Lab practical 3 Flashcards
Experiment 3 outline
Culture E. Coli in sugar solutions of H2O (negative control), glucose (growth, no operon), lactose (growth, operon on), and sucrose.
-37 degrees overnight
-measure cell number through absorbance with spec-20 A600. Convert to get cells/mL.
-use artificial substrate ONPG and measure using spec-20 A420. Convert to get enzyme units/mL
—use cells/mL and enzyme units/mL to get enzyme/cell.
What are artificial substrates used?
- Mimics the structure of lactose
- Makes a good colored product to measure enzyme activity
What is going on in E. Coli with respect to the repressor and activator
In lactose, the cap activator is on while the repressor is off, turning the operon on. In glucose, the cap activator is off while the repressor is on, which doesn’t turn the operon on. In a tube of both lactose and glucose, the repressor and cap activator are both off. There is slight growth up until all glucose is used, leaving just lactose so the operon can turn on.
How is the lac operon working during phase I and phase II?
In phase I there is glucose and lactose present, meaning the repressor and CAP activator are both off. The operon is off but there is still growth until glucose runs out. In phase II the operon is on because only lactose is present, turning the cap activator on. There is exponential growth.
What is enzyme specificity? Why can’t beta-gal be used to metabolize sucrose?
Enzyme specificity is the extent to which an enzymes activity is restricted to a specific substrate. Beta-gal cannot metabolize sucrose because the alpha bond of glucose and fructose cannot be broken down.
Types of SNP’s. Do they always lead to gene expression issues?
Silent mutations, missense mutations, nonsense mutations, frame shift mutations, and small-scale indels. SNP’s do not always lead to gene expression issues because there are times the proteins do not change.
How are SNP’s identified?
SNP’s are identified when a mutation is found that differs from the reference genome. Individuals go through a DNA sequencing to compare the reference genome to spot these mutations.
How is a GWAS performed using a SNP microchip assay? Why are they performed? Do all SNP’s identified have the same level of importance?
A GWAS is performed using a SNP microchip assay by microarray detection. A glass slide of a microchip is immobilized with SNP’s and the SNP’s desired are PCR amplified in order to detect them. The disorder will be labeled in fluorescent light and the control of the PCR Will be labeled in another color. When both are overlapped, a SNP can be associated with a disease where significant differences are found. Not all SNP’s have the same level of importance.
What are the types of genetic tests and what information do they provide?
- Newborn screening assays: mandatory newborn screening occurs right after birth by pricking the heel of the infant and analyzing the blood for disorders. This allows the infant to be diagnosed early enough to alter the course of the disease.
- Comparative genomic hybridization microchip assay (CGH): this profiling type compares a patients dna to a reference genome to determine if there are any abnormalities in their DNA using fluorescence.
- SNP microchip assay: using a glass slide of a microchip, SNP’s are PCR amplified and a GWAS is performed. These show if any SNP’s detected are associated with a disease.
Do predictive genetic tests mean the individual will for sure get that disease?
No, due to environmental and other genetic factors.
What is the difference between genetic tests performed by D2C companies vs medical professionals?
Genetic tests performed by D2C companies are performed by the patient while medical professionals personally deliver the information. Medical professional tests are much more accurate and licensed than the D2C tests as well. D2C tests do not come with any option of clinical suooort or counseling while medical professionals personally deliver the information to you.
What is a karyotype?
An organized preparation of the complete set of stained chromosomes to detect a structural or chromosome number abnormality.
How is a karyotype made?
- Input cells/tissue into a culture dish to stimulate mitosis
- Stop the mitosis and disrupt microtubules
- “Fix” the cells
- Stain chromosomes with color (FISH)
- Observe chromosomes by microscopy
- Capture images
- Rearrange chromosomes into karyotype
Colorimetric stains (G bands) vs FISH
Colorimetric stains is a karyotype that produces light and dark bonding patterns on the chromosome. Each chromosome has a unique pattern of g-bands that can be interpreted with karyotyping. FISH has a much better resolution than G-banding, allowing much smaller defects to be detected. These are enhanced by denaturing DNA and exposing it with a colored fluorescence.
Difference between amniocentesis and CVS?
Amniocentesis is performed at 15-16 weeks of gestation by taking a sample of amniotic fluid out of the amniotic cavidty. There is only a 1% chance that harms the fetus. CVS is performed at 10-12 weeks of gestation transcervically or transabdominally and has a 1-2% chance of harming the fetus. CVS has an advantage because it can be done earlier on, but there is a bigger chance of harming the fetus.
