Generation of DNA Fragments

Although fragments of DNA can be produced by mechanical shearing techniques, this is a haphazard process producing fragments that vary in size. In the early 1970s, it was recognized that certain microbes contain enzymes that cleave double-stranded DNA in or near a particular sequence of nucleotides. These enzymes restrict the entry of foreign DNA into bacterial cells and were therefore called restriction enzymes. They recognize a palindromic nucleotide sequence of DNA of between four and eight nucleotides in length (i.e., the same sequence of nucleotides occurring on the two complementary DNA strands when read in one direction of polarity, e.g., 5′ to 3′) (Table 4.2). The longer the nucleotide recognition sequence of the restriction enzyme, the less frequently that particular nucleotide sequence will occur by chance and therefore the larger the average size of the DNA fragments generated.

More than 300 different restriction enzymes have been isolated from various bacterial organisms. Restriction endonucleases are named according to the organism from which they are derived (e.g., EcoRI is from Escherichia coli and was the first restriction enzyme isolated from that organism).

The complementary pairing of bases in the DNA molecule means that cleavage of double-stranded DNA by a restriction endonuclease always creates double-stranded breaks, which, depending on the cleavage points of the particular restriction enzyme used, results in either a staggered or a blunt end (Figure 4.2).

FIGURE 4.2 The staggered and blunt ends generated by restriction digest of double-stranded DNA by EcoRI and SmaI. Sites of cleavage of the DNA strands are indicated by arrows.

Digestion of DNA from a specific source with a particular restriction enzyme will produce the same reproducible collection of DNA fragments each time the process is carried out.

Recombination of DNA Fragments

DNA from any source, when digested with the same restriction enzyme, will produce DNA fragments with identical complementary ends or termini. When DNA has been cleaved by a restriction enzyme that produces staggered termini, these are referred to as being ‘sticky’ or ‘cohesive’ because they will unite under appropriate conditions with complementary sequences produced by the same restriction enzyme on DNA from any source. Initially the cohesive termini are held together by hydrogen bonding but are covalently attached with the enzyme called DNA ligase. The union of two DNA fragments from different sources produces what is referred to as a recombinant DNA molecule.

Vectors

A vector is the term for the carrier DNA molecule used in the cloning process that, through its own independent replication within a host organism, will allow the production of multiple copies of itself. The incorporation of the target DNA into a vector allows the production of large amounts of that DNA fragment.

For naturally occurring vectors to be used for DNA cloning, they need to be modified to ensure that the target DNA is inserted at a specific location and that recombinant vectors containing target inserted DNA can be detected. Many of the early vectors were constructed so that insertion of the target DNA in a gene for antibiotic resistance resulted in loss of that function (Figure 4.3).

FIGURE 4.3 Two plasmids originally used in recombinant DNA technology showing drug resistance genes (Ap^r, ampicillin resistance; Tc^r, tetracycline resistance) and cleavage sites of restriction endonucleases that are present in the DNA only once for use as a cloning site.

The five main types of vector commonly used include plasmids, bacteriophages, cosmids, and bacterial and yeast artificial chromosomes (BACs and YACs). The choice of vector used in cloning depends on a number of factors, such as the particular restriction enzyme being used and the size of the target DNA to be inserted. Some of the early vectors, such as plasmids and bacteriophages, were very limited in terms of the size of the target DNA fragment that could be inserted. Later generations of vectors, such as cosmids, can take inserts up to approximately 50 kb in size. A cosmid is essentially a plasmid that has had all but the minimum vector DNA necessary for propagation removed (i.e., the cos sequence), to enable insertion of the largest possible foreign DNA fragment and still allow replication.

The development of BACs and YACs allows the possibility of cloning DNA fragments of between 300 kb and 1000 kb in size. YACs consist of a plasmid that contains within it the minimum DNA sequences necessary for centromere and telomere formation plus DNA sequences known as autonomous replication sequences, all of which are necessary for accurate replication within yeast. YACs have the advantage that they can incorporate DNA fragments of up to 1000 kb in size as well as allow replication of eukaryotic DNA with repetitive DNA sequences, which often cannot take place in bacterial cells. Many eukaryotic genes are very large, being up to 2 to 3 million base pairs (bp) in length (p. 388). YACs allow detailed mapping of genes of this size and their flanking regions, whereas the use of conventional vectors would require an inordinate number of overlapping clones.

