Conformations of DNA and DNA denaturation Flashcards
(26 cards)
B Form of DNA
Conformation that the sodium salt of DNA assumes under conditions of relative high humidity ~92% considered “common form”
Base pairs perpendicular to the helical axis.
10 bp per helical turn
pitch of 34 angstroms (pitch=one turn of helix)
A Form of DNA
Humidity reduced to ~75%
Base airs tilted 20 degree angle
11 bp per helical turn
pitch of 28 angstroms
*DNA RNA hybrids
Z DNA
Composed of alternating purines and pyrimidines =left handed helix backbone appeared to zigzag Tilt of 9 degrees 12 bp per helical turn pitch of 46 angstroms
DNA denaturation
melting
presence of organic solvents DMSO
high pH
How does the lowering of the salt concentrations in solution aide DNA denaturation?
Ions are removed that shield the negative charges on DNA strands from each other. The repulsive forces of the negative charges are such that DNA can denature at low temperatures.
DNA Melting
DNA denaturation occurs when the two strands come apart.
Melting Temperature (Tm)
Temperature at which the DNA strands are HALF denatured
Hyperchromic Shift
When two strands separate the quenching of the absorbance is reduced and the absorbance at 260nm INCREASES 30-40%
Measurement of DNA strand separation
Absorbance of DNA solution at 260nm.
Increasing the distance between the bases increases this absorbance.
How does the GC content of DNA effect its stability, Tm, and density?
Increase GC content = linear Increase Tm and density.
GC have a 3rd H-bond that effect DNA Tm
Natural occurring DNA GC content of 22-73%
Renaturation or Annealing
reuniting of DNA strands
What are the 3 factors that contribute to the renaturation efficiency?
Temperature
DNA Concentration
Time
How does temperature effect renaturation?
A Tm of =Tm-25C is best for renaturation to occur.
It is low enough that it does not promote melting but high enough to allow rapid diffusional movement of DNA and weaken transient bond between sequence mismatches.
How does rapid cooling effect DNA renaturation?
Rapid cooling of denatured DNA prevents renaturation and is called quenching.
How does DNA concentration effect DNA renaturation?
The higher the DNA concentration the faster the renaturation.
How does the amount of time effect DNA denaturation?
The longer the time allowed for annealing the more will occur.
Fourth Factor that contributes to DNA renaturation?
DNA Complexity.
The more complex the DNA the slower and less efficient renaturation.
What is the C0t term combine and what are the components measured in?
C0t combines DNA concentration and time in DNA renaturation studies. Concentration multiplied by time.
DNA concentration C0 expressed in moles of nucleotides per liter.
Time t expressed in seconds.
What is “Half C0t”?
C0t is usually expressed as C0t1/2 or half C0t and it represents the C0t at which a DNA sample is half annealed.
Hybridization
A process in which a DNA strand anneals with a complementary strand of RNA to form a HYBRID duplex
DNA size range?
From a few kilobases to hundreds of megabases.
What is TEM?
Transmission electron microscopy.
It is a convenient means to estimate the size of small DNAs and also shows if DNA is linear, circular, or supercoiled.
DNA is more commonly estimated by electrophoresis.
The C-Value Paradox
One would guess that the more DNA an organism has the more complex the organism and the more genes it has. there is a rough correlation between DNA content and number of genes BUT in a number of cases the correlation breaks down.
For Example: mouse and human haploid cells contain 100X more DNA than yeast haploid cells but frogs have 7X and Lilys 100X more than humans.
The C-Value paradox explained by extra DNA in some organisms.
Paradox
A paradox is a statement that apparently contradicts itself and yet might be true.
