Lecture Exam 2 Flashcards
What is agarose gel electrophoresis?
using electricity to seperate molecules by electrophoretic mobility
How does agarose gel electrophoresis separate molecules?
ethidium bromide binds to the DNA and fluoresces under UV light
What types of molecules does agarose gel electrophoresis work best for?
supercoiled DNA runs fastest in the gel
On an agarose gel, how can you tell the difference between supercoiled and relaxed DNA
-supercoiled DNA looks shorter and has very few/no bands
What is a plate reader?
read absorbance (UV/Vis) luminescence
What are some types of readings that plate readers can take?
fluorescence, anisotropy/polarization, FRET
How can fluorescence anisotropy (or fluroescence polarization) help study binding of two molecules?
-there are fluorophores on the ends of a short piece of DNA
-creates a tumbling motion when an enzyme binds which is detected as a change in polarized proximity
-more binding=higher anisotropy
How does FRET work?
fluorescence of the first fluorophore will excite and induce fluorescence in a second one when they are in close proximity
What are restriction enzymes and what can they be used for in research?
-Restriction Enzymes: enzyme that can cleave a DNA molecule at a specific sequence of nucleotides
-THey are used in gel electrophoresis to cleave large pieces of DNA into smaller fragments
What is gel electrophoresis and how does it separate molecules?
-gel electrophoresis is used to separate DNA molecules based on their length
-It separates molecules by applying a voltage across the gel. The DNA migrates towards the positive end of the gel due to the negative charge of DNA
What is molecular cloning and what can it be used to do?
-PCR can be used for diagnostic and forensic application
-PCR uses DNA polymerase and specific DNA primers to amplify DNA sequences in a test tube
What is cDNA?
-Complementary DNA sequence
-just the exons that make the final version of the protein; super condensed version
How is cDNA made?
-made by adding a primer, reverse transcriptase, and DNPTs to cells. Then the strands are separated, and a second primer is added. Then it is amplified using PCR with both primers present
How does Sanger or dideoxy sequencing work?
-It depends on the analysis of DNA chains terminated at every position. It takes a ssDNA needing to be sequenced and adds a primer. Then small amounts of labeled chain-terminating ddNTP’s and excess amounts of unlabeled dNTP’s are added
-This creates a mixture of DNA products; each has a chain-terminating ddNTP labeled with a specific fluorescent marker
-The DNA is loaded onto a capillary gel and, through electrophoresis, separated by size and read in sequence
What is CRISPR-Cas9 and how does it allow editing of a gene/DNA sequence?
-CRISPR is composed of a guide RNA and a Cas9 protein used on a target gene. It creates a double strand break and adds an altered version of the gene (homologous recombination)
-The recombined gene’s Cas9 is catalytically inactive, so it fuses with a transcription activator; the gene is turned on. Then the Cas9 is fused with a transcription repressor and the target gene is turned off
Why are some things not visible using light microscopy?
-Some things are less than half the wavelength of the microscope’s illumination source and are therefore not visible
What components are needed to express a recombinant protein?
-An expression vector is required (a DNA sequence that will promote the production of the protein)
-Requirements for an expression vector:
-Gene of interest with promoter
-origin of replication
-selection gene
What are some examples of host organisms that can be used for recombinant protein expression?
Bacteria, Yeast, SF9 Insect cells, Human or animal cells
What would be an advantage and a disadvantage of expressing a recombinant protein in E.coli?
-Advantage: rapid expression
-Disadvantage: some proteins are not properly folded
What would be an advantage and a disadvantage of expressing a recombinant protein in yeast?
-Advantage: less expensive
-Disadvantage: enhanced safety precautions are required
What is PCR and how does it work?
-PCR uses DNA primers to amplify DNA sequences in a test tube
-Step 1: heat to separate the strands
-Step 2: cool to anneal primers
-Step 3:DNA synthesis (elongation)
-Cycle happens app. 30 times to eventually create only unoriginal DNA. The few remaining original DNA will die out
What are the primers used for in PCR?
-to amplify DNA, primers are needed
-they must be directional and specific (to avoid repeats in the strand) to the DNA strand
How can primers be used to introduce a mutation via PCR?
-two primers are needed that bind to opposite strands; the cell then replicates the inserted primer’s alternate sequence instead of the original sequence
What are some uses of PCR?
Forensics, Cloning, Diagnostics
What is qPCR or Real Time PCR?
allows you to see how much amplification is happening as the machine is running in “real time”
How does qPCR result in a quantitative signal from PCR?
- As it proceeds, DNA levels increase and are picked up as fluorescent signals. The output
is monitored in real time and compared to samples; standard curves are generated to
estimate the amount of sequence in a sample - One-Step: everything is added in at once
- Two-Step: oligodT’s and primers are added, then everything else later
What is reverse transcription? How does coupling reverse transcription with qPCR enable one to study gene expression levels?
- Processing of cell samples to isolate total RNA. Requires RNA transcriptase, DNA Poly,
and primers (primers: random, polydT, or sequence specific) - Converts RNA to DNA
- Combining with PCR:
- Done by taking the reverse transcribed DNA and amplifying sections via PCR
-May be either quantitative or end-point (final). Changes or treatments on target
genes can be monitored by the expression levels. rest
