Restriction Enzymes and Cloning
Name the two types of ends that restriction enzymes can make when they cut DNA
Sticky/Staggered/Overhang Ends
Blunt Ends
Which step of PCR takes place at ~72 degrees Celcius?
Primer Extension
What is cell culture?
the process by which cells are grown under controlled conditions, generally outside of their natural environment
What does CRISPR do?
CRISPR technology can cut/edit DNA in very precise places.
What are the building blocks of proteins?
Amino Acids
What is the name of the circular piece of DNA that we use for cloning?
plasmid
What type of polymerase is needed for PCR and why?
Taq polymerase because it is heat resistant so it won't denature in the 96 degree step.
What is a stem cell? What are the three main types of stem cells?
A cell with the ability to differentiate into many different specialized cell types in the body.
What are the two main types of DNA sequencing, and which is used for high-throughput, efficient, and cheaper results?
Sanger Sequencing and NGS, NGS is high-throughput
What are the four bases of DNA, how do they pair, and what shape does a double stranded DNA make?
Adenine, Thymine, Cytosine, Guanine
A-T, G-C
double helix
Which type of restriction enzyme cuts do we prefer to use in the lab and why?
Sticky Cuts because then we can ligate our own piece DNA into the restriction enzyme cut site
Explain the setup of a DNA gel electrophoresis experiment (wells, electrode placement, which way the DNA will migrate, and which DNA molecules will move furthest)
wells near the positive electrode, DNA migrates towards negative electrode, shortest DNA fragments move the furthest
Name two reasons why we might want to use cell culture.
- Study cells in a way we can't in the body
- Have the cells produce some product for our own use
- test out drugs on cells without safety concerns
What three components are necessary for CRISPR technology to work?
Cas9, guide RNA and PAM sequence
Name three functions of proteins in the body
Structural, mechanical, enzymes, immune system, cell signaling, etc
Explain why restriction enzymes were such an important biological discovery.
Because they gave us an ability to analyze DNA like never before by cutting it in very precise places.
What might occur if we use PCR primers that are too short?
They might amplify the wrong region of the DNA because the sequence wasn't specific enough
How do scientists create induced Stem Cells?
re-introduce the signals that normally tell stem cells to stay as stem cells in the early embryo. These switch off any genes that tell the cell to be specialised, and switch on genes that tell the cell to be a stem cell.
Describe 2 uses of CRISPR.
Use for research (look at specific proteins in cells, how they affect certain processes, edit amino acids in proteins)
Use for therapeutics (correct a mutated gene for someone with genetic disease)
Use for GMOs
What biomolecule makes up cell membranes and why?
Lipids because they are hydrophobic so they form a strong barrier for the cell
Describe one of the real-world applications we went over that used restriction enzymes and recombinant DNA.
Insulin at Genentech
Hepatitis B vaccine at Biogen
Explain why we use gel electrophoresis after finishing a PCR sample.
We use gel electrophoresis to determine whether the PCR worked. If it did work, we would expect a band at the length of our PCR product and nowhere else.
Describe two uses of stem cells.
What are the two types of nucleotides that scientists use in Sanger Sequencing, and what is the purpose of each?
dNTP and fluorescently-labeled ddNTPs. dNTPs to grow the sequences to different lengths, ddNTPs to terminate the PCR at different points and label that base with fluorescence.
Name 3 chemical characteristics that amino acid R groups can have and 2 ways that those R groups could interact with each other or the outside environment.
Charged (positive or negative), Polar (hyrdophilic), nonpolar (hydrophobic)
- Can have ionic interactions between charged R groups, hydrophobic interactions, hydrophilic/polar/H-bonds with each other or outside environment, etc