Lecture 4 Flashcards
If u have two dna strands that have the same sequence, will they come togethr? Why or WHy not
Yes, bc they want to make hydrogen bonds they prefer to be double bonded, wich is why we need energy to split up strands
As soon as you take heat away, what will happen to the two strands, if they are complimentary
They will come back together
if they are not, they will stay single stranded
So what does it mean if you have two sequences and they never come together even when cooled
They are not complimentary sequences
This is a good way for us to analyze dna because if we have a known single strand of dna and an unknown, we can see if they are complimentary if they come together, allows us to figure out the unknown
How does low ionic strength help denature dna
-promotes repulsion
-essentially low ionic strength means that there is low cations (low salts) so that means that the heavy negative charge of the backbones will cause repulsion of the two strands
cations mute out alot of the negatives by bonding to them, allowing for the backbones to come together
How does high pH help denature DNA
-takes away protons (H+) that is shared between electronegative centers so disrupt covalent bonds and hydrogen bonds
Bases are hydrophobic?
Yes, they are trying to hide from water and struff so modifying solubility of hydrophobic substaneces causes denaturing of DNA
CoT stand for
Concentration x Time
when comparing denaturation to renaturation its best to…
keep all factors that you can the same so that u ca n undestand the restuls
What is renaturation dependant on?
1) High DNA concentration: Complimentary strands mist find eachother so the higher the concentration, the more likely for renaturation to occur
2)Salt Concentration: (ionic conditions)- more salt means that the repulsive forces of the backbone are masked so easier to come together
3)Temperature: 20-25 degrees (want it under Tm)
4) Time: longer u leave the reaction to occur, the more chance to have renaturation (longer reaction time is better for this )
5) Size: of dna fragment
6) Complexity: refers to how much unique sequence it has and how much repetitive. Simple sequences renature faster than complex sequences
What does the rate of renaturation equal
measure of complexity of the DNA (genome)
What does re-association kinetics means
The speed at which a single strand sequence is able to find a complementary sequence and base pair with it
In the beginning of a renaturation experiment will concentration be high or low
Low because concentration refers to the concentration of DOUBLE STRANDED DNA
What is concentration in COT dependent on
The amount of double stranded dna present at that time
SO Co varies depending on the time of the experiment
CoT Analysis Conditions
Complexity
-typically bacteria have no repeats because they are small so they dont waste space with repeats so that means that they have less repitions so everything is unique so more complex
What is units of complexity measured in terms of?
nucleotides
hence if a genome is all unique (non-repeating) complexity = # of nucleotides
THe bigger the organism the _____ complex
less
What are the equations for complexity
If genome is all unique:
Complexity = # of nucleotides
If fgenome contains unique and some repeat sequences:
Complexity = # of unique nucleotides + total # of nucleotides from one copy of each repetitive sequence
If genome is all repeats:
complex= #of nucleotides in the repeating Unit ie ACT repeats 10 times, complex = 3
If genome is unique and has similar GC content:
Sizes are proportional to Cot1/2
Which complexity is larger?
1) (AT) repeated 10 times
2) (AT) repeated 2 times
Both are equal. Complexity is equal to 3 for both
Which complexity is larger?
1) (GC) repeated 2 times
2) (AT) repeated 2 times
Both are 2
What is the complexity of 10^5 non repeating nucleotide pairs in length
10^5
non repeating complexity is wqual to the number of units
What is the complexity if DNA is 10^5 non repeating, 100 copies of dAT, and 50 copies of (ATGC)
10^5+2 +4
What is the complexty og this
ATATATATnonrepeating(10^2)AT
2+10^2
just bc the at is at the end doesnt mean it doesnt count as a repeat
Keep in mind though if it feels like te nucleotife being there is random then it doesnt counf as a repeat usually repeats are counted in big amounts
How is CoT analysis carried out
Trying to measure complexity of unkown
1) Have known unique DNA (that is complementary to the other) and unknown dna
2) SHear it into small pieces (200bp)
3) Heat it
4) Cool it (allow it to reanneal)
5)At various time points, mesure the concentration of DS (double stranded) DNA
6) Data plotted as a proportion of ssDNA (or %DsDNA) out of the total DNA
E COLI genome vs Calf Genome
Ecoli:
no repetitibe sequences because it is some, genome is one unique sequece
-difficulft for sequences to find complementary sequences
-once found though, fast reassociatyion
Calf:
Lots of highly repetitive sequwnces, fase reassociation
-some moderately repetitive sequences have slower reassociation
-even less repetitive sequences (unique) take even more times to anneal
-if an organism has all three tiypes, like this, it will be fast first then medium then the slowest near the end
Why use COT insread of time on x axis
lecture 4 minute 40
Since Dna is dependent of time AND concentration, only using one makes u have to take tbe other one into consideration so by using CoT we are multiplying them both so we are taking both into consideration
Rly just know that to get good data, you dont use time by itself u use both
If a genome is very mixed with repeitive sequence types (high, medium, none) what would graph look like
Start off fast, then slow as it goes
What are the highly repetivie sequences like vs moderate
short, but there are many of them so able to find eachother easily
leads to fast renaturation
Moderate:
less numbers but still 10-1000, find eachother relatively fast, some are coding for other gene families, some are non coding
