Lecture 8: nucleic acids Flashcards
What are DNA and RNA made out of?
- Nitrogenous bases
- sugars
- phosphates
What are the purines?
Adenine and guanine
What are the pyrimidines?
Cytosine, thymine and uracil
Are nitrogenous bases planar or non planar?
Planar
What bond is formed between the ribose/deoxyribose sugar and the base?
N-glycosidic
What are other functions of nucleotides than being precursors for nucleic acids?
- ATP/GTP etc: high energy sources for biochemical processes
- cAMP/cGMP: as second messengers in intracellular signaling
- Adenosine: a neurotransmitter that regulates several functions
What is the bond that links nucleotides together?
phosphodiester bond
What are the different interactions that serve to stabilise DNA?
- Hydrophobic interactions: pi-pi stacking between adjacent nitrogenous bases
- Hydrogen bonding between complementary base pairs
- Cumulative effect of Van der Waals interactions between bases
- Electrostatic attraction between the negatively charged sugar phosphate backbone and divalent cations
Each DNA strand usually forms a _______- handed twist
right (B-form)
other forms present:
1. left handed (z form)
2. A-form : right handed but with higher twist
How do alkylating agents act on DNA?
- They covalently bind to DNA
- This causes mutations and disruption of information flow
Which position is most susceptible to attack by alkylating agents?
N7 position of adenine and guanine
What do groove binders do?
They bind (via Van der Waals interactions) to the grooves of the DNA helix
What is the best shape for groove binders?
Crescent shaped because that allows them to fit well into the grooves of DNA
What do intercalators do?
- They insert themselves in between adjacent nucleotides
- They form pi-pi interactions with the aromatic rings of the nucleotide
- They are highly planar
How do nucleoside and base analogs act as therapeutic agents?
- Their structure resembles that of natural nucleosides
- They function as inhibitors –> enzymes recognise them as the substrate instead
What is the name of the base analog that treats gout and how does it help to reduce the amount of uric acid in the body?
- Allopurinol
- It has a similar structure to hypoxanthine and xanthine, 2 substrates of xanthine oxidase
- Allopurinol binds to XO instead of the substrates and gets converted to alloxanthine (instead of hypoxanthine –> xanthine –> uric acid)
How do oligonucleotides act as therapeutic agents?
- They are anti-sense oligonucleotides that form complementary base pairings with target mRNA sequences
- This prevents the mRNA from being translated
How does fomivirsen act as an anti viral agent?
It binds to the sequence of a critical gene in cytomegalovirus –> prevents viral replication
What is cloning?
A technique that allows us to modify the genetic make-up of an organism by introducing foreign/new genes into the organism.
What is the vehicle used to transport the foreign gene into the host cell?
plasmid
What are plasmids?
Extra chromosomal self replicating DNA molecules
What is the bacteria commonly used in molecular cloning?
E. coli
Where are restriction enzymes found and what is their function?
- Found in bacteria
- To destroy incoming foreign DNA in bacteria
What are the features of restriction sites?
- 4-8 bp sequences
- They are generally inverted repeat sequences
- This inverted repeat sequence gives it a 3D structure of a 2-fold rotational symmetry
How frequently do restriction enzymes cut DNA?
Once every 4000bp
What enzyme is used to incorporate the fragment into the plasmid?
DNA ligase
Why is the plasmid cut with 2 restriction enzymes instead of just 1?
- It produces sticky ends that cannot reattach back to each other
- The DNA fragment wont be oriented in the wrong direction
What are polylinkers?
- It is a synthetic multiple cloning site sequence that contains 1 copy of several different restriction sites
- The plasmid vectors are often constructed with this
What is the function of polylinkers?
The presence of multiple restriction sites provides options for accommodating foreign DNAs with different restriction sites on them.
What are the challenges in getting the plasmid into the cell?
The plasmid is negatively charged and so it cannot pass through the phospholipid bilayer as the negative charge on the DNA and the phosphate group repel each other
What is the function of calcium ions in transformation?
The ions interact with the negative charges (on the DNA and the phosphate), creating an electrostatically neutral situation
How do you ensure that the bacterial colonies have taken up the plasmid?
- Grow colonies in a nutrient medium containing antibiotic
- The plasmid has an antibiotic resistance gene, so only the colonies that have taken up the plasmid successfully will survive
How do you ensure that the successfully transformed bacterial colonies have taken up the modified plasmid and not just the empty plasmid?
- Insert lac z gene along with the gene of interest
- Grow the bacteria in a medium containing X gal
- The beta galactosidase enzyme coded for by the lac z gene will digest the x gal to produce a blue compound
- The colonies that have the modified plasmid will be white, the others will be blue
What is added to the bacterial cells to allow for cell lysis to isolate DNA?
- Lysozyme: digests the polymeric component of the cell membrane
- EDTA: removes Mg2+ which is essential to maintain the cell wall + inhibits enzymes that can degrade DNA
- SDS: detergent removes lipids and disrupts membranes
