The essence of cell chemistry is to isolate a particular cellular component and then analyze its chemical structure and activity. In the case of DNA, this is feasible for relatively short molecules such as the genomes of small viruses. But genomes of even the simplest cells are much too large to directly analyze in detail at the molecular level. The problem is compounded for complex organisms. The human genome, for example, contains about 6 × 109 base pairs (bp) in the 23 pairs of chromosomes. Cleavage of human DNA with restriction enzymes that produce about one cut for every 3000 base pairs yields some 2 million fragments, far too many to separate from each other directly. This obstacle to obtaining pure DNA samples from large genomes has been overcome by recombinant DNA technology. With these methods virtually any gene can be purified, its sequence determined, and the functional regions of the sequence explored by altering it in planned ways and reintroducing the DNA into cells and into whole organisms.
The essence of recombinant DNA technology is the prep-aration of large numbers of identical DNA molecules. A DNA fragment of interest is linked through standard 3′ → 5′ phosphodiester bonds to a vector DNA molecule, which can replicate when introduced into a host cell. When a single recombinant DNA molecule, composed of a vector plus an inserted DNA fragment, is introduced into a host cell, the inserted DNA is reproduced along with the vector, producing large numbers of recombinant DNA molecules that include the fragment of DNA originally linked to the vector. Two types of vectors are most commonly used: E. coli plasmid vectors and bacteriophage λ vectors. Plasmid vectors replicate along with their host cells, while λ vectors replicate as lytic viruses, killing the host cell and packaging the DNA into virions (Chapter 6). In this section, the general procedure for cloning DNA fragments in E. coli plasmids is described.
Plasmids are circular, double-stranded DNA (dsDNA) molecules that are separate from a cell’s chromosomal DNA. These extrachromosomal DNAs, which occur naturally in bacteria, yeast, and some higher eukaryotic cells, exist in a parasitic or symbiotic relationship with their host cell. Plasmids range in size from a few thousand base pairs to more than 100 kilobases (kb). Like the host-cell chromosomal DNA, plasmid DNA is duplicated before every cell division. During cell division, at least one copy of the plasmid DNA is segregated to each daughter cell, assuring continued propagation of the plasmid through successive generations of the host cell.
Many naturally occurring plasmids
contain genes that provide some benefit to the host cell, fulfilling the
plasmid’s portion of the symbiotic relationship. For example, some
bacterial plasmids encode enzymes that inactivate antibiotics. Such
drug-resistance plasmids have become a major problem in the treatment of a
number of common bacterial pathogens. As antibiotic use became widespread,
plasmids containing several drug-resistance genes evolved, making their host
cells resistant to a variety of different antibiotics simultaneously. Many of
these plasmids also contain “transfer genes” encoding
proteins that can form a macromolecular tube, or pilus, through
which a copy of the plasmid can be transferred to other host cells of the same
or related bacterial species. Such transfer can result in the rapid spread of
drug-resistance plasmids, expanding the number of antibiotic-resistant bacteria
in an environment such as a hospital. Coping with the spread of drug-resistance
plasmids is an important challenge for modern medicine.
Plasmid vectors are ≈1.2 – 3 kb in length and contain a replication origin (ORI) sequence and a gene that permits selection, usually by conferring resistance to a particular drug. Here the selective gene is ampr; it encodes the enzyme β-lactamase, which inactivates ampicillin. Exogenous DNA can be inserted into the bracketed region without disturbing the ability of the plasmid to replicate or express the ampr gene.
).
The parental strands are shown in blue, and newly synthesized daughter strands are shown in red. The short segments represent the A·T and G·C base pairs connecting the complementary strands. Once DNA replication is initiated at the origin (ORI), it continues in both directions around the circular molecule until the advancing growing forks merge and two daughter molecules are produced. The origin is the only specific nucleotide sequence required for replication of the entire circular DNA molecule.
). Thus any DNA sequence inserted into such
a plasmid is replicated along with the rest of the plasmid DNA; this
property is the basis of molecular DNA cloning.
In 1944, O. T. Avery, C. M. Macleod, and M. McCarty first demonstrated gene transfer with isolated DNA obtained from Streptococcus pneumoniae. This process involved the genetic alteration of a bacterial cell by the uptake of DNA isolated from a genetically different bacterium and its recombination with the host-cell genome. Their experiments provided the first evidence that DNA is the genetic material. Later studies showed that such genetic alteration of a recipient cell can result from the uptake of exogenous extrachromosomal DNA (e.g., plasmids) that does not integrate into the host-cell chromosome. The term transformation is used to denote the genetic alteration of a cell caused by the uptake and expression of foreign DNA regardless of the mechanism involved. (Note that transformation has a second meaning defined in Chapter 6, namely, the process by which normal cells with a finite life span in culture are converted into continuously growing cells similar to cancer cells.)
