Chapter 12 Flashcards
What are the 4 steps of cloning?
1.) Obtain an expression plasmid (vector)
2.) Obtain the human gene or insert
3.) Inter the DNA into the vector
4.) Add the recombinant to the bacteria and clone/replicate
Where do restricition enzymes cut?
They cleave the phosphodiester bonds between nucleotides in the endonuclease manner.
What is palindromic?
Restriction sites are the same sequence 5’ to 3’ and 3’ to 5’.
What can be the product of the restriction enzymes?
1.) Staggered sticky ends
2.) Blunt ends
What does the DNA ligase do?
Pieces of DNA are cut with the same restriction enzymes and can then be ligated together to reform the enzyme cut site sequence.
What is the difference between the blunt and sticky ends?
Blunt ends have a higher likelihood of being ligated compared to sticky ends since the blunt ends have overhangs and the sticky ends do not.
What is DNA cloning?
The creation of recombinant DNA molecules that can be replicated in cells.
What is the recombinant DNA?
Vector + Insert
What is single enzyme cloning?
The same enzyme is used.
What is double enzyme cloning?
2 DIfferent enzymes are used.
What are the 3 essential features of cloning vectors?
1.) Origin of replication which allows the plasmid to replicate in the bacterial host cell
2.) Antibiotic resistance gene allows the selection of cells cotaining the plasmid to grow
3.) Polylinker or multiple cloning site that contains various restriction enzyme cut sites
