Gene Expression 2 - Chapter Three
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You are at the bottom of a Right Handed DNA strand, looking up. Which direction does the DNA spiral in (CW or CCW)
- The DNA spirals in the Clockwise (CW) direction.
What is Nucleic Acid hybridization?
Nucleic Acid Hybridization is the insertion of s third, complimentary strand. Often, this is done with Radioactive Probes.
How might you go about Nucleic Acid Hybridization?
- 1. Melt the DNA to get single Stranded Random Coils.
- - This Can be done by raising temperature and/or increasing H+ concentration.
- 2. Add a Probe to the mix.
- - Probes are often Radioactive.
- 3. Re-Anneal the Strands
- - Re-annealing is done at 25C below the Melting Temperature.
- - Rapid cooling favours probe annealing.
- - Concentration and time also affect annealing.
- - Size and Complexity may also affect annealing.
Explain how the curve on this graph arises.
The curve is the result of the difference in absorbency of 260nm UV light between double and single stranded DNA.
Which type of molecule's electron configuration has the greatest UV260 absorption out of Double and Single stranded DNA?
Single Stranded DNA has the greatest UV absorption.
Define the term, Hyperchromic Shift.
Hyperchromic shift is the change in UV260 absorbency between double and single stranded DNA.
IS DNA density constant?
No, increasing G-C will increase DNA density slightly.
How would a high G-C percentage affect a graph of A260 against Temperature?
High G-C content would cause stronger bonding between the two strands, therefore raising melting temperature and pushing the graph to the right.
How would a drastic change in pH affect a graph of A260 against Temperature?
A change in pH would destabilize the helix, reducing the melting temperature and shifting the graph left.
How would an increase in Anion concentration affect a graph of A260 against Temperature?
An increase in Anion (Negative Ion) concentration would destabilize the helix by reducing cation concentration. Therefore, melting temperature would decrease and the graph would shift left.
If Formanide and/or Urea are added to a DNA solution, how would this affect a graph of A260 against Temperature?
Formanide and Urea compete for Hydrogen Bonding interactions and therefore destabilize the helix. The result would be a decrease in melting point temperature and an increase shift to the left.
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