DNA Structure & Stability
What type of bond links nucleotides together in a DNA strand?
What are phosphodiester bonds between 3' and 5' carbons of neighboring sugars
Which enzyme unwinds the DNA double helix at the replication fork?
What is Helicase
What distinguishes sticky ends from blunt ends after restriction digestion?
Sticky ends have overhangs that can base pair, blunt ends are even cuts with no overhangs
What is the base-pairing rule used during transcription from the template strand?
A pairs with U, T with A, G with C and C with G
What does the term p50 represent in oxygen binding curves?
The partial pressure of O2 when half of the protein's binding sites are occupied
What base pair has three hydrogen bonds and increases DNA stability?
What is G-C base pairs
What is the function of primase in replication?
Synthesizes short RNA primers to act as starting point for DNA synthesis
How many different restriction enzymes should you use for directional cloning?
(don't overthink it)
2 enzymes that can generate sticky ends
What sequence on mRNA signals the start of translation?
Start codon AUG (codes for methionine)
Which protein has a lower p50 and higher affinity for oxygen?
What is Myoglobin
What happens to DNA melting temperature (Tm) when salt concentration increases?
Tm increases because cations stabilize negatively charged backbone
Which enzyme seals nicks on lagging strand?
Bonus question: What are the segments between these gaps called?
What is DNA ligase
(Okazaki fragments)
What is the purpose of the multiple cloning site (MCS) in a plasmid?
It contains several unique restriction sites for inserting genes of interest
A point mutation that changes one base but doesn't alter the amino acid sequence.
What is a silent mutation
What structural feature of hemoglobin allows for cooperative binding?
Its quaternary structure with four subunits that influence each other's O2 binding
How would very high or very low pH affect double stranded DNA?
Extreme pH disrupts hydrogen bonding and causes denaturation
How does topoisomerase prevent DNA ahead of the fork from becoming overwound?
By cutting and rejoining DNA strands to relieve supercoiling tension
What are the two requirements for enzymes to be compatible in a double digest?
Same temperature, same buffer
When using CRISPR to add 100 aspartic acid residues, which DNA repair mechanism should be used and why?
HDR because it allows for precise sequence insertion using a template
CRISPR creates a double-strand break in DNA.
NHEJ (non-homologous end joining) quickly rejoins ends but introduces random insertions/deletions — good for knocking out genes.
HDR (homology-directed repair) uses a provided DNA template to copy in a new sequence exactly — good for adding a specific insert, like those aspartic acids.
Why is hemoglobin better suited than myoglobin for transporting O2 from lungs to tissues?
Hemoglobin's cooperative binding allows it to release O2 where pO2 is low while myoglobin holds O2 tightly.
Both GC content and salt concentration affect DNA stability. Explain how each influences Tm and why DNA melts at a higher temp that AT-rich DNA
Higher salt concentration shields the negative phosphate backbone, reducing repulsion and stabilizing the helix, further increasing Tm.
DNA polymerase has proofreading ability through which enzymatic activity and what error does it correct?
3'-->5' exonuclease activity; it removes mispaired nucleotides during replication
If a plasmid is cut with two different restriction enzymes and produces two fragments, what does this tell you about the cut sites and digestion success?
Both enzymes each successfully cut once on the plasmid so the double digest worked and the insert region was released/opened
What would happen to the resulting protein if translation started in a different reading frame than the start codon?
(bonus question: how many reading frames are there?)
The amino acid sequence would be completely different, likely resulting in a nonfunctional protein
At high altitudes, how does the fractional saturation of hemoglobin in the lungs change, and what physiological response compensates?
Fractional saturation decreases due to lower pO2, body compensates by increasing RBC production and 2,3-BPG levels