Nucleic Acids

Question 1

Chargaff’s rule

Answer 1

In DNA the number of
A = T
G = C

Question 2

Nucleases cleave ….
Endonuclease -
Exonuclease -

Answer 2

phosphodiester bonds
Endonuclease - cleaves bonds internally
Exonuclease starts at one end and cleaves bonds
one at a time

Question 3

Restriction endonucleases (type II)

Answer 3

Cleave both strands of dsDNA at specific sequences. Cuts can be staggered.
Recognition sites are palindromic.

Question 4

EcoR1 =

Answer 4

Escherichia coli R1
5’ to 3’: G AATTC
5’ overhangs

Question 5

Pst1 =

Answer 5

Pseudomonas stuartii 1
5’ to 3’: CTGCA G
3’ overhangs

Question 6

Hpa1 =

Answer 6

Haemophilus parainfluenzae 1
5’ to 3’: GTT AAC
blunt ends

Question 7

DNA ligases - they…

Answer 7

join DNA
DNA ligase will catalyse the formation of a phosphodiester link (bond) between the 5’-phosphate and 3’-OH of adjoining nucleotides in duplex DNA.

Question 8

Mechanism of ligation by DNA ligase:

Answer 8

Step 1: Adenylation of ligase
lysine residue on ligase
needs energy: (AMP from ATP or NAD+)

(NAD -bacterial ligase; ATP - eukaryotes,viruses, phage ligases)

Step 2:Activation of 5’-phosphate at 5’ terminus
(transfer of AMP moiety)
AMP is transferred to the 5’ phosphate

Step 3: Displacement of AMP seals the nick

Question 9

Alkaline phosphatase:

Calf Intestinal Phosphatase, (CIP):

Answer 9

removes 5’-phosphate from DNA or RNA
(DNA ends become resistant to ligation)
Used in DNA cloning

Question 10

Hyperchromicity

Answer 10

When double-stranded DNA goes from an ‘ordered’
to a single-stranded ‘disordered’ structure the
Absorbance (A) at 260 nm increases.

Tm = melting temperature Tm=~ 85 Celcius

Poly AT (curve to the left, less energy required)~70
Poly GC (curve to the right, more energy required)~ 100

Question 11

Topoisomerases -

Answer 11

can relax supercoiled DNA by introducing transient single-stranded or double-stranded breaks into DNA e.g. DNA replication

Supercoiled (cccDNA)

Question 12

Ethidium bromide -

Answer 12

intercalates between stacked bases in DNA

on agarose gel DNA is visible because ethidium
bromide fluoresces when exposed to UV light

Question 13

Summary:

Answer 13

Textbooks
Molecular Biology of the Gene (MGB) 6th or 7th Edition
Molecular Cell Biology (MCB) 6th or 7th Edition
Note: where figures from these texts (and others) have been used (or adapted for use) acknowledgement is given on each figure. Earlier editions of these textbooks will include key information on nucleic acids.

2 lectures on the following topics

1. The building blocks of DNA (this is revision from year 1).
2. DNA double helix – structure and stability
3. Enzymes that break and form phosphodiester bonds in DNA
4. The enzymes you will use in the DNA cloning research project
5. Melting and re-annealing of the DNA helix
6. DNA supercoiling
7. Visualing DNA (research project)