Figure 4.1
.Examples of the manipulations that can be carried out with DNA molecules
 |
The toolkit of techniques used by molecular biologists to study DNA molecules was assembled during the 1970s and 1980s. Before then, the only way in which individual genes could be studied was by classical genetics, using the procedures that we will examine in Chapter 5. Classical genetics is a powerful approach to gene analysis and many of the fundamental discoveries in molecular biology were made in this way. The operon theory proposed by Jacob and Monod in 1961 (Section 9.3.1), which describes how the expression of some bacterial genes is regulated, was perhaps the most heroic achievement of this era of genetics. But the classical approach is limited because it does not involve the direct examination of genes, information on gene structure and activity being inferred from the biological characteristics of the organism being studied. By the late 1960s these indirect methods had become insufficient for answering the more detailed questions that molecular biologists had begun to ask about the expression pathways of individual genes. These questions could only be addressed by examining directly the segments of DNA containing the genes of interest. This was not possible using the current technology, so a new set of techniques had to be invented.
In this example, the fragment of DNA to be cloned is inserted into a plasmid vector which is subsequently replicated inside a bacterial host.
). These manipulations, which are described in Section 4.1, form the basis of recombinant DNA technology, in which new or ‘recombinant’ DNA molecules are constructed in the test tube from pieces of naturally occurring chromosomes and plasmids. Recombinant DNA methodology led to the development of DNA or gene cloning, in which short DNA fragments, possibly containing a single gene, are inserted into a plasmid or virus chromosome and then replicated in a bacterial or eukaryotic host (Figure 4.2). We will examine exactly how gene cloning is performed, and the reasons why this technique resulted in a revolution in molecular biology, in Section 4.2.
In this example, a single gene is copied.
) - but it has become immensely important in many areas of biological research, not least the study of genomes. PCR is covered in detail in Section 4.3.
Recombinant DNA technology was one of the main factors that contributed to the rapid advance in knowledge concerning gene expression that occurred during the 1970s and 1980s. The basis of recombinant DNA technology is the ability to manipulate DNA molecules in the test tube. This, in turn, depends on the availability of purified enzymes whose activities are known and can be controlled, and which can therefore be used to make specified changes to the DNA molecules that are being manipulated. The enzymes available to the molecular biologist fall into four broad categories:
In (A), the activity of a DNA-dependent DNA polymerase is shown on the left and that of an RNA-dependent DNA polymerase on the right. In (B), the activities of endonucleases and exonucleases are shown. In (C) the red DNA molecule is ligated to itself on the left, and to a second molecule on the right.
);
);
);End-modification enzymes (Section 4.1.4), which make changes to the ends of DNA molecules, adding an important dimension to the design of ligation experiments, and providing one means of labeling DNA molecules with radioactive and other markers (Technical Note 4.1).
New nucleotides are added on to the 3′ end of the growing polynucleotide, the sequence of this new polynucleotide being determined by the sequence of the template DNA.
). The mode of action is very similar to that of a template-dependent RNA polymerase (Section 3.2.2), the new polynucleotide being synthesized in the 5′→3′ direction: DNA polymerases that make DNA in the other direction are unknown in nature.
(A) A DNA polymerase requires a primer in order to initiate the synthesis of a new polynucleotide. (B) The sequence of this oligonucleotide determines the position at which it attaches to the template DNA and hence specifies the region of the template that will be copied. When a DNA polymerase is used to make new DNA in vitro, the primer is usually a short oligonucleotide made by chemical synthesis. For details of how DNA synthesis is primed in vivo, see Figure 13.12).
). The way in which this requirement is met in living cells when the genome is replicated is described in Chapter 13. In the test tube, a DNA copying reaction is initiated by attaching to the template a short synthetic oligonucleotide, usually about 20 nucleotides in length, which acts as a primer for DNA synthesis. At first glance, the need for a primer might appear to be an undesired complication in the use of DNA polymerases in recombinant DNA technology, but nothing could be further from the truth. Because annealing of the primer to the template depends on complementary base-pairing, the position within the template molecule at which DNA copying is initiated can be specified by synthesizing a primer with the appropriate nucleotide sequence (Figure 4.6B). A short specific segment of a much longer template molecule can therefore be copied, which is much more valuable than the random copying that would occur if DNA synthesis did not need to be primed. You will fully appreciate the importance of priming when we deal with PCR in Section 4.3.
All DNA polymerases can make DNA and many also have one or both of the exonuclease activities.
):
A 3′→5′ exonuclease activity enables the enzyme to remove nucleotides from the 3′ end of the strand that it has just synthesized. This is called the proofreading activity because it allows the polymerase to correct errors by removing a nucleotide that has been inserted incorrectly.
