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What is recombinant DNA technology?
It is the strategy to capture specific DNA like:
-a gene of interest
-a gene that encodes a protein
-DNA that has polymorphisms
-A fragment of DNA that codes for the phenotype
What is the source of DNA used for cloning?
-Usually genomic DNA (coding DNA)
-also called cDNA
What is genomic DNA?
-Genomic DNA is the DNA inside a eukaryote cell
-mRNA (cDNA) codes for phenotype
-DNA = 100% of genome
-cDNA = 10% of genome
What are restriction enzymes (restriction endonucleases)?
-They are the molecular scissors
-Their function is to cut DNA
How do restriction enzymes discern between bacterial DNA and foreign DNA?
-Restriction enzymes target foreign DNA because they are not methylated
-Restriction enzymes can only bind to certain sites. If these sites are methylated, the enzyme cannot bind there.
Where does the restriction enzymes bind to the DNA?
-Restriction enzymes travel in pairs and bind to one strand of DNA each
-Restriction enzymes can only cut one DNA strand
What are the two types of cuts restriction enzymes can do?
1) Restriction enzymes can do blunt cuts, where both DNA strands are cut evenly
2) Restriction enzymes can also cut to leave sticky ends, where one of the strands extends three or four basepairs further than the other.
When a restriction enzyme cuts DNA, what stops the DNA from simply rejoining again?
-The restriction enzymes take the 5´ phospho group.
-DNA Ligase is able to reseal this gap.
What is DNA ligase?
-The molecular glue
-DNA ligase can join two pieces of DNA together by forming the missing phosphodiester
-This allows the recombination of DNA
What are the three features of cloning vectors?
1) Origin of replication = directs self replication
2) Dominant selectable marker = usually drug resistance of host cell
3) At least one EcoRO (cleavage site)
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