The phenomenon of transformation permits plasmid vectors to be introduced into and expressed by E. coli cells. In order to be useful in DNA cloning, however, a plasmid vector must contain a selectable gene, most commonly a drug-resistance gene encoding an enzyme that inactivates a specific antibiotic. As we’ve seen, the ampicillin-resistance gene (ampr) encodes β-lactamase, which inactivates the antibiotic ampicillin. After plasmid vectors are incubated with E. coli, those cells that take up the plasmid can be easily selected from the larger number of cells that do not by growing them in an ampicillin-containing medium. The ability to select transformed cells is critical to DNA cloning by plasmid vector technology because the transformation of E. coli with isolated plasmid DNA is inefficient.
Normal E. coli cells cannot take up plasmid DNA from the medium. Exposure of cells to high concentrations of certain divalent cations, however, makes a small fraction of cells permeable to foreign DNA by a mechanism that is not understood. In a typical procedure, E. coli cells are treated with CaCl2 and mixed with plasmid vectors; commonly, only 1 cell in about 10,000 or more cells becomes competent to take up the foreign DNA. Each competent cell incorporates a single plasmid DNA molecule, which carries an antibiotic-resistance gene. When the treated cells are plated on a petri dish of nutrient agar containing the antibiotic, only the rare transformed cells containing the antibiotic-resistance gene on the plasmid vector will survive. All the plasmids in such a colony of selected transformed cells are descended from the single plasmid taken up by the cell that established the colony.
Although not indicated by color, the plasmid contains a replication origin and ampicillin-resistance gene. Uptake of plasmids by E. coli cells is stimulated by high concentrations of CaCl2. Even in the presence of CaCl2, transformation occurs with a quite low frequency, and only a few cells are transformed by incorporation of a single plasmid molecule. Cells that are not transformed die on ampicillin-containing medium. Once incorporated into a host cell, a plasmid can replicate independently of the host-cell chromosome. As a transformed cell multiplies into a colony, at least one plasmid segregates to each daughter cell.
). The
inserted DNA is replicated along with the rest of the plasmid DNA and segregates
to daughter cells as the colony grows. In this way, the initial fragment of DNA
is replicated in the colony of cells into a large number of identical copies.
Since all the cells in a colony arise from a single transformed parental cell,
they constitute a clone of cells. The initial fragment of DNA inserted into the
parental plasmid is referred to as cloned DNA, since it can be
isolated from the clone of cells.
Four distinct DNA fragments, depicted in different colors, are inserted into plasmid cloning vectors, yielding a mixture of recombinant plasmids each containing a single DNA fragment. E. coli cells treated with CaCl2 are incubated with the mixture of recombinant plasmids and then plated on nutrient agar containing ampicillin. Each colony of transformed, antibiotic-resistant cells that grows (represented by a group of cells) arises from a single cell that took up one or another of the recombinant plasmids; all the cells in a given colony thus carry the same DNA fragment. Overnight incubation of E. coli at 37 °C produces visible colonies containing about a million cells. Since the colonies are separated from one another on the culture plate, copies of the DNA fragments in the original mixture are isolated in the individual colonies. Although it’s not shown here, the transformed cells contain multiple copies of a given plasmid.
). Each fragment type is individually inserted into a
plasmid vector. The resulting mixture of recombinant plasmids is incubated with
E. coli cells under conditions that facilitate
transformation; the cells then are cultured on antibiotic selective plates.
Since each colony that develops arose from a single cell that took up a single
plasmid, all the cells in a colony harbor the identical type of plasmid
characterized by the DNA fragment inserted into it. As a result, copies of the
DNA fragments in the initial mixture are isolated from one another in the
separate bacterial colonies. DNA cloning thus is a powerful, yet simple method
for purifying a particular DNA fragment from a complex mixture of fragments and
producing large numbers of the fragment of interest.
To clone specific DNA fragments in a plasmid vector, as just described, or in other vectors discussed in later sections, the fragments must be produced and then inserted into the vector DNA. As noted in the introduction, restriction enzymes and DNA ligases are utilized to produce such recombinant DNA molecules.
(a) EcoRI, a restriction enzyme from E. coli, makes staggered cuts at the specific 6-bp inverted repeat sequence shown. This cleavage yields fragments with single-stranded, complementary “sticky” ends. Many other restriction enzymes also produce fragments with sticky ends. (b) Bacterial cells with restriction endonucleases also contain corresponding modification enzymes that methylate bases in the restriction-recognition site. For example, E. coli cells containing the EcoRI restriction enzyme also contain EcoRI methylase, a modification enzyme that catalyzes addition of a methyl group to two adenines in the EcoRI recognition sequence. The methylated restriction site is not cleaved by EcoRI, assuring that a cell making this restriction enzyme does not destroy its own DNA.
