L2 Restriction enzymes and cloning

Question 1

What are restriction enzymes?

Answer 1

Enzymes that hydrolyze phosphodiester bonds in the sugar-phosphate backbone. Needs Mg as co-factor.

These are used for large molecules e.g human genome, E.coli.

The fragments can be rejoined

Question 2

Role of restriction enzymes

Answer 2

Natural: Protect bacteria from infection by viruses by cleaving non-host DNA whilst its own DNA is protected by methylation.

Question 3

Types of restriction enzyme

Answer 3

Type I - Multisubunit, randomly cut DNA from recognition sequence

Type II- Cut DNA at defined positions within their recognition system.

Type III- Restriction and modification enzyme complex.
Cleave DNA outside of recognition sequence.

Question 4

Restriction enzymes nomenclature

Answer 4

1) Name of host (EcoLi)
2) Strain type (R)
3) Number (1)

Hence, EcoRI

Question 5

Type 2 restriction enzyme

Answer 5

• Always cuts at specific nucleotide sequence
• Recognition can be continous or discontinous and can vary in length
• Some may recognize same sequence but cut differently (blunt,sticky)

Question 6

Joining DNA fragments - Ligase

Answer 6

Ligase catalyze formation of phosphodiester bonds

It requires a free OH group at the 3’ end of DNA chain and phosphate group on the 5’ end of the other end.

Ligates sticky and blunt

Question 7

Chemistry of Ligase

Answer 7

reacts with ATP to form covalent enzyme-ADP complex

Ligase AMP activates phosphate group at 5’ end

Nucleophilic attack by OH at 3’ end, phosphodiester bond is formed, AMP released

Question 8

DNA cloning principles

Answer 8

Requirements:

• Insert DNA, DNA generated by enzymatic activity or physical treatment
• Vector, a DNA molecule that provides the information necessary to propagate cloning.

Question 9

Religation of the vector

Answer 9

Alkaline phosphatase treatment.
-removes phosphate from 5’ nucleic acids

This prevents the vector ligating with itself.

Question 10

Blunt > Sticky end conversion

Answer 10

Ligation of Sticky is a lot easier, hence:

S1 DNA nuclease is used to convert blunt to sticky

Question 11

New restriction sites - Oligonucleotides linkers

Answer 11

contains endonuclease cleavage site and can be ligated to create new restriction site.

e.g
Blunt DNA > Oligo link added > (longer length) > restriction sites are cut > New sticky ends

Question 12

DNA into bacteria method

Answer 12

1) Bacteria treated with salt
2) Heat shocked to induce DNA uptake

OR

1) Treated with salt
2) Electric pulse induces opening of pores

OR

1) Remove bacteria wall by enzyme
2) DNA enters and regeneration of wall