A 5′→3′ exonuclease activity is less common, but is possessed by some DNA polymerases whose natural function in genome replication requires that they must be able to remove at least part of a polynucleotide that is already attached to the template strand that the polymerase is copying.
| Polymerase | Description | Main use | Cross reference |
|---|---|---|---|
| DNA polymerase I | Unmodified E. coli enzyme | DNA labeling | Technical Note 4.1 |
| Klenow polymerase | Modified version of E. coli DNA polymerase I | DNA labeling | Technical Note 4.1 |
| Sequenase | Modified version of phage T7 DNA polymerase I | DNA sequencing | Box 6.1 |
| Taq polymerase | Thermus aquaticus DNA polymerase I | PCR | Section 4.3 |
| Reverse transcriptase | RNA-dependent DNA polymerase, obtained from various retroviruses | cDNA synthesis | Section 5.3.3 |
Of the two exonuclease activities, it is the 5′→3′ version that causes most problems when a DNA polymerase is used to manipulate molecules in the test tube. This is because an enzyme that possesses this activity is able to remove nucleotides from the 5′ ends of polynucleotides that have just been synthesized (Figure 4.8
). It is unlikely that the polynucleotides will be completely degraded, because the polymerase function is usually much more active than the exonuclease, but some techniques will not work if the 5′ ends of the new polynucleotides are shortened in any way. In particular, DNA sequencing is based on synthesis of new polynucleotides, all of which share exactly the same 5′ end, marked by the primer used to initiate the sequencing reactions. If any nibbling of the 5′ ends occurs, then it is impossible to determine the correct DNA sequence. When DNA sequencing was first introduced in the late 1970s, it made use of a modified version of the Kornberg enzyme called the Klenow polymerase. The Klenow polymerase was initially prepared by cutting the natural E. coli DNA polymerase I enzyme into two segments with a protease. One of these segments retained the polymerase and 3′→5′ exonuclease activities, but lacked the 5′→3′ exonuclease of the untreated enzyme. The enzyme is still often called the Klenow fragment in memory of this old method of preparation, but nowadays it is almost always prepared from E. coli cells whose polymerase gene has been engineered so that the resulting enzyme has the desired properties. But in fact the Klenow polymerase is now rarely used in sequencing and has its major application in DNA labeling (see Technical Note 4.1). This is because an enzyme called Sequenase (see Table 4.1), which has superior properties as far a sequencing is concerned, was developed in the 1980s. We will return to the features of Sequenase, and why they make the enzyme ideal for sequencing, in Box 6.1
The E. coli DNA polymerase I enzyme has an optimum reaction temperature of 37 °C, this being the usual temperature of the natural environment of the bacterium, inside the intestines of mammals such as humans. Test-tube reactions with either the Kornberg or Klenow polymerases, and with Sequenase, are therefore incubated at 37 °C, and terminated by raising the temperature to 75 °C or above, which causes the protein to unfold or denature, destroying its activity. This regimen is perfectly adequate for most molecular biology techniques but, for reasons that will become clear in Section 4.3, PCR requires a thermostable DNA polymerase - one that is able to function at temperatures much higher than 37 °C. Suitable enzymes can be obtained from bacteria such as Thermus aquaticus, which live in hot springs at temperatures up to 95 °C, and whose DNA polymerase I enzyme has an optimum working temperature of 72 °C. The biochemical basis of protein thermostability is not fully understood, but probably centers on structural features that reduce the amount of protein unfolding that occurs at elevated temperatures.
One additional type of DNA polymerase is important in molecular biology research. This is reverse transcriptase, which is an RNA-dependent DNA polymerase and so makes DNA copies of RNA rather than DNA templates. Reverse transcriptases are involved in the replication cycles of retroviruses (Section 2.4.2), including the human immunodeficiency viruses that cause AIDS, these having RNA genomes that are copied into DNA after infection of the host. In the test tube, a reverse transcriptase can be used to make DNA copies of mRNA molecules. These copies are called complementary DNAs (cDNAs). Their synthesis is important in some types of gene cloning and in techniques used to map the regions of a genome that specify particular mRNAs (Section 7.1.2).
| Nuclease | Description | Main use | Cross reference |
|---|---|---|---|
| Restriction endonucleases | Sequence-specific DNA endonucleases, from many sources | Many applications | Section 4.1.2 |
| S1 nuclease | Endonuclease specific for single-stranded DNA and RNA, from the fungus Aspergillus oryzae | Transcript mapping | Section 7.1.2 |
| Deoxyribonuclease I | Endonuclease specific for double-stranded DNA and RNA, from Escherichia coli | Nuclease footprinting | Section 9.1.1 |
A restriction endonuclease is an enzyme that binds to a DNA molecule at a specific sequence and makes a double-stranded cut at or near that sequence. Because of the sequence specificity, the positions of cuts within a DNA molecule can be predicted, assuming that the DNA sequence is known, enabling defined segments to be excised from a larger molecule. This ability underlies gene cloning and all other aspects of recombinant DNA technology in which DNA fragments of known sequence are required.
In the top part of the diagram, the DNA is cut by a Type I or Type III restriction endonuclease. The cuts are made in slightly different positions relative to the recognition sequence, so the resulting fragments have different lengths. In the lower part, a Type II enzyme is used. Each molecule is cut at exactly the same position to give exactly the same pair of fragments.
