7.1 DNA Modification
Recombinant DNA technology relies on the ability to manipulate DNA using nucleic acid–
modifying enzymes. The isolation of these enzymes followed shortly after James Watson
and Francis Crick’s description of the double helical structure of DNA in 1953. Recall
that DNA is made up of two twisting complementary strands, comprising alternating
units of deoxyribose sugar and phosphates that run in opposite directions. Attached to
each deoxyribose sugar is a nitrogen-rich base. The bases, adenine (A), thymine (T),
guanine (G), and cytosine (C), on opposite strands are held together by hydrogen bonds
to form base pairs (bp); A with T and G with C. The complementary nature of the
strands means that each strand provides a template for the synthesis of the other (Fig. 7.1).
In 1955, Arthur Kornberg and colleagues isolated DNA polymerase I, an enzyme
capable of using this template to synthesize DNA in vitro in the presence of the four
bases, in the form of deoxribonucleoside triphosphates (dNTPs). Although this was the
first enzyme to be discovered that had the required polymerase activities, the primary
enzyme involved in DNA replication is DNA polymerase III.
While DNA polymerases can replicate a second strand of DNA, they cannot join the
ends of DNA together. The discovery of circular DNA molecules (plasmids, discussed
later) suggested that such an enzyme must exist. In 1966, Bernard Weiss and Charles
Richardson isolated DNAligase, an enzyme that allowed DNA to be “glued” together, cat-
alyzing the formation of a phosphodiester bond (Fig. 7.2).
Soon after this discovery, investigations into bacterial resistance that “restricted” viral
growth revealed thatendonucleaseswithin the cells could destroy invading foreign DNA
molecules. Among the first “restriction enzymes” to be purified were EcoRI from
Escherichia coli, andHindIII fromHaemophilus influenzae. Restriction enzymes went
on to become one of the most useful tools available to molecular biologists and deserve
special consideration.
Restriction enzymes (restriction endonucleases) are produced by a wide variety of pro-
karyotes. These enzymes identify specific nucleotide sequences in DNA of 4–8 bp, usually
palindromes, and cleave specific phosphodiester bonds in each strand of the DNA. The
methylation of these specific nucleotide sequences in the host DNA protects the cell
from attack by its own restriction enzymes. There are many different site-specific restriction
enzymes. These are named after the bacterial species and strain of origin. The restriction
endonucleaseEcoRI, for example, was the first restriction endonuclease identified from
the bacteriumEscherichia coli, strainRY13 (other examples are shown in Table 7.1).
Such enzymes recognize a specific double-stranded DNA sequence and cleave the
strands to produce either a 5^0 overhang, a 3^0 overhang, or blunt ends (Fig. 7.3).
DNA fragments that contain single-stranded overhangs (“sticky ends”) are the easiest to
join together. Two DNA molecules, with compatible single-stranded overhangs, can hybri-
dize to bring the 5^0 phosphate and 3^0 hydroxyl residues together, allowing DNA ligase to
catalyze the formation of phosphodiester bonds (recall Fig. 7.2). In this way, two DNA mol-
ecules from different sources can be combined to produce an artificial or “recombinant”
DNA molecule (Fig. 7.4). All of biotechnology hinges on recombinant DNA—combining
DNA from various sources to do something new. Using two restriction enzymes with differ-
ent recognition sequences, one can combine two DNA molecules in a predetermined orien-
tation (Fig. 7.5).
The first recombinant DNA molecule was created in Paul Berg’s lab in 1972. This pio-
neering work formed the basis of the recombinant DNA revolution; however, it was not
160 RECOMBINANT DNA, VECTOR DESIGN, AND CONSTRUCTION