Exam 1 (Ppt 3)

Question 1

Identify differences between bases

Answer 1

Base: Residual structure + functional group

A: 2 cyclic rings + Amino (NH2)
G: 2 cyclic rings+ Amino + Carbonyl

C: 1 cyclic ring + amino + carbonyl
T: 1 cyclic ring + 2 carbonyls + 1 methyl group

Question 2

Base, nucleoside, nucleotide

Answer 2

Base: Purine (AG) or Pyrimidine (CT)
NucleoSide: Base + Sugar
NucleoTide: Nucleoside + phosphate group

Question 3

What catalyzes DNA extension?

Answer 3

DNA Polymerase

Question 4

What kind of forces stabilize DNA structures?

Answer 4

1) H bond pairing

2) ∏-stacking forces

Question 5

∏-stacking forces

Answer 5

“base pairs lie vertically one above the other, an arrangement that maximises hydrophobic interactions and in addition, maximises van der Waals attractive forces between them” -open.edu
Stacking forces dependent on what base pairs are vertically near each other to interact

Question 6

Base Flipping

Answer 6

May be correctly paired but one base just “decides” to protrude outwardly

Question 7

Is DNA usually R or L handed

Answer 7

Right handed

Question 8

What is the difference of angle b/w major and minor grooves due to?

Answer 8

glycosidic bonds
Major => 240 deg
Minor => 120 deg

Question 9

What are the different 3D forms of DNA and how can you identify them?

Answer 9

B: R handed, Longer and thinner, 10 bp/turn
A: R handed, shorter and broad, 11 bp/turn ( Thick Ass)
Z: L handed, slim and elongated, 12 bp/turn

Question 10

Which one DNA 3D forms is the most prominent?

Are they always exactly same in solution?

Answer 10

B form is most common (https://bio.libretexts.org/)

Not sure if they are the same in soln but I would figure it is not

Question 11

Nucleic Acid Sugar Puckering

Answer 11

B form has C2 atom out of plane, P groups 7 A apart
A form has C3 atom out of plane, P groups 6 A apart

*answered why DNA has so many forms, mostly A and B form

Question 12

Denaturation:

Answer 12

Complementary strands of the DNA double helix can come apart due to (e.g.) heat, high pH (renaturation is the opposite of this).

PCR denatures DNA to anneal a primer then replicate

Question 13

Hybridization

Answer 13

The ability of DNA to form hybrid structures between two complementary strands.

Question 14

Absorbance

what wavelength is DNA’s highest optical density?
Which type of DNA (ds or ssDNA) has a greater absorbance?

Answer 14

A measure the optical density of a molecule at a certain wavelength

260 nm

Absorbance comparison:
single nucleotide > ssDNA > dsDNA

Question 15

Melting point (Tm)
Which type of DNA (high A-T content or high G-C content) would you predict has a higher Tm?
how does this impact primer design

Answer 15

The temperature at which DNA is ½ ds and ½ ss in a solution.
G-C b/c of the additional H bond (3 in total) compared to A-T H bonds (2 in total)
Primers would then be designed to anneal the A-T rich areas b/c it would open up “faster” than G-C rich areas

Question 16

3 basic steps of PCR process

Answer 16

1) Denaturation - 95 deg
2) Annealing - 50-68 deg
3) hybridization - 72 deg

Question 17

Hyperchromicity

Answer 17

“When the temperature of a solution of DNA is raised to near the boiling point of water, the optical density (called absorbance) at 260 nm markedly increases” - Prof Ye

• near bp of water (Tm), absorbance of DNA exponentially increases. This is where DNA is half ds half ss

Question 18

If a dsDNA stock was diluted for 100 times and the OD260 of the diluted DNA is 1.4, please calculate the concentration of the dsDNA stock.