| Enzyme | Recognition sequence | Type of ends | End sequences |
|---|---|---|---|
| AluI | 5′-AGCT-3′ | Blunt | 5′-AG CT-3′ |
| 3′-TCGA-5′ | 3′-TC GA-5′ | ||
| Sau3AI | 5′-GATC-3′ | Sticky, 5′ overhang | 5′- GATC-3′ |
| 3′-CTAG-5′ | 3′-CTAG -5′ | ||
| HinfI | 5′-GANTC-3′ | Sticky, 5′ overhang | 5′-G ANTC-3′ |
| 3′-CTNAG-5′ | 3′-CTNA G-5′ | ||
| BamHI | 5′-GGATCC-3′ | Sticky, 5′ overhang | 5′-G GATCC-3′ |
| 3′-CCTAGG-5′ | 3′-CCTAG G-5′ | ||
| BsrBI | 5′-CCGCTC-3′ | Blunt | 5′- NNNCCGCTC-3′ |
| 3′-GGCGAG-5′ | 3′- NNNGGCGAG-5′ | ||
| EcoRI | 5′-GAATTC-3′ | Sticky, 5′ overhang | 5′-G AATTC-3′ |
| 3′-CTTAAG-5′ | 3′-CTTAA G-5′ | ||
| PstI | 5′-CTGCAG-3′ | Sticky, 3′ overhang | 5′-CTGCA G-3′ |
| 3′-GACGTC-5′ | 3′-G ACGTC-5′ | ||
| NotI | 5′-GCGGCCGC-3′ | Sticky, 5′ overhang | 5′-GC GGCCGC-3′ |
| 3′-CGCCGGCG-5′ | 3′-CGCCGG CG-5′ | ||
| BglI | 5′-GCCNNNNNGGC-3′ | Sticky, 3′ overhang | 5′-GCCNNNN NGGC-3′ |
| 3′-CGGNNNNNCCG-5′ | 3′-CGGN NNNNCCG-5′ |
N = any nucleotide.
Note that most, but not all, recognition sequences have inverted symmetry: when read in the 5′→3′ direction, the sequence is the same in both strands.
). For example, the Type II enzyme called EcoRI (isolated from E. coli) cuts DNA only at the hexanucleotide 5′-GAATTC-3′. Digestion of DNA with a Type II enzyme therefore gives a reproducible set of fragments whose sequences are predictable if the sequence of the target DNA molecule is known. Over 2500 Type II enzymes have been isolated and more than 300 are available for use in the laboratory (Brown, 1998). Many enzymes have hexanucleotide target sites, but others recognize shorter or longer sequences (Table 4.3). There are also examples of enzymes with degenerate recognition sequences, meaning that they cut DNA at any of a family of related sites. HinfI (from Haemophilus influenzae), for example, recognizes 5′-GANTC-3′, where ‘N’ is any nucleotide, and so cuts at 5′-GAATC-3′, 5′-GATTC-3′, 5′-GAGTC-3′ and 5′-GACTC-3′. Most enzymes cut within the recognition sequence, but a few, such a BsrBI, cut at a specified position outside of this sequence.
(A) Blunt ends and sticky ends. (B) Different types of sticky end: the 5′ overhangs produced by BamHI and the 3′ overhangs produced by PstI. (C) The same sticky ends produced by two different restriction endonucleases: a 5′ overhang with the sequence 5′-GATC-3′ is produced by both BamHI (recognizes 5′-GGATCC-3′) and Sau3AI (recognizes 5′-GATC-3′).
). Some sticky-end cutters give 5′ overhangs (e.g. Sau3AI, HinfI) whereas others leave 3′ overhangs (e.g. PstI) (Figure 4.10B). One feature that is particularly important in recombinant DNA technology is that some pairs of restriction enzymes have different recognition sequences but give the same sticky ends, examples being Sau3AI and BamHI, which both give a 5′-GATC-3′ sticky end even though Sau3AI has a 4-bp recognition sequence and BamHI recognizes a 6-bp sequence (Figure 4.10C).
The range of fragment sizes that can be resolved depends on the concentration of agarose in the gel. Electrophoresis has been performed with three different concentrations of agarose. The labels indicate the sizes of bands in the left and right lanes. Photo courtesy of BioWhittaker Molecular Applications.
). Fragments less than 150 bp can be separated in a 4% or 5% agarose gel, making it possible to distinguish bands representing molecules that differ in size by just a single nucleotide. With larger fragments, however, it is not always possible to separate molecules of similar size, even in gels of lower agarose concentration. If the starting DNA is long, and so gives rise to many fragments after digestion with a restriction enzyme, then the gel may simply show a smear of DNA because there are fragments of every possible length that all merge together. This is the usual result when genomic DNA is restricted.
(A) Transfer of DNA from the gel to the membrane. (B) The membrane is probed with a radioactively labeled DNA molecule. On the resulting autoradiograph, one hybridizing band is seen in lane 2, and two in lane 3.
). This process results in the DNA bands becoming immobilized in the same relative positions on the surface of the membrane.
the probe is radioactively labeled and the signal is detected by autoradiography. The band that is seen on the autoradiograph is the one that corresponds to the restriction fragment that hybridizes to the probe and which therefore contains the gene that we are searching for.DNA fragments that have been generated by treatment with a restriction endonuclease can be joined back together again, or attached to a new partner, by a DNA ligase. The reaction requires energy, which is provided by adding either ATP or NAD to the reaction mixture, depending on the type of ligase that is being used.
(A) In living cells, DNA ligase synthesizes a missing phosphodiester bond in one strand of a double-stranded DNA molecule. (B) To link two DNA molecules in vitro, DNA ligase must make two phosphodiester bonds, one in each strand. (C) Ligation in vitro is more efficient when the molecules have compatible sticky ends, because transient base-pairing between these ends holds the molecules together and so increases the opportunity for DNA ligase to attach and synthesize the new phosphodiester bonds. For the role of DNA ligase during DNA replication in vivo, see Figures 13.17 and 13.19.
