BLGY1232 Gene cloning and sequencing
Molecular analysis requires the use of cloned genes
The genetic engineer’s toolkit
Restriction enzymes - cut DNA in a reproducible way
DNA ligase - an enzyme that joins DNA fragments end-to-end makes a
phosphodiester bond between the terminal 5’-phosphate on one DNA molecule
and the terminal 3-OH on another
A vector - a DNA molecule that will replicate autonomously (e.g. a plasmid and
bacteriophages)
A host - an organism that will grow quickly and propagate the vector// an
organism in which the vector will replicate (e.g. E. coli)
Plasmid vectors
Small - thus containing very few unwanted restriction sites
Multicopy - so a single cell will contain lots (100+) plasmid copies
Selectable - usually by carrying an antibiotic resistance gene to ensure that only
bacteria carrying the vector are propagated.
Will contain several unique restriction sites - for the insertion of foreign DNA
fragments
Will carry a marker allowing easy identification of foreign DNA
e.g. “pBluescript”; 2.9kb, 100+ copies per cell, Ampicillin resistance, a “multiple
cloning site”, Insertional inactivation of LacZ
Cloning
1. Digest your target DNA with a suitable restriction enzyme
2. Digest your vector with the same restriction enzyme
3. Digest vector with alkaline phosphatase as this prevents self-ligation
4. Mix the two together and add DNA ligase
5. Introduce the recombinant plasmids to E. coli cells by transformation:
treatments of bacteria with CaCl2 causes them to take up DNA readily
6. Select cells containing plasmid by plating on medium containing ampicillin
Only transformed cells will grow
7. Distinguish the cells containing insertless, non-recombinant plasmid from cells
containing recombinant (insert-containing) plasmid by including a chromogenic
substrate in the growth medium The lacZ gene encodes the enzyme beta-
Molecular analysis requires the use of cloned genes
The genetic engineer’s toolkit
Restriction enzymes - cut DNA in a reproducible way
DNA ligase - an enzyme that joins DNA fragments end-to-end makes a
phosphodiester bond between the terminal 5’-phosphate on one DNA molecule
and the terminal 3-OH on another
A vector - a DNA molecule that will replicate autonomously (e.g. a plasmid and
bacteriophages)
A host - an organism that will grow quickly and propagate the vector// an
organism in which the vector will replicate (e.g. E. coli)
Plasmid vectors
Small - thus containing very few unwanted restriction sites
Multicopy - so a single cell will contain lots (100+) plasmid copies
Selectable - usually by carrying an antibiotic resistance gene to ensure that only
bacteria carrying the vector are propagated.
Will contain several unique restriction sites - for the insertion of foreign DNA
fragments
Will carry a marker allowing easy identification of foreign DNA
e.g. “pBluescript”; 2.9kb, 100+ copies per cell, Ampicillin resistance, a “multiple
cloning site”, Insertional inactivation of LacZ
Cloning
1. Digest your target DNA with a suitable restriction enzyme
2. Digest your vector with the same restriction enzyme
3. Digest vector with alkaline phosphatase as this prevents self-ligation
4. Mix the two together and add DNA ligase
5. Introduce the recombinant plasmids to E. coli cells by transformation:
treatments of bacteria with CaCl2 causes them to take up DNA readily
6. Select cells containing plasmid by plating on medium containing ampicillin
Only transformed cells will grow
7. Distinguish the cells containing insertless, non-recombinant plasmid from cells
containing recombinant (insert-containing) plasmid by including a chromogenic
substrate in the growth medium The lacZ gene encodes the enzyme beta-
