Lab: Plasmid vs. Lambda DNA

Function of a restriction enzyme in nature + benefit to biotech

Function in nature:

→ Restriction enzymes protect bacteria by cutting up invading viral DNA (bacteriophages).

Benefit to biotech:

→ They allow scientists to cut DNA at specific sequences, enabling cloning, recombinant DNA, gene insertion, DNA mapping, and genetic engineering.

How are restriction enzymes named?

Based on the bacterium they were isolated from:

• 1st letter: genus

• Next 2 letters: species

• Strain (optional)

• Roman numeral: order of discovery

Example: EcoRI = Escherichia coli strain RY13, enzyme I.

Which type of DNA has more restriction sites: chromosomal or plasmid DNA?

Chromosomal DNA has more restriction sites.

Why: It is much larger and therefore contains many more sequences that match restriction enzyme recognition sites.

Plasmid DNA with 3 EcoRI sites → how many fragments?

3 sites in circular DNA = 3 fragments

(Circular DNA fragments = number of cut sites)

Linear DNA with 3 EcoRI sites → how many fragments?

3 sites in linear DNA = 4 fragments

(Linear DNA fragments = sites + 1)

Why do partial digests occur?

Not enough enzyme, incomplete reaction time, incorrect temperature, or enzyme inhibited → only some sites get cut instead of all.

Define: Catenanes, Supercoiled DNA, Nicked DNA

Catenanes

→ Two or more circular DNA molecules linked like chain links.

Supercoiled DNA

→ Tightly twisted plasmid form; compact; moves fastest in a gel.

Nicked DNA

→ One strand cut; loses supercoiling; becomes relaxed/open circle.

Order of migration (fastest → slowest)

1. Supercoiled DNA (smallest/most compact)

2. Nicked circular DNA (open circle, slower)

3. Catenanes (largest and linked, slowest)

Size of the supercoiled plasmid for the experiment

4500 BP