). In order to join together two restriction fragments, the ligase has to synthesize two phosphodiester bonds, one in each strand (Figure 4.13B). This is by no means beyond the capabilities of the enzyme, but the reaction can occur only if the ends to be joined come close enough to one another by chance - the ligase is not able to catch hold of them and bring them together. If the two molecules have complementary sticky ends, and the ends come together by random diffusion events in the ligation mixture, then transient base pairs might form between the two overhangs. These base pairs are not particularly stable but they may persist for sufficient time for a ligase enzyme to attach to the junction and synthesize phosphodiester bonds to fuse the ends together (Figure 4.13C). If the molecules are blunt ended, then they cannot base-pair to one another, not even temporarily, and ligation is a much less efficient process, even when the DNA concentration is high and pairs of ends are in relatively close proximity.
In this example, each linker contains the recognition sequence for the BamHI restriction endonuclease. DNA ligase attaches the linkers to the ends of the blunt-ended molecule in a reaction that is made relatively efficient because the linkers are present at a high concentration. The restriction enzyme is then added to cleave the linkers and produce the sticky ends. Note that during the ligation the linkers ligate to one another, so a series of linkers (a concatamer) is attached to each end of the blunt molecule. When the restriction enzyme is added, these linker concatamers are cut into segments, with half of the innermost linker left attached to the DNA molecule. Adaptors are similar to linkers but each one has one blunt end and one sticky end. The blunt-ended DNA is therefore given sticky ends simply by ligating it to the adaptors; there is no need to carry out the restriction step. For more details, see Brown (2001).
In this example, a poly(G) tail is synthesized at each end of a blunt-ended DNA molecule. Tails comprising other nucleotides are synthesized by including the appropriate dNTP in the reaction mixture.
). Another way to create a sticky end is by homopolymer tailing, in which nucleotides are added one after the other to the 3′ terminus at a blunt end (Figure 4.15). The enzyme involved is called terminal deoxynucleotidyl transferase, which we will meet in the next section. If the reaction contains the DNA, enzyme, and only one of the four nucleotides, then the new stretch of single-stranded DNA that is made consists entirely of just that single nucleotide. It could, for example, be a poly(G) tail, which would enable the molecule to base-pair to other molecules that carry poly(C) tails, created in the same way but with dCTP rather than dGTP in the reaction mixture.
), obtained from calf thymus tissue, is one example of an end-modification enzyme. It is, in fact, a template- independent DNA polymerase, because it is able to synthesize a new DNA polynucleotide without base-pairing of the incoming nucleotides to an existing strand of DNA or RNA. Its main role in recombinant DNA technology is in homopolymer tailing, as described above.
See the text for details.
). Imagine also that a small E. coli plasmid has been purified and treated with BamHI, which cuts the plasmid in a single position. The circular plasmid has therefore been converted into a linear molecule, again with 5′-GATC-3′ sticky ends. Mix the two DNA molecules together and add DNA ligase. Various recombinant ligation products will be obtained, one of which comprises the circularized plasmid with the animal gene inserted into the position originally taken by the BamHI restriction site. If the recombinant plasmid is now re-introduced into E. coli, and the inserted gene has not disrupted its replicative ability, then the plasmid plus inserted gene will be replicated and copies passed to the daughter bacteria after cell division. More rounds of plasmid replication and cell division will result in a colony of recombinant E. coli bacteria, each bacterium containing multiple copies of the animal gene. This series of events, as illustrated in Figure 4.16, constitutes the process called DNA or gene cloning.
, the plasmid acts as a cloning vector, providing the replicative ability that enables the cloned gene to be propagated inside the host cell. Plasmids replicate efficiently in bacterial hosts because each plasmid possesses an origin of replication which is recognized by the DNA polymerases and other proteins that normally replicate the bacterium's chromosomes (Section 13.2.1). The host cell's replicative machinery therefore propagates the plasmid, plus any new genes that have been inserted into it. Bacteriophage genomes can also be used as cloning vectors because they too possess origins of replication that enable them to be propagated inside bacteria, either by the host enzymes or by DNA polymerases and other proteins specified by phage genes. The next two sections describe how plasmid and phage vectors are used to clone DNA in E. coli.
Plasmids are uncommon in eukaryotes, although Saccharomyces cerevisiae possesses one that is sometimes used for cloning purposes; most eukaryotic vectors are therefore based on virus genomes. Alternatively, with a eukaryotic host the replication requirement can be bypassed by performing the experiment in such a way that the DNA to be cloned becomes inserted into one of the host chromosomes. These approaches to cloning in eukaryotic cells are described later in the chapter.
The easiest way to understand how a cloning vector is used is to start with the simplest E. coli plasmid vectors, which illustrate all of the basic principles of DNA cloning. We will then be able to turn our attention to the special features of phage vectors and vectors used with eukaryotes.
The map shows the positions of the ampicillin-resistance gene (amp R), the tetracycline-resistance gene (tet R), the origin of replication (ori) and the recognition sequences for seven restriction endonucleases.
). This means that cells containing a pBR322 plasmid can be distinguished from those that do not by plating the bacteria onto agar medium containing ampicillin and/or tetracycline. Normal E. coli cells are sensitive to these antibiotics and cannot grow when either of the two antibiotics is present. Ampicillin and tetracycline resistance are therefore selectable markers for pBR322.
