Gel Electrophoresis
What does it do?
Separates DNA by size
Shorter DNA travels further through gel and utilizes Agarose
What is the use and steps?
Use: Make MANY copies of a region of DNA to study
Steps:
Denaturation
Annealing
Extension
What are the cons of CRISPR?
Ethics (how far is too far?)
Potentially wiping out species, creating bacterial resistance
Mutations at the cut site or other places in the genome
Why should we dilute samples?
We can protect against false positives and also show disease progression
Describe the northern blot?
Very similar process to the Western Blot, but this test looks for RNA, not protein
What are the uses?
Crime scenes
Paternal/Maternal testing
Comparing species
Identifying/observing certain diseases
Why do we use PCR?
PCR is used because it lets us take a small section of DNA and make hundreds of copies. This small section can be amplified and examined through many processes.
What are the pros of CRISPR?
Scientists can accelerate research, there can be rapid diagnostic and they can correct mutations.
Look for color change; AMOUNT of color change can be used to signify the rate of disease progression. (More marker detected = More color change)
Why the membrane?
The gel is too fragile for next steps!
Antibodies could become stuck in the gel and give false results
Once proteins have moved onto the membrane, we add antibodies to locate the proteins we want to see
The antibodies act like a flag on the target protein
When separating DNA, does it have the same charge? Where will it move?
Yes! Same charge. The DNA will move from negative to positive ends.
What is PCR 20x-30x?
When these 3 steps are repeated many, many times! DNA made from a previous cycle is used as a template. This ensures that the desired DNA is the only thing getting replicated.
What does CRISPR stand for?
Clustered Regularly Interspaced Short Palindromic Repeats
What does ELISA stand for? What does it do? Uses?
Stands for: Enzyme-Linked Immunosorbent Assay
Does: Measures antibodies, antigens, proteins, and glycoproteins in a biological sample
Uses: Diagnosis, pregnancy tests, test for contamination, allergens, disease progression
What is a reporter enzyme?
The reporter enzyme allows us to locate the proteins!
By adding the antibodies, we only see the proteins we’re looking for on the final membrane
How does it work?
Each line on the gel is a band of DNA.
DNA samples that have similar banding patterns will be more closely related
Describe step 1
Denaturation:
Heat the strands of DNA to break (denature) them apart into two separate strands and give room to replicate the desired gene.
What does Cas 9 do?
Cas9 will cut the DNA, shutting the targeted gene off
Research suggests CRISPR-Cas9 can be used to target and modify “typos” (aka mutations) in the human genome
Describe:
Step 1: Antibody coating
Step 2: Protein capture
Step 1: The specific capture antibody is immobilized on high protein-binding plates.
Step 2: Samples and standard dilutions are added to wells and will be captured by bound antibodies
What is a western blot?
It’s a form of gel electrophoresis to detect specific proteins.
Detect protein of interest
Size of proteins
Amount of protein expressed
Are banding patterns similar if organisms are related?
Yes! Closely related organisms have similar DNA, therefore their banding patterns will also be similar.
Describe steps 2 & 3
Step 2: Primer annealing
Step 3: Primer Extension
Step 2: Primer annealing:
Cools DNA so primers can bind. Primers bind to areas of the DNA that we want to amplify. And two primers are needed for each side of DNA.
Step 3: Primer extension:
Heat up DNA again to use Taq Polymerase and Taq Polymerase adds nucleotides to extend the primers and replicate DNA. This enzyme is adapted to work in high temps!
What does Cas9 bind to? Describe it.
Cas9 will bind to the DNA using a "spacer." A spacer is a short RNA sequence that guides the CRISPR system to the target DNA.
Describe:
Step 3: Detection antibody
Step 4: Detection conjugate
Step 5: Substrate added
Step 3: Specific detection antibody is added to enable detection of captured protein. (If protein is absent, this antibody will attach to nothing.)
Step 4: Binds to detection antibody (like a signal)
Step 5: Changes color to analyze results
How does the western blot work?
Proteins in the sample are coated with a detergent to denature (unfold) them AND give them a negative charge. (We need linear proteins to move through the gel.)
Perform gel electrophoresis. (Once complete, move proteins onto a membrane)
Take completed gel & membrane and sandwich between filter paper
Negatively charged proteins will be drawn to the positive end
The membrane is between the positive end of the chamber and the gel, so the proteins will be drawn onto the membrane
Once the membrane copies the gel, it’s moved to a chamber and exposed to primary antibodies
This primary antibody specifically attaches to the protein we’re looking for
Once attached, the membrane is washed so extra antibodies don’t stick around
A secondary antibody is then added to the membrane
This secondary antibody attaches to the primary antibody, not the protein
Contains a reporter enzyme that will produce light or color