Recombinant DNA Recombinant DNA
Recombining DNA, or “fiddling” with DNA to put it together in different, usual new, ways. Changing the genetic information in a cell Specific trait of one organism may be isolated, cut, and “pasted” into the cell of another organism
How do we do this? Restriction Enzymes y
Restriction enzymes
• Also called restriction endonucleases • Occur naturally in bacteria • Hundreds are purified and available commercially • Named for bacterial genus, species, strain, and type: Example: EcoRI Genus: Escherichia Species: coli Strain: R
What do restriction enzymes do?
Recognize specific base sequences in DNA Cut DNA at those recognition sites
Why would bacteria have Restriction Enzymes?
bacterial "immune system": destroy any "non-self" DNA called "restriction enzymes“ because restriction restrict host range for certain bacteriophage methylase recognizes same sequence in host DNA and protects it by methylating it; restriction enzyme destroys unprotected (non-self) DNA
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The same in pictures…
Viral DNA
Restriction Enzymes
Hundreds of restriction enzymes have been identified. Most recognize and cut palindromic sequences Many leave staggered (sticky) ends by choosing correct enzymes can cut DNA very precisely
Bacterial DNA
Restriction Enzyme Recognition Site
• Enzymes recognize specific 4-8 bp sequences
EcoRI 5’…GAATTC…3’ 3’…CTTAAG…5’
• Recognition sites have symmetry • Some enzymes cut in a staggered fashion • Some enzymes cut in a direct fashion
PvuII 5’…CAGCTG…3’ 3’…GTCGAC…5’
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Q: How Frequently Will a Restriction Enzyme Cut DNA? A: It Depends of the Length of the Recognition Sequence
Average distance between cuts is: 4n where “n” is number of bp’s in recognition site. “n • 4-base cutter: • 5-base cutter: • 6-base cutter: • 8-base cutter: 256 bp 44 = 45 = 1,024 bp 46 = 4,096 bp 48 = 65,536 bp
Why would (should) this interest me?
Important for molecular biologists because restriction enzymes...