18 
This was the first breakthrough, the development of a simple procedure 
for joining together DNA segments from very different sources. Ordinarily, 
when one carries out such joinings, one produces very complex mixtures of 
DNA segments and hybrids, often unresolvable, and therefore useless for 
experimental purposes. 
Therefore, one is confronted with the question of obtaining a single 
pure type of recombinant DNA and this proposed a second challenge. In 
fact, it required a second breakthrough. 
It was known that some bacteria contain plasmids. Plasmids are small, 
extra chromosomes that occur in cells in addition to their major chromo- 
somes. A plasmid is also a piece of DNA, and it exists and propogates as 
a circular molecule. Such plasmids can be readily isolated in pure form 
and then reintroduced into other bacteria, where they can multiply auto- 
nomously — that is, independent of the cellular chromosome. 
A plasmid which turned out to be very useful was one carrying genes 
that confer resistance to the antibiotic tetracycline. Thus, the entry 
and the establishment of such a plasmid in a bacterium like li. coli can be 
monitored and, in fact, selected for by the use of tetracycline resistance 
for the modified bacteria. 
May I have the next slide (5), please? 
This is a strategy which was developed by Stanley Cohen at Stanford, 
and it involves the following. It is to obtain such a plasmid, and this is 
a particular plasmid which he used. That plasmid contains a sequence which 
can be cut by the enzyme I mentioned a moment ago, to produce a linear 
structure with short cohesive ends. It is then possible to take foreign 
DNA, and by foreign DNA I mean DNA from any source, any type of cell on 
this earth, cut that DNA with the same enzyme to produce short bits which 
have the same type of cohesive ends. It is only necessary to mix these two 
to produce a hybrid structure in which the ends of the plasmid are bridged 
by the segment of foreign DNA. 
What Stanley Cohen showed, which was most important, was that these 
fused elements could be reintroduced into virgin bacteria where they can 
take up residence and multiple as the cell divides. The modified cells 
can grow on a very simple solution containing some sugar, a source of ni- 
trogen, and a few minerals. But because they can divide roughly every 20 
minutes, within a day one can obtain billions of organisms carrying the 
new DNA segment that they acquired, and this can be produced in pure form 
in large quantities. Therefore, it is a very simple matter to obtain seg- 
ments of any DNA in large quantities in pure form. 
[159] 