Of the three: high, moderate and unique, which ones are the ones tha are most likely to code for proteins
unique
if a genome does not contain repeitive sequences, the complexity is the same as genome size therefore,
reassociation is decreased as complexity increases
reassociation is ________ to the genome size
inversely proportional
How to determine genome size by COT analysis
We are trying to find size of unknown dna
first must find size of unique portion:
1) Look at the known sample and find what Cot1/2 is
2) Look for where the unique part begins for the unknown (look for when the line turns to unique
3) determine what value of genome association that is (y value) this will give us the percentage of genome that is unique
4) find half of that value (half of x) and add it to the value (x), and look at the cot value at that new point. THis will be the cot1/2 of the unique portion of the unknown
5) do Cot1/2 of known / cot1/2 unknown = size of known genome / size of unknown genome (a)
solve for a
a will be the size of the UNIQUE portion of UNKNOWN genome
Now must find the rest:
so we know what percentage of the genome is unique ( determined that in step 3
do x/100 = unique genome size /percentage in full number
look at the articles from lecture 4
Does an organism that is more complex necessarily have more complex DNA
no complex phenotypes does not mean complex genome
lots of plants have more dna than we do but are not as complex
What is C value paradox
C Value Paradox
no correlation between the amount of DNA (size of genome) and the apparent complexity of organisms
What do prokaryotic cells carry
Only non repetitive DNA
Circular dna
-two strands of DNa that form a closed struction (without free ends) = “ double circle”
the higher the organisms the more linear it is
What are some examples of things that are circular
Prokarotic genome. plasmids, viral dna, choroplast genome, mitochondria genomes
What is endosymbiotic theory
theory that mitochondira and chlroplasts were bacteria that came into cell and they went in and realized that it was beneficial for them to live there and they formed a symbiotic relationship
-both of them have double membranes
Denaturation of circular dna
-the two strands cannot unqind and seperate like linear dna
-hard to serperate think like seperated a hair tie of knots, its hard because hte more u do ut, the more tangled on itself it gets so even if u heat it, cant seperate (they are tangeld together even though they are indicidual strands now (think two rubber bands knotted together
SO
in vivo, nicking occurs naturally during dna replication and can be induced experimentally by using an enzmyme
so need to cut one or both to work it
the nick is what really prevents it from supercoiliong
What is the primary structure of DNA like for both circular and linear dna
sugar-phosphate chain with purine and pyrimidine bases as side chains
What is the secondary structure of dna for circular and linear dna
Double helical structure, (hydrogen bonding between At Gc, stacking interactions, phosphate backbone is outisde
What is the tertiary strucuture of dna for circular and linear dna
Double stranded dna makes complexes with proteins (SUPERCOIL) esscentially is the coiling of a coil, the helix itself wraprs around eachother due to bonds hydrgeon bonding, twisting of it twists upon themselves
Supercoiling
-used by chromosomes to become self-compacts, important for the packing of dna
-reduces stress on the dna helix by twisting and untwisting on itself
-becomes more compact
What is a topological Isomer
- DNA differing only in state of SUPERCOILING
same molecule but just supercoiled differently
What is a relaxed molecule
Circular dna without any supercoiling in itself (no superhelical turn)
When does a DNA helix become topographically lionearized
- essentially asking when does it uncoil?
-during replication and transcription
Base paring is interrupted
DNA molecule exhibits supercoiling.
How do we open (unwind a dna)
two ways :
1) If one end of dna is free (essentially if dna is NOT a circle and NOT LONG linear dna molecule)
-just untwist it, one rotation
2) If both ends are FIXED (so a circle or long dna molecule (bound by chromatin scaffold), cant rotate one against the other like abox so
-strain is releaed by the formation of a supercoil (make superhelical turns)
One dna supercoil forms in the double helix for every ______ bp (for B dna)
10
Positive and Negative supercoils
-Remeber, dna is righthanded, so if u add twists to more right handed one, u are making it more Stressful (POSITIVE SUPERCOIL)
-if u make supercoils the other way, u are making Negative supercoils, relaxing it
Twist number
the number of times the individual strands in the double helxi cross over eachother
-the crossing of one strand of dsDNA over the other
measures how tightly the helix is wound
T= total # of base pairs / # base pairs per turn
ie if a 2000 bp B DNA was there
T=200 because 2000/10
When would T (Twisting #) be positive
When it is a right handed helix (ie B dna or A DNA)
negative when left handed helix (Z DNA)
THe longer the strand the ______ it would twist up with eachother
more
Writhing #
Number of superhelical turns (How many times the duplex dna crosses over itself)
-number of times the double strand crosses over eachother
Relaxed DNA. (W=0)
Negative supercoil : W is neg
pos: W is pos
Twist dna vs W #
Twist dna ( how many times two dna strands cross over eachother
Writhing (how many times the whole thing crosses over eachother
Linking #
Linking # = total # of tiemes one strand of closed molecule of DsDNA encircles the other strand
L =T + W
How can L be changed>
By breaking one or both strands of DN, winding them tighter or loose and rejoining the ends = chnage W
What happens if a dna becomes too tightened overall
Stops the dna polymerase from moving
Negative supercoiling prevents what?
Prevents more twisting later on;
they store energy which can be converted into untwisting. of double helix