Transformation of the Host Organism

After introducing the target DNA fragment into the vector, the recombinant vector is introduced into specially modified bacterial or yeast host cells. The bacterial cell membrane is not normally permeable to large molecules such as DNA fragments but can be made permeable by a variety of different methods, including exposure to certain salts or high voltage; this is known as becoming competent. Usually only a single DNA molecule is taken up by a host cell undergoing the process known as transformation. If the transformed cells are allowed to multiply, large quantities of identical copies of the original single target DNA or clones will be produced (Figure 4.4).

FIGURE 4.4 Generation of a recombinant plasmid using EcoRI and transformation of the host bacterial organism.

(From Emery AE 1981 Recombinant DNA technology. Lancet ii:1406–1409, with permission.)

Screening for Recombinant Vectors

After the transformed cells have multiplied in culture medium, they are plated out on a master plate of nutrient agar in a Petri dish. Recombinant vectors can be screened for by a detection system; for example, loss of antibiotic resistance can be screened for by replica plating on agar containing the appropriate antibiotic (see Figure 4.3). Thus, if the enzyme PstI were used to generate DNA fragments and to cut the plasmid pBR322, any recombinant plasmids produced would make the bacterial host cells they transform sensitive to ampicillin, as this gene would no longer be functional, but they would remain resistant to tetracycline. Replica plating of the master plates from the cultures allows identification of individual specific recombinant clones.

Selection of Specific Clones

Several techniques have been developed to detect the presence of clones with specific DNA sequence inserts. The most widely used method is nucleic acid hybridization (p. 57). Colonies of transformed host bacteria with recombinant clones are used to make replica plates that are lyzed and then blotted on to a nitrocellulose filter to which nucleic acid binds. The DNA of the replica blot is then denatured to make the DNA single stranded, which will allow it to hybridize with single-stranded, radioactively labeled DNA or RNA probes (p. 58), which can then be detected by exposure to an x-ray film, or what is known as autoradiography. In this way, a transformed host bacterial colony containing a sequence complementary to the probe can be detected and, from its position on the replica plate, the colony containing that clone can be identified on the master plate, ‘picked’, and cultured separately (Figure 4.5).

FIGURE 4.5 Identification of recombinant DNA clones with specific DNA inserts by loss of antibiotic resistance, nucleic acid hybridization, and autoradiography.

The PCR

DNA sequence information is used to design two oligonucleotide primers (amplimers) of approximately 20 bp in length complementary to the DNA sequences flanking the target DNA fragment. The first step is to denature the double-stranded DNA by heating. The primers then bind to the complementary DNA sequences of the single-stranded DNA templates. DNA polymerase extends the primer DNA in the presence of the deoxynucleotide triphosphates (dATP, dCTP, dGTP, and dTTP) to synthesize the complementary DNA sequence. Subsequent heat denaturation of the double-stranded DNA, followed by annealing of the same primer sequences to the resulting single-stranded DNA, will result in the synthesis of further copies of the target DNA. Some 30 to 35 successive repeated cycles results in more than 1 million copies (amplicons) of the DNA target, sufficient for direct visualization by ultraviolet fluorescence after ethidium bromide staining, without the need to use indirect detection techniques (Figure 4.6).

FIGURE 4.6 Diagram of the polymerase chain reaction showing serial denaturation of DNA, primer annealing, and extension with doubling of the target DNA fragment numbers in each cycle.

PCR allows analysis of DNA from any cellular source containing nuclei; in addition to blood, this can include less invasive samples such as buccal scrapings or pathological archival material. It is also possible to start with quantities of DNA as small as that from a single cell, as is the case in preimplantation genetic diagnosis (p. 335). Great care has to be taken with PCR, however, because DNA from a contaminating extraneous source, such as desquamated skin from a laboratory worker, will also be amplified. This can lead to false-positive results unless the appropriate control studies are used to detect this possible source of error.

Another advantage of PCR is the rapid turnaround time of samples for analysis. Use of the heat-stable Taq DNA polymerase isolated from the bacterium Thermophilus aquaticus, which grows naturally in hot springs, generates PCR products in a matter of hours rather than the days or weeks required for cell-based in-vivo DNA cloning techniques.

Real-time PCR machines have reduced this time to less than 1 hour, and fluorescence technology is used to monitor the generation of PCR products during each cycle, thus eliminating the need for gel electrophoresis.

DNA cloning by PCR, in contrast to in-vivo cell-based techniques, has the disadvantage that it requires knowledge of the nucleotide sequence of the target DNA fragment and is best used to amplify DNA fragments of up to 1 kb, although long-range PCR allows the amplification of larger DNA fragments of up to 20 kb to 30 kb.