Lecture 5: DNA methods Flashcards
(40 cards)
what are the basic methodologies for DNA? (3)
Denaturation and renaturation
– ds → ss → ds (GC content, repeat content)
Gel electrophoresis
– size, shape, supercoiling
– Detection methods
Density gradient centrifugation
– supercoiling
– GC content
in cells, important the DNA strands to _________ during transcription
and replication. In the lab, many methods take advantage of this property of
DNA
“melt” or separate
what types of things can we conclude from denaturation/renaturation experiments in lab?
-estimate GC content – more GC pairs, need more energy to denature
(ie, look at their structure- 3 H-bonds, vs 2)
-estimate “genome” complexity: renaturation – more complex DNA molecules
or genomes take more time to “find” their complement after denaturation
basically just examining the time component of the experiment!
what type of graph do we make from denaturation/renaturation data? (not really used anymore, except in qPCR)
simple DNA melting curves
______ = the increase in absorbance of a material, e.g.
dsDNA melts and becomes more opaque to 260 nm UV light
Hyperchromicity
how do we measure Tm?
easy to measure via optical density
______ – is OD at 50 % denaturation and is dependent on GC
content and salt concentration (ionic strength)
Tm (melting temp)
how can we estimate the melting temp we’ll need for DNA? what is the parameter this rule needs to follow?
Tm = 2x(A+T) + 4x(G+C)
only for SHORT DNA segments (~20 bps)
how does DNA respond to ionic strength of a solution?
(DNA physical properties are dependent on the
properties of the solution they are in)
Tm increases as ionic strength increases
Tm decreases as ionic strength decreases
-change ionic strength with salt concentration
HOW does the ionic strength of a soln change the TM?
sodiu ions can hide phsophate chrages and protect them, making them happier to stay together and not melt
In theory the negative charges on the Phosphate groups
repel the two strands -ve charges are “neutralized” by the
presence of positive ions (Na+, Mg++, etc.)
At 0.2 M NaCl (physiological condition) all phosphates are
“shielded”
_____ have been historically used to compare genome sizes and decipher genome complexity
Cot curves
CoT is a measure of…
concentration and time (Co + T)
what is the principle of CoT?
repetitive (high copy) DNA should reassociate more quickly compared to unique (low copy) DNA sequences
- Based on the probability of two complementary strands
meeting! (per mg of DNA)
what would reanneal fastest? virus, E coli, Yeast
Virus – overall renatures the fastest of these “simple genomes”
T/F: in a cot curve, Heterogenous DNA may renature irregularly due to variable complexity
true! highly repetitive sequences may reanneal faster (more copies to choose from), but single copy sequences will be super slow
T/F: Generally, you will not encounter CoT in the wild now
true!!
HOWEVER- a variation of melting curves are still routinely run
after _________ to verify you have amplified
the correct product (e.g., in a diagnostic or research lab)
qPCR (quantitative PCR)
T/F: Also, several common DNA purification techniques use
differential melting and renaturation to selectively purify
nucleic acids
true!!
______: Separation of DNA fragments based on Molecular
Weight (or physical size of the strand – length and
conformation)
electrophoresis
where do DNA/RNA go towards on an electrophoresis?
towards the positive, since they carry a negative charge
what matrix do we use for electrophoresis?
Agarose or Polyacrylamide (small molecules)
principle of electrophoresis?
set up a mesh - or sieve - through which the nucleic acids migrate
Large molecules move slowly
Small molecules move fast
polyacrylamide is a…
polymer of acrylamide
agarose is extracted from… and is an….
seaweed, disaccharide
