¢ The first step is to isolate all of the RNA from the cell and make copies of the RNA through the process of PCR. These RNA pieces are then fragmented and labeled with biotin. (12) What is the purpose of the biotin and the fluorescent molecules? What happens when the RNA sample is washed over the microarray? The biotin attaches to each RNA fragment and acts like a molecular glue for fluorescent molecules that will be washed over the array The fluorescent molecules will then glow and show specifically which feature the RNA has hybridized to. †¢ When the sample is washed over the array, there are millions upon millions of tagged RNA pieces floating around and coming in contact with the million of probes on each feature. Most will not find a match, but somewhere a match may be made and that specific RNA will stick to the probes on the feature. (13) How can you tell if the sample matches a probe? What if it doesn’t? To look for a match, the excess sample is washed off the array, which leaves only the attached RNA (stuck to the probe it matches). To visualize which feature the RNA stuck to and in what amount, a fluorescent molecule is washed over the array and will stick to the biotin on the RNA fragments. The feature that the RNA has combined with will then fluoresce or glow when shined on with a laser from the scanner. †¢ If there is no match between the RNA and the feature probes, all the RNA will wash away and there will be no biotin for the fluorescent dye to stick to. Thus, when it is hit with a laser, it will not fluoresce or glow. (14) How can you tell if the gene was highly expressed? A gene that is highly expressed will create more RNA copies. If this occurs, then many RNA molecules will stick to the probes and the feature will shine brightly when scanned. Those genes expressed at a low level will create a small amount of RNA which will stick to the probes on the feature but will shine with less intensity. (15) How can a Gene Expression microarray be used to determine which genes are taking part in the disease or trait being studied? What can scientists do once they have identified the specific genes responsible? Scientists can look at which genes are expressed in people or organisms with a specific trait or disease. For example, they could identify which genes are expressed specifically in people that are loud speakers and not expressed in those that are not loud speakers. †¢ They can then do further studies to look at what is the function of the proteins created by these genes and find out how they result in the disease or trait. Scientists could do comparison studies using multiple chips to give them even more data. (16) Using heat map results, how could a scientist classify a disease based on genetics? What advantage does that give the researchers? Scientists compare gene expression patterns (heat maps) from people with the disease they are studying to patterns from people with similar diseases or no diseases at all. They do this to look for expression patterns for each situation. This way, they are specifically classifying each disease by the genetics behind it. †¢ This has an advantage because it allows researchers to develop therapies or drugs targeted at the specific genetics that cause the disease and not a general target for a group of similar diseases. (17) In the black and white gene expression image, what colors represent a strong intensity? What does that tell you about the gene expression level of the gene the feature represents? In a colors display image, what gene expression level does each color indicate? white and grey features represent a strong intensity. †¢ This tell you that the genes represented by features express a high level which results in lots of RNA from that gene. The color image display goes from the highest expression level to lowest level expression level. The expressed colors are white, red, yellow, green, light blue, dark blue, and black. (18) Before developing a treatment, what must a scientist who has identified a disease pathway do? How can Gene Expression microarrays be used for this? The scientist need to be able to tell if they block or disrupt the gene pathway in any way with the disease be disrupted and stop progression. This can be tested by blocking the function of the specific gene and evaluating the cells response. Micorarrays can determine which gene or combination of genes should be blocked to treat the disease. (19) How could Gene Expression microarrays be used to find a successful drug? What else can microarrays tell scientists about the effects of the drug? Gene expression microarrays can be used to find a successful drug by taking lagre numbers of chemical compounds and evaluating the effect of the expression of the genes on each compound. Seeing the effect the drug has on the gene expression of a particular organism is how this process occurs. †¢ Visualizing the effects the compounds have on other genes in the genome, giving clues to possible side effects. (20) What is personalized medicine? How can these microarrays be used for this? Personalized medicine is choosing