DNA fragments from different sources can be linked together to make recombinant DNA. DNA can be cut with a restriction enzyme and then rejoined with DNA ligase. There are many different restriction enzymes, each with its own recognition sequence.
learning outcomes
You should be able to:
Show how staggered cuts in DNA made by restriction enzymes lead to the creation of sticky ends.
Summarize the Cohen and Boyer experiments on creating recombinant DNA.
How does a staggered cut in DNA create a sticky end?
The sequences for recognition by a restriction enzyme often occur near one another on the opposite strands of DNA. For example,
5'….GAATTC….3'
3'….CTTAAG….5'
The top and bottom strands have the same 5'-to 3'-sequence. A restriction enzyme recognizes this and cuts between the G and A:
5'….GAATTC….3'
3'….CTTAAG….5'
Note that the cuts are staggered, not directly opposite each other.
How did Cohen and Boyer make the first recombinant DNA?
DNA from two genetically different strains of bacteria, each carrying a different antibiotic resistance gene, was cut with the restriction enzyme EcoRI and then spliced together using DNA ligase.
Recombinant DNA has no biological significance until it is inserted inside a living cell, which can replicate and transcribe the transplanted genetic information. How can recombinant DNA made in the laboratory be inserted and expressed in living cells?