The manipulations shown in Figure 4.16
, resulting in construction of a recombinant plasmid, are carried out in the test tube with purified DNA. Pure pBR322 DNA can be obtained quite easily from extracts of bacterial cells (Technical Note 4.2), but how can the manipulated plasmids be re-introduced into the bacteria? The answer is to make use of the natural processes for transformation of bacteria, which result in the uptake of ‘naked’ DNA by a bacterial cell. This is the process studied by Avery and his colleagues in the experiments which showed that bacterial genes are made of DNA (Section 1.1.1). Transformation is not a particularly efficient process in many bacteria, including E. coli, but the rate of uptake can be enhanced significantly by suspending the cells in calcium chloride before adding the DNA, and then briefly incubating the mixture at 42 °C. Even after this enhancement, only a very small proportion of the cells take up a plasmid. This is why the antibiotic-resistance markers are so important - they allow the small number of transformants to be selected from the large background of non-transformed cells.
See text for details. The inset shows how replica plating is performed.
indicates the positions of the recognition sequences for seven restriction enzymes, each of which cuts the plasmid at just one location. Note that six of these sites lie within one or other of the genes for antibiotic resistance. This means that if a new fragment of DNA is ligated into one of these six sites, then the antibiotic-resistance properties of the plasmid become altered - the plasmid loses the ability to confer either ampicillin or tetracycline resistance on the host cells. This is called insertional inactivation of the selectable marker and is the key to distinguishing a recombinant plasmid - one that contains an inserted piece of DNA - from a non-recombinant plasmid that has no new DNA. Identifying recombinants is important because the manipulations illustrated in Figure 4.16 result in a variety of ligation products, including plasmids that have recircularized without insertion of new DNA. To identify recombinants, the resistance properties of colonies are tested by transferring cells from agar containing one antibiotic onto agar containing the second antibiotic. For example, if the BamHI site has been used then recombinants will be ampicillin resistant but tetracycline sensitive, because the BamHI site lies within the region that specifies resistance to tetracycline. After transformation, cells are plated onto ampicillin agar (Figure 4.18). All cells that contain a pBR322 plasmid, whether recombinant or not, divide and produce a colony. The colonies are then transferred onto tetracycline agar by replica plating, which results in the colonies on the second plate retaining the relative positions that they had on the first plate. Some colonies do not grow on the tetracycline agar because their cells contain recombinant pBR322 molecules with a disrupted tetracycline-resistance gene. These are the colonies we are looking for because they contain the cloned gene, so we return to the ampicillin plate, from which samples of the cells can be recovered.
See the text for details.
; Vieira and Messing, 1982). This vector carries the ampicillin-resistance gene from pBR322, along with a second gene, called lacZ′, which is part of the E. coli gene for the enzyme β-galactosidase. The remainder of the lacZ gene is located in the chromosome of the special E. coli strain that is used when cloning genes with pUC8. The proteins specified by the gene segments on the plasmid and on the chromosome are able to combine to produce a functional β-galactosidase enzyme. The presence of functional β-galactosidase molecules in the cells can be checked by a histochemical test with a compound called X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside), which the enzyme converts into a blue product. The lacZ′ gene contains a cluster of unique restriction sites; insertion of new DNA into any one of these sites results in insertional inactivation of the gene and hence loss of β-galactosidase activity. Recombinants and non-recombinants can therefore be distinguished simply by plating the transformed cells onto agar containing ampicillin and X-gal (Figure 4.19). All colonies that grow on this medium are made up of transformed cells because only transformants are ampicillin resistant. Some colonies are blue and some are white. Those that are blue contain cells with functional β-galactosidase enzymes and hence with undisrupted lacZ′ genes; these colonies are therefore non-recombinants. The white colonies comprise cells without β-galactosidase activity and hence with disrupted lacZ′ genes; these are recombinants.
Compare with the lytic infection cycle of T2 bacteriophage, shown in Figure 1.4B. The special feature of the lysogenic cycle is the insertion of the phage genome into the bacterium's chromosomal DNA, where it can remain quiescent for many generations.
).
(A) In the λ genome, the genes are arranged into functional groups. For example, the region marked as ‘protein coat’ comprises 21 genes coding for proteins that are either components of the phage capsid or are required for capsid assembly, and ‘cell lysis’ comprises four genes involved in lysis of the bacterium at the end of the lytic phase of the infection cycle. The regions of the genome that can be deleted without impairing the ability of the phage to follow the lytic cycle are indicated in green. (B) The differences between a λ insertion vector and a λ replacement vector.
). These segments can therefore be deleted without impairing the ability of the phage to infect bacteria and direct synthesis of new λ particles by the lytic cycle. Two types of vector have been developed (Figure 4.21B):
Insertion vectors, in which part or all of the optional DNA has been removed and a unique restriction site introduced at some position within the trimmed down genome;
Replacement vectors, in which the optional DNA is contained within a stuffer fragment, flanked by a pair of restriction sites, that is replaced when the DNA to be cloned is ligated into the vector.