the best treatment for a specific patient by identifying which drugs would work best on them based on their specific genetic pattern. †¢ Microarrays can be used to determine how people will react to specific drug if they have the same genetic pattern. Microarrays can identify specific genetic differences allows educated guesses on how a person will respond to a particular drug treatment. Part II – The GeneChip Genotype Microarray for SNPs (21) Define genotype. Explain what a person’s genotype is and give an example. A genotype is the combination a person has for a section of DNA or genes on the two chromosomes. One chromosome is given from each parent allowing ever person to have a pair of chromosomes. The combination of two alleles makes a genotype. If a Dd allele was crossed with the Ff allele the genotype would be D/F, d/F, F/f, d/f. (22) What is a SNP? Explain. †¢ A SNP is a single nucleotide polymorphism. It is a single base pair difference between two people. One person’s DNA might be AGC at a specific location and another person’s DNA might read ACG at the exact same location, (23) How can genotyping SNPs be used to find a disease gene? Researchers can determine which SNPs are found with people of the disease. For example, studies may show that 500 people with the disease share the same dozen or so SNPs. This helps the scientists to pinpoint the areas of the genome to focus their studies and look for the disease gene(s). (24) Why won’t a DNA with the sequence ATCATG bind to DNA with the sequence TATGAC? The base C in the first DNA sequence will not match up to the second T in the bottom DNA sequence. The strands do not match 100%. (25) How does knowing the sequence of one DNA strand help you to determine the SNP genotype the person has? How are probes built to find this out? The probes of a SNP genotyping microarray are designed to detect the SNP by having the middle base on the probe be variable. For example, the first probe may read ATTCATG while the second probe may read ATTTATG. These two probes are the exact same except for the middle base of the 7. The middle is used because that is the exact spot where the SNP has been identified. All people have the exact same DNA in the area this probe represents except for the middle base. †¢ If the person’s DNA sticks to first probe, you know that they must have G in their DNA at that spot (and C on their opposite strand, thus the person has the C/G SNP genotype). If the DNA sticks to the second probe, you know they must have an A at that exact spot (and T on their opposite strand, thus the person has the A/T SNP genotype). (26) How many SNPs are on the newest Genotyping arrays? 500,000 SNPs or more on a single array for the newest genotyping arrays. (27) Where is the SNP found on the 25 base long probe? Either base pair number 13 out of the 25 bases of the probe, or the middle position represents the place of the SNP. (28) Once the probes and the microarray are made, what is the first step to genotyping a sample with a genotyping array? How is this different from the use of gene expression arrays? First you would have to extract the DNA from the subjects’ blood or saliva. †¢ These two differ because Microarray’s need DNA samples as to gene expression arrays who need RNA from the sample to be tested. The amount of RNA needed for each sample varies because not all genes are expressed in all cells but the amount of DNA used is the exact same every time because the amount of DNA is the exact same in every cell. (29) What are the rest of the steps to get a DNA sample ready for genotype analysis? 1. After extracting the DNA, it must be amplified into large amounts by PCR, then labeled with Biotin. 2. The labeled DNA is then randomly fragmented into pieces. 3. The labeled fragments are washed over the array and those DNA fragments that are complementary to a probe will stick to the array. 4. The array is then washed to remove those fragments not bound and then washed with a fluorescent dye which sticks only to the Biotin-labeled DNA fragments. 5. The array is then scanned to look for which features show matches, indicating which type of SNP. (30) Why does a sample that binds to an ATTCATG probe have the C/G genotype? The middle base is a C so the complementary DNA strand that binds to it must have a G at the same spot where the C is. This makes the genotype C/G. (31) Explain the difference between someone heterozygous for a genotype versus someone who is homozygous for a genotype. Which is implicated in causing more diseases? Why? If a person inherited the A/t genotype for a SNP from both parents, the sequence does not vary making it exactly the same making them homozygous. If a person has one C/G genotype SNP from one parent and one A/T genotype from another, they are heterozygous for this SNP. †¢ Diseases will develop more so on a persons’ homozygous SNP genotype. This is due to the fact if a disease gene attached to the SNP C/G genotype and you are homozygous for that SNP genotype then you now have double the amount of the diseased gene compared to a person with a heterozygous SNP.