TTAATTAA
AATT
AATTAATTTTAAAATT
TTAAGene for
replicationGene for
tetracycline
resistanceCut with
EcoRIInsulin geneDNA
ligaseCut with
EcoRIPlasmid DNAHuman DNARecombinant
DNACutting DNA and Making Recombinant DNA
An example of how restriction enzymes work is shown in Figure 3.
The enzyme recognizes a specific sequence of DNA. The sequence
the enzyme recognizes and the sequence on the complementary
DNA strand are palindromes—they read the same backward as they
do forward (such as the word noon).
The cuts of most restriction enzymes produce pieces of DNA with
short single strands on each end that are complementary to each
other. The ends are called sticky ends. As illustrated in Figure 3, the
vectors that are used contain only one nucleotide sequence that the
restriction enzyme recognizes. Thus, vectors such as the circular
plasmids “open up” with the same sticky ends as those of the cut
human DNA. The two DNA molecules bond together by means of
complementary base pairing at the sticky ends. The plasmid DNA
has both the gene for plasmid DNA replication and the gene that
makes the cell carrying the plasmid resistant to the antibiotic tetra-
cycline.Cloning, Selecting, and Screening Cells
One difficult part in a genetic engineering experiment is find-
ing and isolating the cells that contain the gene of interest.
First, the cells that have taken up the plasmid must be identi-
fied. The bacterial cells that have taken up the plasmid are
identified by growing the bacteria on plates that contain the
antibiotic tetracycline. As shown in Figure 4,only the cells
that have taken up the vectors (which contain the gene for
tetracycline resistance) survive when exposed to tetracycline.
Each surviving cell makes a copy of the vector every time the
cell reproduces. Eventually, each surviving cell forms a colony
of genetically identical cells, or clones. Some vectors contain
the gene of interest, and some do not.230 CHAPTER 11Gene Technology
The restriction enzyme EcoRI recognizes the nucleotide sequence
GAATTC and makes its cut between the G and the A.
Figure 3 Restriction enzymes cut DNA
Figure 4 Screening.
Only the cells that take up
the vectors are resistant
to tetracycline and survive
when tetracycline is added.