Exam 1 (Ppt 3) Flashcards
Identify differences between bases
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
Base, nucleoside, nucleotide
Base: Purine (AG) or Pyrimidine (CT)
NucleoSide: Base + Sugar
NucleoTide: Nucleoside + phosphate group
What catalyzes DNA extension?
DNA Polymerase
What kind of forces stabilize DNA structures?
1) H bond pairing
2) ∏-stacking forces
∏-stacking forces
“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
Base Flipping
May be correctly paired but one base just “decides” to protrude outwardly
Is DNA usually R or L handed
Right handed
What is the difference of angle b/w major and minor grooves due to?
glycosidic bonds
Major => 240 deg
Minor => 120 deg
What are the different 3D forms of DNA and how can you identify them?
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
Which one DNA 3D forms is the most prominent?
Are they always exactly same in solution?
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
Nucleic Acid Sugar Puckering
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
Denaturation:
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
Hybridization
The ability of DNA to form hybrid structures between two complementary strands.
Absorbance
what wavelength is DNA’s highest optical density?
Which type of DNA (ds or ssDNA) has a greater absorbance?
A measure the optical density of a molecule at a certain wavelength
260 nm
Absorbance comparison:
single nucleotide > ssDNA > dsDNA
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
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
3 basic steps of PCR process
1) Denaturation - 95 deg
2) Annealing - 50-68 deg
3) hybridization - 72 deg
Hyperchromicity
“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
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.
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
UV spectrophotometer and NanoDrop
Advantages vs Disadvantages
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
What is DNA Denaturation dependent on?
1) G-C content
2) salt conc.
How does salt conc. affect on DNA Tm? Why?
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/)
Plasmid
“Many bacteria have small autonomously replicating genetic elements” Prof Ye
cccDNA
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)
1) Are all bacteria genomic DNA circular?
2) How about Eukaryotic genomic DNA?
1) Most are circular
2) Mostly Linear (butttt Euk. have something called eccDNA that’s derived from gDNA, containing repetitive sequences of DNA)
Twist Number (Tw)
The number of helical turns of one strand about the other.
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.
Linking Number (LK)
Dependent on both twist and writhe.
LK=Tw+Wr
Lk°
The linking number of a free supercoiling, relaxed B-DNA in the physiological condition, is assigned the symbol LK°.
What are the two forms of writhe of supercoiled DNA?
interwound:
Toroidal: Protein in middle of supercoiled DNA (circular old school phone cord)
How Can We Remove Supercoils from cccDNA if It Is Not Already Relaxed?
DNaseI (Topoisomerase 1)
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?)
Topoisomerase 2 basic steps
1) Making a transient break of duplex
2) Uncut duplex passes the cut
3) Need ATP (to reseal the break?)
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
Topoisomerase stabilizes the (de)supercoiling process via..
Enzyme bridge
Electrophoretic separation of DNA topoisomers
Distance travelled from shortest to longest
Relaxed or nicked circular DNA < Linear DNA < supercoiled DNA< Highly supercoiled cccDNA
Ethidium Ions Cause DNA to…?
Unwind
