Exam 1 (Ppt 3) Flashcards

1
Q

Identify differences between bases

A

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

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2
Q

Base, nucleoside, nucleotide

A

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

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3
Q

What catalyzes DNA extension?

A

DNA Polymerase

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4
Q

What kind of forces stabilize DNA structures?

A

1) H bond pairing

2) ∏-stacking forces

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5
Q

∏-stacking forces

A

“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

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6
Q

Base Flipping

A

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

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7
Q

Is DNA usually R or L handed

A

Right handed

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8
Q

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

A

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

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9
Q

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

A

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

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10
Q

Which one DNA 3D forms is the most prominent?

Are they always exactly same in solution?

A

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

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11
Q

Nucleic Acid Sugar Puckering

A

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

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12
Q

Denaturation:

A

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

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13
Q

Hybridization

A

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

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14
Q

Absorbance

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

A

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

260 nm

Absorbance comparison:
single nucleotide > ssDNA > dsDNA

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15
Q
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
A

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

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16
Q

3 basic steps of PCR process

A

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

17
Q

Hyperchromicity

A

“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
18
Q

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.

A

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

19
Q

UV spectrophotometer and NanoDrop

Advantages vs Disadvantages

A

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

20
Q

What is DNA Denaturation dependent on?

A

1) G-C content

2) salt conc.

21
Q

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

A

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/)

22
Q

Plasmid

A

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

23
Q

cccDNA

A

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)

24
Q

1) Are all bacteria genomic DNA circular?

2) How about Eukaryotic genomic DNA?

A

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

25
Twist Number (Tw)
The number of helical turns of one strand about the other.
26
Writhe Number (Wr)
The number of times the double helix is either twisted around itself or twisted around (e.g. a protein) in a cylindrical manner.
27
Linking Number (LK)
Dependent on both twist and writhe. | LK=Tw+Wr
28
Lk°
The linking number of a free supercoiling, relaxed B-DNA in the physiological condition, is assigned the symbol LK°.
29
What are the two forms of writhe of supercoiled DNA?
interwound: Toroidal: Protein in middle of supercoiled DNA (circular old school phone cord)
30
How Can We Remove Supercoils from cccDNA if It Is Not Already Relaxed?
DNaseI (Topoisomerase 1)
31
Topoisomerase 1 vs Topoisomerase 2 and Mechanisms of Topoisomerase Cleavage: Types I and II
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?)
32
Topoisomerase 2 basic steps
1) Making a transient break of duplex 2) Uncut duplex passes the cut 3) Need ATP (to reseal the break?)
33
Topoisomerase I can only catenate/decatenate the DNA with a nick. why?
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
34
Topoisomerase stabilizes the (de)supercoiling process via..
Enzyme bridge
35
Electrophoretic separation of DNA topoisomers
Distance travelled from shortest to longest Relaxed or nicked circular DNA < Linear DNA < supercoiled DNA< Highly supercoiled cccDNA
36
Ethidium Ions Cause DNA to...?
Unwind