Double-stranded DNA is represented by single black lines in this figure. Digestion of Ad2 DNA (center) with EcoRI generates 6 EcoRI fragments (top); these result from cleavage at each EcoRI restriction site (GAATTC) in the Ad2 sequence. Digestion with HindIII cleaves the Ad2 DNA at each HindIII site (AAGCTT), generating 11 specific fragments (bottom), all different from the EcoRI fragments. By convention, restriction fragments are labeled A – Z in order of decreasing size. By techniques described later, the order of fragments in the original DNA can be determined, thus mapping the restriction sites on the uncut DNA (indicated by short arrows). Such a “restriction-site map” for various restriction enzymes is a unique characteristic of each DNA.
, are short inverted repeat
sequences; that is, the restriction-site sequence is the same on each DNA strand
when read in the 5′ → 3′
direction. Because the DNA isolated from an individual organism has a specific
sequence, restriction enzymes cut the DNA into a reproducible set of fragments
called restriction fragments (Figure 7-6).
| Source Microorganism | Enzyme* | Recognition Site (↓)† | Ends Produced |
|---|---|---|---|
| Arthrobacter luteus | AluI | AG↓CT | Blunt |
| Bacillus amyloliquefaciens H | BamHI | G↓GATCC | Sticky |
| Escherichia coli | EcoRI | G↓AATTC | Sticky |
| Haemophilus gallinarum | HgaI | GACGC+5↓ | ‡ |
| Haemophilus influenzae | HindIII | A↓AGCTT | Sticky |
| Haemophilus parahaemolyticus | HphI | GGTGA+8↓ | ‡ |
| Nocardia otitiscaviaruns | NotI | GC↓GGCCGC | Sticky |
| Staphylococcus aureus 3A | Sau3AI | ↓GATC | Sticky |
| Serratia marcesens | SmaI | CCC↓GGG | Blunt |
| Thermus aquaticus | TaqI | T↓CGA | Sticky |
Enzymes are named with abbreviations of the bacterial strains from which they are isolated; the roman numeral indicates the enzyme’s priority of discovery in that strain (for example, AluI was the first restriction enzyme to be isolated from Arthrobacter luteus).
.The cleavage sites for HphI and HgaI occur several nucleotides away from the recognition sequence. HgaI cuts five nucleotides 3′ to the GACGC sequence on the top strand and ten nucleotides 5′ to the complementary GTGCG sequence on the bottom strand. HphI cuts eight nucleotides 3′ to the GGTGA sequence on the top strand and seven nucleotides 5′ to the complementary CCACT sequence on the bottom strand.
SOURCE: R. J. Roberts, 1988, Nucl. Acids Res. 16(suppl):271.
). Together with the
restriction endonuclease, the methylating enzyme forms a
restriction-modification system that protects the host DNA while it destroys
foreign DNA. Restriction enzymes have been purified from several hundred
different species of bacteria, allowing DNA molecules to be cut at a large
number of different sequences corresponding to the recognition sites of these
enzymes (Table 7-1).
,
EcoRI makes staggered cuts in the two DNA strands. Many
other restriction enzymes make similar cuts, generating fragments that have a
single-stranded “tail” at both ends. The tails on the
fragments generated at a given restriction site are complementary to those on
all other fragments generated by the same restriction enzyme. At room
temperature, these single-stranded regions, often called “sticky
ends,” can transiently base-pair with those on other DNA fragments
generated with the same restriction enzyme, regardless of the source of the DNA.
This base pairing of sticky ends permits DNA from widely differing species to be
ligated, forming chimeric molecules.
In this example, EcoRI fragments from DNA I (left) are mixed with several different restriction fragments, including EcoRI fragments, produced from DNA II (right). The short DNA sequences composing the sticky ends of each fragment type are shown. The complementary sticky ends on the two types of EcoRI fragments, (a′) and (a), can transiently base-pair, whereas the TaqI fragments (b) and HindIII fragments (c) with noncomplementary sticky ends do not base-pair to EcoRI fragments. The adjacent 3′-hydroxyl and 5′-phosphate groups (red) on the base-paired fragments then are covalently joined (ligated) by T4 DNA ligase. One ATP is consumed for each phosphodiester bond (red) formed.
). When DNA
ligase and ATP are added to a solution containing restriction fragments with
sticky ends, the restriction fragments are covalently ligated together through
the standard 3′ → 5′
phosphodiester bonds of DNA.