The linear form of the vector is shown at the top of the diagram. Treatment with the appropriate restriction endonuclease produces the left and right arms, both of which have one blunt end and one end with the 12-nucleotide overhang of the cos site. The DNA to be cloned is blunt ended and so is inserted between the two arms during the ligation step. These arms also ligate to one another via their cos sites, forming a concatamer. Some parts of the concatamer comprise left arm-insert DNA-right arm and, assuming this combination is 37–52 kb in length, will be enclosed inside the capsid by the in vitro packaging mix. Parts of the concatamer made up of left arm ligated directly to right arm, without new DNA, are too short to be packaged.
. The concatamers are then added to an in vitro packaging mix, which contains all the proteins needed to make a λ phage particle. These proteins form phage particles spontaneously, and will place inside the particles any DNA fragment that is between 37 and 52 kb in length and is flanked by cos sites. The packaging mix therefore cuts left arm-new DNA-right arm combinations of 37–52 kb out of the concatamers and constructs λ phages around them. The phages are then mixed with E. coli cells, and the natural infection process transports the vector plus new DNA into the bacteria.
After infection, the cells are spread onto an agar plate. The objective is not to obtain individual colonies but to produce an even layer of bacteria across the entire surface of the agar. Bacteria that were infected with the packaged cloning vector die within about 20 minutes because the λ genes contained in the arms of the vector direct replication of the DNA and synthesis of new phages by the lytic cycle, each of these new phages containing its own copy of the vector plus cloned DNA. Death and lysis of the bacterium releases these phages into the surrounding medium, where they infect new cells and begin another round of phage replication and lysis. The end result is a zone of clearing, called a plaque, which is visible on the lawn of bacteria that grows on the agar plate (Figure 4.23
). With some λ vectors, all plaques are made up of recombinant phages because ligation of the two arms without insertion of new DNA results in a molecule that is too short to be packaged. With other vectors it is necessary to distinguish recombinant plaques from non-recombinant ones. Various methods are used, including the β-galactosidase system described above for the plasmid vector pUC8 (see Figure 4.19), which is also applicable to those λ vectors that carry a fragment of the lacZ gene into which the DNA to be cloned is inserted.
| Number of clones* | |||
|---|---|---|---|
| Type of vector | Insert size (kb) | P = 95% | P = 99% |
| λ replacement | 18 | 532 500 | 820 000 |
| Cosmid, fosmid | 40 | 240 000 | 370 000 |
| P1 | 125 | 77 000 | 118 000 |
| BAC, PAC | 300 | 32 000 | 50 000 |
| YAC | 600 | 16 000 | 24 500 |
| Mega-YAC | 1400 | 6850 | 10 500 |
Calculated from the equation:
where N is the number of clones required, P is the probability that any given segment of the genome is present in the library, a is the average size of the DNA fragments inserted into the vector, and b is the size of the genome.
pJB8 is 5.4 kb in size and carries the ampicillin-resistance gene (amp R), a segment of λ DNA containing the cos site, and an Escherichia coli origin of replication (ori).
). Concatamers of cosmid molecules, linked at their cos sites, act as substrates for in vitro packaging because the cos site is the only sequence that a DNA molecule needs in order to be recognized as a ‘λ genome’ by the proteins that package DNA into λ phage particles. Particles containing cosmid DNA are as infective as real λ phages, but once inside the cell the cosmid cannot direct synthesis of new phage particles and instead replicates as a plasmid. Recombinant DNA is therefore obtained from colonies rather than plaques. As with other types of λ vector, the upper limit for the length of the cloned DNA is set by the space available within the λ phage particle. A cosmid can be 8 kb or less in size, so up to 44 kb of new DNA can be inserted before the packaging limit of the λ phage particle is reached. This reduces the size of the human genomic library to about a quarter of a million clones, which is an improvement compared with a λ library, but is still a massive number of clones to have to work with.
The first major breakthrough in attempts to clone DNA fragments much longer than 50 kb came with the invention of yeast artificial chromosomes or YACs (Burke et al., 1987). These vectors are propagated in S. cerevisiae rather than in E. coli and are based on chromosomes, rather than on plasmids or viruses. The first YACs were constructed after studies of natural chromosomes had shown that, in addition to the genes that it carries, each chromosome has three important components:
The centromere, which plays a critical role during nuclear division (see Figure 2.7);
The telomeres, the special sequences which mark the ends of chromosomal DNA molecules (see Figure 2.10);
One or more origins of replication, which initiate synthesis of new DNA when the chromosome divides (Section 13.2.1).
(A) The cloning vector pYAC3. (B) To clone with pYAC3, the circular vector is digested with BamHI and SnaBI. BamHI restriction removes the stuffer fragment held between the two telomeres in the circular molecule. SnaBI cuts within the SUP4 gene and provides the site into which new DNA will be inserted. Ligation of the two vector arms with new DNA produces the structure shown at the bottom. This structure carries functional copies of the TRP1 and URA3 selectable markers. The host strain has inactivated copies of these genes, which means that it requires tryptophan and uracil as nutrients. After transformation, cells are plated onto a minimal medium, lacking tryptophan and uracil. Only cells that contain the vector, and so can synthesize tryptophan and uracil, are able to survive on this medium and produce colonies. Note that if a vector comprises two right arms, or two left arms, then it will not give rise to colonies because the transformed cells will still require one of the nutrients. The presence of insert DNA in the cloned vector molecules is checked by testing for inactivation of SUP4. This is done by a color test: on the appropriate medium, colonies containing recombinant vectors (i.e. with an insert) are white; non-recombinants (vector but no insert) are red.
