Lecture 8: nucleic acids

Question 1

What are DNA and RNA made out of?

Answer 1

1. Nitrogenous bases
2. sugars
3. phosphates

Question 2

What are the purines?

Answer 2

Adenine and guanine

Question 3

What are the pyrimidines?

Answer 3

Cytosine, thymine and uracil

Question 4

Are nitrogenous bases planar or non planar?

Answer 4

Planar

Question 5

What bond is formed between the ribose/deoxyribose sugar and the base?

Answer 5

N-glycosidic

Question 6

What are other functions of nucleotides than being precursors for nucleic acids?

Answer 6

1. ATP/GTP etc: high energy sources for biochemical processes
2. cAMP/cGMP: as second messengers in intracellular signaling
3. Adenosine: a neurotransmitter that regulates several functions

Question 7

What is the bond that links nucleotides together?

Answer 7

phosphodiester bond

Question 8

What are the different interactions that serve to stabilise DNA?

Answer 8

1. Hydrophobic interactions: pi-pi stacking between adjacent nitrogenous bases
2. Hydrogen bonding between complementary base pairs
3. Cumulative effect of Van der Waals interactions between bases
4. Electrostatic attraction between the negatively charged sugar phosphate backbone and divalent cations

Question 9

Each DNA strand usually forms a _______- handed twist

Answer 9

right (B-form)

other forms present:
1. left handed (z form)
2. A-form : right handed but with higher twist

Question 10

How do alkylating agents act on DNA?

Answer 10

1. They covalently bind to DNA
2. This causes mutations and disruption of information flow

Question 11

Which position is most susceptible to attack by alkylating agents?

Answer 11

N7 position of adenine and guanine

Question 12

What do groove binders do?

Answer 12

They bind (via Van der Waals interactions) to the grooves of the DNA helix

Question 13

What is the best shape for groove binders?

Answer 13

Crescent shaped because that allows them to fit well into the grooves of DNA

Question 14

What do intercalators do?

Answer 14

1. They insert themselves in between adjacent nucleotides
2. They form pi-pi interactions with the aromatic rings of the nucleotide
3. They are highly planar

Question 15

How do nucleoside and base analogs act as therapeutic agents?

Answer 15

1. Their structure resembles that of natural nucleosides
2. They function as inhibitors –> enzymes recognise them as the substrate instead

Question 16

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?

Answer 16

1. Allopurinol
2. It has a similar structure to hypoxanthine and xanthine, 2 substrates of xanthine oxidase
3. Allopurinol binds to XO instead of the substrates and gets converted to alloxanthine (instead of hypoxanthine –> xanthine –> uric acid)

Question 17

How do oligonucleotides act as therapeutic agents?

Answer 17

1. They are anti-sense oligonucleotides that form complementary base pairings with target mRNA sequences
2. This prevents the mRNA from being translated

Question 18

How does fomivirsen act as an anti viral agent?

Answer 18

It binds to the sequence of a critical gene in cytomegalovirus –> prevents viral replication

Question 19

What is cloning?

Answer 19

A technique that allows us to modify the genetic make-up of an organism by introducing foreign/new genes into the organism.

Question 20

What is the vehicle used to transport the foreign gene into the host cell?

Answer 20

plasmid

Question 21

What are plasmids?

Answer 21

Extra chromosomal self replicating DNA molecules

Question 22

What is the bacteria commonly used in molecular cloning?

Answer 22

E. coli

Question 23

Where are restriction enzymes found and what is their function?

Answer 23

1. Found in bacteria
2. To destroy incoming foreign DNA in bacteria

Question 24

What are the features of restriction sites?

Answer 24

1. 4-8 bp sequences
2. They are generally inverted repeat sequences
3. This inverted repeat sequence gives it a 3D structure of a 2-fold rotational symmetry

Question 25

How frequently do restriction enzymes cut DNA?

Answer 25

Once every 4000bp

Question 26

What enzyme is used to incorporate the fragment into the plasmid?

Answer 26

DNA ligase

Question 27

Why is the plasmid cut with 2 restriction enzymes instead of just 1?

Answer 27

1. It produces sticky ends that cannot reattach back to each other 2. The DNA fragment wont be oriented in the wrong direction

Question 28

What are polylinkers?

Answer 28

1. It is a synthetic multiple cloning site sequence that contains 1 copy of several different restriction sites 2. The plasmid vectors are often constructed with this

Question 29

What is the function of polylinkers?

Answer 29

The presence of multiple restriction sites provides options for accommodating foreign DNAs with different restriction sites on them.

Question 30

What are the challenges in getting the plasmid into the cell?

Answer 30

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

Question 31

What is the function of calcium ions in transformation?

Answer 31

The ions interact with the negative charges (on the DNA and the phosphate), creating an electrostatically neutral situation

Question 32

How do you ensure that the bacterial colonies have taken up the plasmid?

Answer 32

1. Grow colonies in a nutrient medium containing antibiotic 2. The plasmid has an antibiotic resistance gene, so only the colonies that have taken up the plasmid successfully will survive

Question 33

How do you ensure that the successfully transformed bacterial colonies have taken up the modified plasmid and not just the empty plasmid?

Answer 33

1. Insert lac z gene along with the gene of interest 2. Grow the bacteria in a medium containing X gal 3. The beta galactosidase enzyme coded for by the lac z gene will digest the x gal to produce a blue compound 4. The colonies that have the modified plasmid will be white, the others will be blue

Question 34

What is added to the bacterial cells to allow for cell lysis to isolate DNA?

Answer 34

1. Lysozyme: digests the polymeric component of the cell membrane 2. EDTA: removes Mg2+ which is essential to maintain the cell wall + inhibits enzymes that can degrade DNA 3. SDS: detergent removes lipids and disrupts membranes