The Collaboration Begins
In the collaborative work we planned, fragments produced by cutting DNA with the EcoR1 enzyme that Herb's lab had purified would be joined to plasmids which would then be introduced into bacteria by the transformation procedure my lab had developed. While the concept was straightforward, no one knew at the time whether DNA molecules constructed artificially would be biologically functional and capable of being propagated in living cells. The experimental part of the collaboration began right after the Hawaii meeting and by March 1973 we had established the feasibility of DNA cloning.
In our collection at Stanford was a small plasmid, pSC101, that carried a gene and coding resistance to the antibiotic Tetracycline. We found that this plasmid was cut only once by the Eco R1 enzyme. Because the cleavage site did not interrupt either the replication region of the plasmid or its Tetracycline resistance gene, pSC101 could be used as a cloning vector to carry DNA fragments that lack their own replication region. The plasmid was opened up by cutting with the eco R1 enzyme and mixed with DNA from a large plasmid that had been cleaved into multiple fragments by the same enzyme. The complimentary ends on both kinds of DNA molecules held the fragments together so that they could be spliced efficiently by DNA ligase. Plasmids containing inserted DNA fragments were introduced into bacteria by the transformation procedure and clones of bacteria that acquired recombinant plasmids were selected by culturing them on agar plates containing Tetracycline, which prevented the growth of bacteria lacking the plasmids.
  
|
Winding Your Way Through DNA Resource Book Index
