Recombinant DNA technology Flashcards
What is gene cloning?
Gene cloning involves isolating a single gene of interest and inserting it into a vector so that it can be copied quickly and its function and structure can be studied.
Where is the gene of interest located?
In the host chromosome
How can the gene be isolated?
Either using restriction enzymes or PCR
What is a plasmid?
A segment of DNA separate from the chromosome capable of replication.
When can you amplify a gene?
If the gene has been well studied, and it is under 5 kb, using PCR
If you can’t amplify a gene what happens instead?
The Genome can be digested with restriction enzymes, individual segments cloned and then reassembled.
What two DNA molecules are able to act as a cloning vector?
Plasmids and Bacteriophages
What do the cloning vehicles do?
Enter a host cell and, once inside, replicate to produce multiple copies of itself.
What is a vector?
A segment of DNA separate from the chromosome capable of replication.
What are the features needed for any plasmid that’s being used as a cloning vector
An origin of replication: Required for independent replication of the plasmid.
A dominant selectable marker: Required to select for bacteria carrying the desired plasmid.
Unique restriction endonuclease cleavage sites: Essential to permit the insertion of foreign DNA fragments into the plasmid.
What are the two enzymes involved in gene cloning?
Nucleases and Ligases
What do nucleases do?
Cut, shorten or degrade nucleic acids
What do ligases do?
Join nucleic acid molecules together
What happens if a molecule is cut more than once
It will be broken into two or more fragments and will be useless as a cloning vector.
What are restriction sites?
Short palindromic sequences.