). All of these components can be contained in a DNA molecule of 10–15 kb. Natural yeast chromosomes range from 230 kb to over 1700 kb, so YACs have the potential to clone Mb-sized DNA fragments. This potential has been realized, standard YACs being able to clone 600 kb fragments, with special types able to handle DNA up to 1400 kb in length. Currently this is the highest capacity of any type of cloning vector, and several genome projects have made extensive use of YACs. Unfortunately, with some types of YAC there have been problems with insert stability, the cloned DNA becoming rearranged into new sequence combinations (Anderson, 1993). For this reason there is also great interest in other types of vectors, ones that cannot clone such large pieces of DNA but which suffer less from instability problems. These vectors include the following:
Bacteriophage P1 vectors (Sternberg, 1990) are very similar to λ vectors, being based on a deleted version of a natural phage genome, the capacity of the cloning vector being determined by the size of the deletion and the space within the phage particle. The P1 genome is larger than the λ genome, and the phage particle is bigger, so a P1 vector can clone larger fragments of DNA than a λ vector, up to 125 kb using current technology.
Bacterial artificial chromosomes or BACs (Shizuya et al., 1992) are based on the naturally occurring F plasmid of E. coli. Unlike the plasmids used to construct the early cloning vectors, the F plasmid is relatively large and vectors based on it have a higher capacity for accepting inserted DNA. BACs can be used to clone fragments of 300 kb and longer.
P1-derived artificial chromosomes or PACs (Ioannou et al., 1994) combine features of P1 vectors and BACs and have a capacity of up to 300 kb.
Fosmids (Kim et al., 1992) contain the F plasmid origin of replication and a λ cos site. They are similar to cosmids but have a lower copy number in E. coli, which means that they are less prone to instability problems.
Cloning is not merely an aid to DNA sequencing: it also provides a means of studying the mode of expression of a gene and the way in which expression is regulated, of carrying out genetic engineering experiments aimed at modifying the biological characteristics of the host organism, and of synthesizing important animal proteins, such as pharmaceuticals, in a new host cell from which the proteins can be obtained in larger quantities than is possible by conventional purification from animal tissue. These multifarious applications demand that genes must frequently be cloned in organisms other than E. coli.
(A) YIp5, a typical yeast integrative plasmid. The plasmid contains the ampicillin-resistance gene (amp R), the tetracycline-resistance gene (tet R), the yeast gene URA3, and an Escherichia coli origin of replication (ori). The presence of the E. coli ori means that recombinant YIp5 molecules can be constructed in E. coli before their transfer into yeast cells. YIp5 is therefore a shuttle vector - it can be shuttled between two species. (B) YIp5 has no origin of replication that can function inside yeast cells, but can survive if it integrates into the yeast chromosomal DNA by homologous recombination between the plasmid and chromosomal copies of the URA3 gene. The chromosomal gene carries a small mutation which means that it is non-functional and the host cells are ura3 -. One of the pair of URA3 genes that are formed after integration of the plasmid DNA is mutated, but the other is not. Recombinant cells are therefore ura + and can be selected by plating on to minimal medium, which does not contain uracil.
). What is the logic behind the construction of YIp5? When used in a cloning experiment, the vector is initially used with E. coli as the host, up to the stage where the desired recombinant molecule has been constructed by restriction and ligation. The recombinant vector is then purified from E. coli and transferred into S. cerevisiae, usually by mixing the DNA with protoplasts - yeast cells whose walls have been removed by enzyme treatment. Without an origin of replication the vector is unable to propagate independently inside yeast cells, but it can survive if it becomes integrated into one of the yeast chromosomes, which can occur by homologous recombination (Section 7.2.2) between the URA3 gene carried by the vector and the chromosomal copy of this gene (Figure 4.26B). ‘YIp’ in fact stands for ‘yeast integrative plasmid’. Once integrated the YIp, plus any DNA that has been inserted into it, replicates along with the host chromosomes.
), the kanamycin-resistance gene (kan
R), an Escherichia coli origin of replication (ori), and the two boundary sequences from the T-DNA region of the Ti plasmid. These two boundary sequences recombine with plant chromosomal DNA, inserting the segment of DNA between them into the plant DNA. The orientation of the boundary sequences in pBIN19 means that the lacZ′ and kan
R genes, as well as any new DNA ligated into the restriction sites within lacZ′, are transferred to the plant DNA. Recombinant plant cells are selected by plating onto kanamycin agar, and then regenerated into whole plants. Note that pBIN19 is another example of a shuttle vector, recombinant molecules being constructed in E. coli, using the lacZ′ selection system, before transfer to Agrobacterium tumefaciens and thence to the plant. For more details, see Brown (2001).
) have been designed to make use of this natural genetic engineering system (Bevan, 1984). The recombinant vector is introduced into A. tumefaciens cells, which are allowed to infect a cell suspension or plant callus culture, from which mature transformed plants can be regenerated.
In essence, DNA cloning results in the purification of a single fragment of DNA from a complex mixture of DNA molecules. Cloning is a powerful technique and its impact on our understanding of genes and genomes has been immeasurable. Cloning does, however, have one major disadvantage: it is a time-consuming and, in parts, difficult procedure. It takes several days to perform the manipulations needed to insert DNA fragments into a cloning vector and then introduce the ligated molecules into the host cells and select recombinants. If the experimental strategy involves generation of a large clone library, followed by screening of the library to identify a clone that contains a gene of interest (see Technical Note 4.3), then several more weeks or even months might be needed to complete the project.