Answer 18

OD260 of the diluted DNA x O.D value of dsDNA x dilution factor (ng/microliter = conc of ds

1.4× 50 ng/μL× 100 =7000 ng/μL or 7 μg/μL

O.D values (ng/microL)
dsDNA = 50, ssDNA= 20-33, RNA= 40

Question 19

UV spectrophotometer and NanoDrop

Advantages vs Disadvantages

Answer 19

NanoDrop = UV spec
Pro: more accurate for samples of high concentration, relatively affordable, widespread use, quick and efficient data
Con: doesnt automatically know if the sample is DNA, RNA, or protein. sometimes the readings are not precise but this can be troubleshooted

Question 20

What is DNA Denaturation dependent on?

Answer 20

1) G-C content

2) salt conc.

Question 21

How does salt conc. affect on DNA Tm? Why?

Answer 21

Positive correlation
Increase salt conc. => increase Tm
Why? DNA is polyanionic and salt essentially shields this negative charge, making it energetically unfavorable for the strands to seperate (http://oregonstate.edu/)

Question 22

Plasmid

Answer 22

“Many bacteria have small autonomously replicating genetic elements” Prof Ye

Question 23

cccDNA

Answer 23

differs from cDNA

two ends of DNA are covalently linked to form a circular DNA molecule

if there are no interruptions in the sugar-phosphate backbones of the two strands, then the absolute number of times the chains can twist about each other cannot change. (This is how they differ)

Question 24

1) Are all bacteria genomic DNA circular?

2) How about Eukaryotic genomic DNA?

Answer 24

1) Most are circular
2) Mostly Linear (butttt Euk. have something called eccDNA that’s derived from gDNA, containing repetitive sequences of DNA)

Question 25

Twist Number (Tw)

Answer 25

The number of helical turns of one strand about the other.

Question 26

Writhe Number (Wr)

Answer 26

The number of times the double helix is either twisted around itself or twisted around (e.g. a protein) in a cylindrical manner.

Question 27

Linking Number (LK)

Answer 27

Dependent on both twist and writhe.

LK=Tw+Wr

Question 28

Lk°

Answer 28

The linking number of a free supercoiling, relaxed B-DNA in the physiological condition, is assigned the symbol LK°.

Question 29

What are the two forms of writhe of supercoiled DNA?

Answer 29

interwound:
Toroidal: Protein in middle of supercoiled DNA (circular old school phone cord)

Question 30

How Can We Remove Supercoils from cccDNA if It Is Not Already Relaxed?

Answer 30

DNaseI (Topoisomerase 1)

Question 31

Topoisomerase 1 vs Topoisomerase 2
and
Mechanisms of Topoisomerase Cleavage: Types I and II

Answer 31

TOPO 1: nick made on single strand of dsDNA. Prok. have special one called DNA gyrase that introduces supercoil
TOPO 2: nick made on both strands.

Topoisomerases decatenate, disentangle, and unknot DNA

Mechanism: TOPO comes in, cleaves strand(s) and binds 5’ phosphate group = Tyrosine DNA intermediate, then rejoins the 3’ OH group that was cleaved, resulting in another coil (or minus a coil too?)

Question 32

Topoisomerase 2 basic steps

Answer 32

1) Making a transient break of duplex
2) Uncut duplex passes the cut
3) Need ATP (to reseal the break?)

Question 33

Topoisomerase I can only catenate/decatenate the DNA with a nick. why?

Answer 33

Not sure but probably b/c there’s only one free 3’ OH group to do work on

catenate: the bonding of atoms of the same element into a series, called a chain

Question 34

Topoisomerase stabilizes the (de)supercoiling process via..

Answer 34

Enzyme bridge

Question 35

Electrophoretic separation of DNA topoisomers

Answer 35

Distance travelled from shortest to longest

Relaxed or nicked circular DNA < Linear DNA < supercoiled DNA< Highly supercoiled cccDNA

Question 36

Ethidium Ions Cause DNA to…?

Answer 36

Unwind