(a) Sequence of a polylinker that includes one copy of the recognition site, indicated by brackets, for each of the 10 restriction enzymes indicated. Polylinkers are chemically synthesized and then are inserted into a plasmid vector. Only one strand is shown. (b) Insertion of genomic restriction fragments into the pUC19 plasmid vector, which contains the polylinker shown in (a). (The length of the polylinker in relation to the rest of the plasmid is greatly exaggerated here.) One of the restriction enzymes whose recognition site is in the polylinker is used to cut both the plasmid molecules and genomic DNA, generating singly-cut plasmids and restriction fragments with complementary sticky ends (letters at ends of green fragments). By use of appropriate reaction conditions, insertion of a single restriction fragment per plasmid can be maximized. Note that the restriction sites are reconstituted in the recombinant plasmid. [See C. Yanisch-Perron, J. Vieira, and J. Messing, 1985, Gene 33:103.]
). When
such a vector is treated with a restriction enzyme that recognizes a recognition
sequence in the polylinker, it is cut at that sequence, generating sticky ends.
In the presence of DNA ligase, DNA fragments produced with the same restriction
enzyme will be inserted into the plasmid (Figure 7-8b). The ratio of DNA fragments to be inserted to cut
vectors and other reaction conditions are chosen to maximize the insertion of
one restriction fragment per plasmid vector. The recombinant plasmids produced
in in vitro ligation reactions then can be used to transform
antibiotic-sensitive E. coli cells as shown in Figure 7-4. All the cells in each
antibiotic-resistant clone that remains after selection contain plasmids with
the same inserted DNA fragment, but different clones carry different
fragments.
was chemically synthesized and
then inserted into plasmid vectors to facilitate the cloning of fragments
generated by different restriction enzymes. This example illustrates the use of
synthetic DNAs to add convenient restriction sites where they otherwise do not
occur. As described later in the chapter, synthetic DNAs are used in sequencing
DNA and as probes to identify clones of interest. Synthetic DNAs also can be
substituted for natural DNA sequences in cloned DNA to study the effects of
specific mutations; this topic is examined in Chapter 8.
The first nucleotide (monomer 1) is bound to a glass support by its 3′ hydroxyl; its 5′ hydroxyl is available for addition of the second nucleotide. The second nucleotide in the sequence (monomer 2) is derivatized by addition of 4′,4′-dimethoxytrityl (DMT) to its 5′ hydroxyl, thus blocking this hydroxyl from reacting; in addition, a highly reactive methylated diisopropyl phosphoramidite group (red letters) is attached to the 3′ hydroxyl. When the two monomers are mixed in the presence of a weak acid, they form a 5′ → 3′ phosphodiester bond with the phosphorus in the trivalent state. Oxidation of this intermediate with iodine (I2) increases the phosphorus valency to 5, and subsequent removal of the DMT group by detritylation with zinc bromide (ZnBr2) frees the 5′ hydroxyl. Monomer 3 then is added, and the reactions are repeated. Repetition of this process eventually yields the entire oligonucleotide. Finally, all the methyl groups on the phosphates are removed at the same time at alkaline pH, and the bond linking monomer 1 to the glass support is cleaved. [See S. L. Beaucage and M. H. Caruthers, 1981, Tetrahedron Lett. 22:1859.]
. Note that chains grow in the
3′ → 5′ direction,
opposite to the direction of DNA chain growth catalyzed by DNA polymerases. Once
the chemistry for producing synthetic DNA was standardized, automated
instruments were developed that allow researchers to program the synthesis of
oligonucleotides of specific sequences up to about 100 nucleotides long.
In DNA cloning, recombinant DNA molecules are formed in vitro by inserting DNA fragments of interest into vector DNA molecules. The recombinant DNA molecules are then introduced into host cells, where they replicate, producing large numbers of recombinant DNA molecules that include the fragment of DNA originally linked to the vector.
The most commonly used cloning vectors are E. coli plasmids, small circular DNA molecules that include three functional regions: (1) an origin of replication, (2) a drug-resistance gene, and (3) a region where DNA can be inserted without interfering with plasmid replication or expression of the drug-resistance gene.
).
). All the cells in each colony that
grows on this medium contain identical plasmids descended from the
single plasmid that entered the founder cell of the colony. Isolated
colonies thus represent clones of the different restriction fragments
originally inserted into the plasmid vector.Polylinkers are synthetic oligonucleotides composed of one copy of several different restriction sites. Plasmid vectors that contain a polylinker will be cut only once by multiple restriction enzymes, each acting at its own site. Inclusion of a polylinker in a plasmid vector thus permits cloning of restriction fragments generated by cleavage of DNA with multiple different restriction enzymes.
Single-stranded DNA containing up to 100 nucleotides of any desired sequence can be chemically synthesized using automated instruments. Synthetic dsDNAs are produced by synthesizing complementary ssDNAs and then hybridizing them.
