Lecture 2 - DNA Denaturation and Hybridization techniques Flashcards
why are cells different in multicellular organisms despite having the same DNA?
Some genes are expressed in certain cells so there are different mRNA and proteins in each type of cell.
DNA is highly viscous at pH7 and room temp, but becomes liquid at higher temp, why?
Denaturation occured. This can happen in extreme conditions such as high temp or pH. It is caused by breaking of H bonds; covalent bonds are intact along with the code.
what happens to UV absorbance in denaturation?
UV absorbance increases because there is more space for the electrons to move around when light interacts with it. Less chance of photon emission.
What is the basis for PCR?
thermal DNA denaturation at high temps cause duplex DNA to dissociate and at lower temps the strands can anneal.
what is hyperchromicity? why does it happen in DNA denaturation?
Hyperchromicity is the increase of absorbance of a substance (DNA). it happens because base stacking decreases the ability of duplex DNA to absorb light since there is more space for the P-electrons to move around.
what does the curve of the melting point of DNA tell us about what kind of process it is?
cooperative process - DNA more likely to dissociate into separate strands as more strands separate; zipper analogy need 12 at least to make DNA stable
what is the melting temperature?
melting temperature is based on the midpoint when 50% of absorbance is reached which is dependent on the amount of DNA being measured.
what affects the melting temperature of DNA?
G and C base pairs: the higher amount = the higher temp (because they form 3 H bonds compared to A and T which only form 2); DNA length(longer DNA means more bonds); ionic strength (ions stabilize the negative charges on the backbone); pH of the solution (can help stabilize charges on backbone with postive charges; lower pH = higher temp)
what are the x and y axis of the melting temperature graph?
X axis is temperature, y axis is the relative absorbance
why doesn’t DNA denature uniformly?
there are A-T rich regions that are weaker so the bonds break there easily. They are also sites for DNA replication and transcription.
what is DNA hybridization?
DNA hybridization is when two strands of DNA pair with each other if they complementary and can be used to label specific sequences.
compare low and high stringency
Low stringency is when there is imperfect matching during hybridizations. high stringency is when it is perfect. Low stringency requires high salt and low temp while high stringency requires high temp and low salt. high temp is for breaking the H bonds, H bonds are stronger when there are more perfect matches whereas in low stringency there are imperfect matches
What is required when trying to hybridize DNA from 2 different species?
Low temps allow for slow annealing to occur so that you can get a mix of hybrids and non hybrids
Why is annealing so slow?
DNA have to collide with each other randomly and also there will be errors when pairing so it takes awhile.
What is nucleic acid hybridization (NAH)?
purpose is to identify how closely DNA molecules relate to each other. Factors that need to be considered are: strand length, base composition, chemical environment (charges or ions), melting temperature, hybridization stringency
