Test 1: Nucleic Acid Acidity and Acid/Base Reactions

Question 1

pH =

Answer 1

-log([H+])

Question 2

Henderson Hasselbach equation:

Answer 2

pH = pK + log(base/acid)

Question 3

In an equilibrium base/acid the amount of acid and the amount of base must equal what?

Answer 3

pka = 1 - x / x; x = acid
Use one above
pka = 1 / 1 - x; x = base

Question 4

Steps to calculate the charge of a nucleic acid using Henderson Hasselbach equation.
- Do several practice problems

Answer 4

1: identify ionizable groups
2: determine pH of solution, and pKa of each ionizable group
3: determine protonation status of ionizable groups
4: Add up the charges of ionizable groups to determine the net charge of the nucleic acid
5: remember phosphate (pKa 1, unless it has 5’OH, which has pKa 6. (negative if not protonated)

Question 5

Three methods of Nucleic Acid Isolation by charge:

Answer 5

Phenol extraction:
Diethylaminoethyl (DEAE) chromatography:
Electrophoresis:

Question 6

Phenol Extraction:

Answer 6

proteins go to phenol, DNA goes to H20

Question 7

Diethylaminoethyl (DEAE) chromatography:

Answer 7

Ionic chromatography separation with a positively charged DEAE resin.

Question 8

Electrophoresis:

Answer 8

Separation based on size, not base composition

Question 9

Explain ionic chromatography:

Answer 9

A chamber is filled with negatively or positively charged beads. They are coated with ions of the opposite charge. These ions are displaced by your sample (which should have charges). Substances with stronger charges will hold on longer to the beads. Beads in this case are coated with DEAE.

Question 10

What is chromatography resin?

Answer 10

The stationary phase. What interacts with the sample and does or does not slow it.

Question 11

When does alkali cleavage occur?

Answer 11

In an alkali environment, if there is a missing base (a nucleotide - guanine). Aka an exposed 1’ OH.

Question 12

Alkali cleavage of RNA (RNA scission):

Answer 12

Phosphate in RNA chain attacked. it is cleaved from its 5’ carbon, and it attaches to 2’ or 3’ carbon.

Question 13

Acid denaturation of A or G (A + G), what occurs?

Answer 13

Protonated nitrogen pulls electrons through purine, breaks connection to ribose/deoxyribose.
N3 for problem child for adenine
N7 for problem child for guanine

Question 14

At what pH does acid denaturation of A and G (purines) occur?

Answer 14

<= 2.5

Question 15

Cleavage “in-trans”:

Cleavage “In-cis”:

Answer 15

It is cut by another molecule

It is cut by itself

Question 16

What can happen to purines under a pH of 2.5?

Answer 16

A nitrogen is protonated, causing the ring to change its resonant structure and break from its ribose.
Random Aside: If this was added to an alkali afterwards we would see alkali cleavage of DNA with a missing base.

Question 17

Dimethyl sulfate:

Answer 17

Sn2 reaction, (nucleophiles attack opposite group of leaving group. Methylates G ~10 times more rapidly as A

Question 18

Look up Sn2 reaction

Answer 18

Are bimolecular, and therefor do not proceed with an intermediate.
Give inversion of stereochemistry at reaction center.
A nucleophilic substitution (so a proton loving reaction, an attack by a nucleophile like OH-