NUCLEIC ACIDS 59
isolated from bacteria, that recognize and cut specifi c sequences — restriction
sites — in DNA. One restriction enzyme, BamHI, locates and cuts any occur-
rence of
(^) 53-GGATCC-′′
(^) 35-CCTAGG-′′
The enzyme clips after the fi rst G in each strand. Both strands contain the
sequence GGATCC but in antiparallel orientation. This type of recognition
site is calledpalindromic. Another sequencing method, Maxam – Gilbert (also
called the chemical degradation method), uses chemicals to cleave DNA at
specifi c bases, resulting in fragments of different lengths. The resulting DNA
restriction fragments are separated by electrophoresis on an agarose gel in a
method similar to that described for proteins in Section 2.2.3. Even fragments
that differ in size by a single nucleotide can be resolved. The gel - separated
fragments are converted to single - stranded DNA (ssDNA) by treatment with
strong base. In the Southern blot technique, the gel is then covered with nitro-
cellulose paper and compressed with a heavy plate. The nitrocellulose paper
preferentially takes up the ssDNA, producing a pattern of DNA identical to
that on the gel. A DNA probe (either a ssDNA or RNA fragment with a
complementary base sequence to the DNA of interest) then identifi es the
desired single strands of DNA through use of a^32 P radioactively tagged nucle-
otide probe. For eukaryotic DNA, one usually begins with an mRNA template
that will synthesize the desired DNA by treatment with reverse transcriptase,
an enzyme that transcribes RNA to DNA, the reverse of the normal transcrip-
tion process. The synthesized DNA is called cDNA because it carries a comple-
mentary base sequence to the mRNA template. In this cloning method, only
the DNA exon portion that codes for the desired protein is transcribed, the
intron portion that does not code for protein is not transcribed.
Once the desired cDNA or gene fragment has been obtained, it is produced
(expressed) in large quantity through the cloning process. DNA cloning in vivo
(in a living cell) can be carried out in a unicellular prokaryote such as the
bacteriumEscherichia coli , in a unicellular eukaryote such as yeast, or in mam-
malian cells grown in tissue culture. In any case, the recombinant DNA must
be taken up by the cell in a form that can be replicated and expressed. This is
accomplished by incorporating the cDNA into a vector, often a plasmid. Plas-
mids are small (a few thousand base pairs) circular DNA molecules that are
found in bacteria separate from the bacterial chromosome. Usually they carry
only one, or at most a few, genes. The same restriction enzyme that yielded the
cDNA of interest can be used to cleave the plasmid, and then the enzyme
DNA ligase is used to splice the cDNA of interest into the plasmid. The result
is an edited or recombinant DNA molecule. When the recombinant DNA
plasmid is inserted into the host, say E. coli , the E. coli cells will produce many
copies of the DNA, which will in turn be transcribed and translated into the
desired protein. The process, much simplifi ed, is illustrated in Figure 2.19.