3.1.4 microarrays
After you put the DNA in the scanner..what does the red color indicate?
-cancer..usually called oncogene -produce more mRNA in cancer cells than healthy cells...."turned up" in cancer
p53 tumor suppressor
-decreased in the lungs cells of lung cancer patients This gene codes for a protein that's located in the mitochondria and in the nucleus. This protein is involved with cell cycle checkpoints. This gene is a tumor suppressor gene and is thought to be the "Guardian of the Genome"
Human carcinoembryonic antigen (CEACAM6)
-increased in the lung cells of lung cancer patients -This gene codes for a protein that is located in the extracellular matrix. This protein is involved with cell cycle regulation, particularly with adhesion between cells. It is thought to be a proto-oncogene. When overexpressed, it becomes an oncogene, because it leads to unregulated cell division and inhibits cellular death.
Cytochrome P450 (CYP1A1)
-increased in the lungs cells of lung cancer patients This gene codes for a protein that's located in the endoplasmic reticulum. This protein catalyzes reactions involved in drug metabolism and synthesizes cholesterol, steroids, and other lipids. The expression of this protein is induced by some polycyclic aromatic hydrocarbons (PAHs), some of which are found in cigarette smoke.
SRY
-no expression in the lung cells of lung cancer patients This gene codes for a protein that is located in the nucleus. The protein that this gene codes for is a testis-determining factor (TDF), which initiates male sex determination. This protein has no function in lung cells.
Surfactant protein B (SFTPB)
-normal in the lung cells of lung cancer patients This gene codes for proteins that assist breathing and isn't involved in the regulation of the cell cycle
steps to a microarray (normal, not the soybean)
1. collect tissue 2. isolate RNA 3. isolate mRNA 4. make labeled DNA copy 5. Apply DNA 6. scan microarray 7. analyze data
soybean steps to microarray
1. grow soybeans in high CO2 and normal atmospheric conditions for 1 month 2. Spot soybean gene sequences onto a glass slide 3. isolate mRNA from soybeans grown in high CO2 levels and in normal air 4. produce fluorescent-labeled cDNAs by reverse transcription 5. bind cDNAs to complementary gene sequences on a glass slide 6. Analyze results by comparing colors of the spots on the microarray 7. identify genes that are expressed or repressed by high CO2 levels.
How are DNA microarrays made?
1. primer pairs are created for PCR to copy all 20,000 genes 2. use PCR to make copies of every gene in the human genome 3. separate the double-stranded DNA from each gene copy into single strands 4. place microscopic droplets of each single-stranded DNA sample into ordered rows and columns on a glass, silicon, or plastic slide
3 things DNA microanalysis can't do
1. tell you which gene(s) "went bad" to cause a disease 2. cure a disease 3. identify every gene that's behaving inappropriately
What is "hybridization"?
2 complementary DNA strands from different sources can pair with one another
how many genes do we have?
20,000-25,000
Why is looking at normal cells/cancer cells under a microscope not always the best way to tell the difference between them?`
Because not only do they not look too different from each other but looking at appearance doesn't explain why the cells are different
DNA microarray is also known as..
DNA chip or gene chip
What would be in each spot on the microarray? Why are there so many spots?
Each spot contains multiple copies of a unique DNA sequence which corresponds to a single gene. There's so many spots because each one represents one gene
After you centrifuge the solvent/cell mixture, how come the DNA doesn't stay in solution and is found at the bottom of the tube?
It's a lot longer than RNA molecules
Why don't you make your own microarray? Where can you get them from?
It's too much to do for most individual scientists and research labs. You can get them from biotechnology companies
Why are tissue samples from healthy and cancer cells taken from the same patient?
We can look at differences in gene expression in cells that have the exact same genetic blueprint
Why is it necessary to make a cDNA ("complementary DNA") copy? Why is mRNA not used?
We incorporate a fluorescent label in the cDNA label that allows for cDNA visualization later on -DNA is much more stable than RNA
What's a DNA microarray?
a power tool that can identify genes that are expressed differently in 2 different cell types by comparing every single gene in a single experiment
why aren't all genes turned on, or "expressed" in all cells?
because different cells have different functions
How do DNA microarrays work?
by measuring the amount of mRNA for every gene that's present in a cell sample
What does the yellow color indicate?
cancer + healthy...not important because it means that the gene acts the same even when cancerous
What is a major limitation of DNA microarray technology?
defects can't be detected --can't say cause of disease
What does the black color indicate?
genes that weren't transcribed
What does the green color indicate?
genes whose expression is "turned down" in cancer cells -healthy...usually called tumor suppressor genes
What is the green and red "labeling mix"? What's the purpose of each?
green-healthy sample red-cancer sample The labelling mix will assemble a complementary DNA strand opposite every RNA molecule inside labelling mix: poly-T primer, reverse transcriptase (mRNA --> DNA), labeled nucleus
Why does the presence of mRNA tell you if a gene is turned on or off?
mRNA leads to protein production and if a gene makes proteins, it's turned on.
What conclusions can you make from the microarray data?
protein activity
What happens once you apply the DNA from the two samples to the DNA microarray?
they hybridize (some don't) the ones that don't get washed off
Describe the process used to isolate mRNA from the other types of RNA
wash RNA samples with solution filled with beads that will bind only to RNA strands that have a poly-A-tail (only mRNA does) -other strands will wash away
