18 +19. Restriction Endonucleases Flashcards
what are the four general tools used for molecular cloning?
Vector - plasmid or a phage
Host
restriction enzyme
dna ligase
what is a vector and how can it be used for DNA cloning?
vectors are small independant replicating DNA molecules.
They are modified so that foreign DNA can be inserted, then the foreign DNA will be copied with the vector DNA for uses in cloning.
how are plasmids taken up by bacteria in nature?
plasmid DNA is taken up readily by other bacteria by transformation.
plasmids can only replicate in bacteria
what is the purpose of the host in molecular cloning? what is a preferred host?
the host is required as it contains the machinery for gene expression.
bacteria is a preferred host for lab cultures
explain what would occur in a medium that had antibiotics in it with E.coli grown. some of the ecoli has antibiotic resistance (plasmid) and some has no plasmid.
most bacteria in the culuture will not take up the plasmid and will die. The bacteria with the antibiotic resistance will be sucessful at growing.
explain what would occur in a medium that had no antibiotics in it with E.coli grown. some of the ecoli has antibiotic resistance (plasmid) and some has no plasmid.
the e.coli with the plasmid would grow very slowly because having the plasmid requires more energy. This means the bacteria without a plasmid would outgrow them, and the plasmid bactera (GMO one) will die out.
therefore antibiotics must be kept in the medium
what is the function of the restriction endonuclease
restriction endonuclease cuts DNA at specific sites - called restriction sites
what does DNA ligase do in molecular cloning?
joins the fragments together. Creates covalent phosphodiester bonds.
& seals gaps in the sugar-phosphate backbone
what are the steps in a basic DNA cloning strategy?
look at image carefully
- plasmid is isolated
- plasmid is cut with restriction enzyme
- get the gene of interest and cut with the same enzyme. Isolate the insert fragment
- combine the gene with plasmid
- recombinant plasmid complete - add DNA ligase to create covalent bonds
- introduce plasmid into bacterium and plate for colonies
what is the purpose of restriction endonucleases in nature?
Part of the bacterial defense system against invading DNA from bacteriophage
what are the two enzyme activities of restriction enzymes? what are three features of each of these?
Endonuclease:
- Destroys foreign DNA.
- Sequence-specific cleavage of DNA.
- Prevents infection by invading viruses (phage)
Methylase
- Modifies & protects host DNA.
- Sequence-specific methylation of DNA (-CH3)
- Prevents cleavage of host DNA by endonuclease.
what is the symmetry of restirction sites of type 2 restirciton enzyme like?
type 2 enzymes are inverted palindrones which means they read the same sequence forwards and backwards (like the word civic)
why are type 2 enzymes useful for DNA manipulation?
because they recognise DNA at a specific site and cleave within that site.
gets predictable and producable fragments.
what are the two types of ends that can be created by restriction enzymes?
blunt ends and sticky ends
what is the reaction carried out by restirction enzymes and DNA ligase?
restriction enzymes are hydrolases which produce ends with a 5- phosphate and 3- OH group
water molecule is made!
then DNA ligase can join these ends together. this looses a water molecule and is a phosphodiester bond
