Recombinant DNA

Question 1

Applications

Answer 1

DNA cloning: iso of a DNA fragment and its propagation without alteration of the original DNA sequence.
To generate many copies for analysis.
Protein production, certain bacterial systems are engineered to express proteins of interest.
Genetic mod, foreign genes into organisms to study gene function, improve agri, or develop gene therapies.

Question 2

DNA cloning workflow

Answer 2

Isolation of target DNA fragments (inserts)
Ligation of inserts into an appropriate cloning vector, creating recombinant molecules (plastids).
Transformation of recombinant plasmids into bacterial or other host for propagation.
Selection of successful transformants identified and isolated.

Question 3

Vector

Answer 3

DNA molecule that can be used to transfer a DNA fragment into a host cell.
Provides control elements for replication and expression (autonomous replication)
Bacterial plasmids are the most commonly used vectors to clone DNA fragments.

Question 4

Restriction

Answer 4

Have a DNA sequence formed by combination of two-non homologous DNA molecules.
Restriction of DNA fragment, create blunt or sticky ends.
Enzymes are essential for generating compatible fragments that can be inserted into vectors.

Question 5

DNA ligase/ligation

Answer 5

Catalyses the covalent ligation between the 3’ and 5; ends of DNA strands, securing the insert into the vector.

Question 6

Identification of host cells

Answer 6

Selection of bacterial clones that have incorporated via antibiotic resistance screening.

Question 6

White/blue screening

Answer 6

Chromogenic substrate (X-gal) added to bacterial growth substrate.
If the plasmid remains undisturbed, enzymatic reaction causes the production of a blue pigment, resulting in blue bacterial colonies.
The insertion of a foreign DNA fragment in the MCS prevents the enzyme production, white.

Question 6

Transformation of host cells

Answer 6

Chemical transformation
Electroporation
Addition of plasmids into cell.