PCR complements DNA cloning in that it enables the same result to be achieved - purification of a specified DNA fragment - but in a much shorter time, perhaps just a few hours (Saiki et al., 1988). PCR is complementary to, not a replacement for, cloning because it has its own limitations, the most important of which is the need to know the sequence of at least part of the fragment that is to be purified. Despite this constraint, PCR has acquired central importance in many areas of molecular biology research. We will examine the technique first and then survey its applications.
). Unlike cloning, PCR is a test-tube reaction and does not involve the use of living cells: the copying is carried out not by cellular enzymes but by the purified, thermostable DNA polymerase of T. aquaticus (Section 4.1.1). The reason why a thermostable enzyme is needed will become clear when we look in more detail at the events that occur during PCR.
). They must attach to the target DNA at either side of the segment that is to be copied; the sequences of these attachment sites must therefore be known so that primers of the appropriate sequences can be synthesized.
See the text for details.
are shown at the top. The next cycle of denaturation-annealing-synthesis leads to four products, two of which are identical to the first cycle products and two of which are made entirely of new DNA. During the third cycle, the latter give rise to ‘short’ products which, in subsequent cycles, accumulate in an exponential fashion.
). The temperature is then reduced to 50–60 °C, which results in some rejoining of the single strands of the target DNA, but also allows the primers to attach to their annealing positions. DNA synthesis can now begin, so the temperature is raised to 72 °C, the optimum for Taq polymerase. In this first stage of the PCR, a set of ‘long products’ is synthesized from each strand of the target DNA. These polynucleotides have identical 5′ ends but random 3′ ends, the latter representing positions where DNA synthesis terminates by chance. When the cycle of denaturation-annealing- synthesis is repeated, the long products act as templates for new DNA synthesis, giving rise to ‘short products’ whose 5′ and 3′ ends are both set by the primer annealing positions (Figure 4.29). In subsequent cycles, the number of short products accumulates in an exponential fashion (doubling during each cycle) until one of the components of the reaction becomes depleted. This means that after 30 cycles, there will be over 250 million short products derived from each starting molecule. In real terms, this equates to several micrograms of PCR product from a few nanograms or less of target DNA.
The PCR has been carried out in a microfuge tube. A sample is loaded into lane 2 of an agarose gel. Lane 1 contains DNA size markers, and lane 3 contains a sample of a PCR done by a colleague. After electrophoresis, the gel is stained with ethidium bromide (see Technical Note 2.1). Lane 2 contains a single band of the expected size, showing that the PCR has been successful. In lane 3 there is no band - this PCR has not worked.
). Alternatively, the sequence of the product can be determined, using techniques described in Section 6.1.1.
PCR is such a straightforward procedure that it is sometimes difficult to understand how it can have become so important in modern research. First we will deal with its limitations. In order to synthesize primers that will anneal at the correct positions, the sequences of the boundary regions of the DNA to be amplified must be known. This means that PCR cannot be used to purify fragments of genes or other parts of a genome that have never been studied before. A second constraint is the length of DNA that can be copied. Regions of up to 5 kb can be amplified without too much difficulty, and longer amplifications - up to 40 kb - are possible using modifications of the standard technique. However, the >100 kb fragments that are needed for genome sequencing projects are unattainable by PCR.
What are the strengths of PCR? Primary among these is the ease with which products representing a single segment of the genome can be obtained from a number of different DNA samples. We will encounter one important example of this in the next chapter when we look at how DNA markers are typed in genetic mapping projects (Section 5.2.2). PCR is used in a similar way to screen human DNA samples for mutations associated with genetic diseases such as thalassemia and cystic fibrosis. It also forms the basis of genetic profiling, in which variations in microsatellite length are typed (see Figure 2.25).
A second important feature of PCR is its ability to work with minuscule amounts of starting DNA. This means that PCR can be used to obtain sequences from the trace amounts of DNA that are present in hairs, bloodstains and other forensic specimens, and from bones and other remains preserved at archaeological sites. In clinical diagnosis, PCR is able to detect the presence of viral DNA well before the virus has reached the levels needed to initiate a disease response. This is particularly important in the early identification of viral-induced cancers because it means that treatment programs can be initiated before the cancer becomes established.
The above are just a few of the applications of PCR. The technique is now a major component of the molecular biologist's toolkit and we will discover many more examples of its use in the study of genomes as we progress through the remaining chapters of this book.
Give short definitions of the following terms:
2 μm circle
Bacterial artificial chromosome (BAC)
Complementary DNA (cDNA)
cos site
Denaturation of protein
Hybridization probe
In vitro packaging
Knockout mice
Linker
P1-derived artificial chromosome (PAC)
Polymerase chain reaction (PCR)
Yeast artificial chromosome (YAC)
Draw diagrams that outline the events that occur during (a) DNA cloning, and (b) PCR. What are the limitations of each of these two techniques?
List the types of enzyme used in recombinant DNA research.
Distinguish between the two types of exonuclease activity that can be possessed by a DNA polymerase, and explain how these activities influence the potential applications of individual DNA polymerases in recombinant DNA research.
Using examples, describe the various types of end produced after digestion of DNA with a restriction endonuclease.
How are agarose gel electrophoresis and Southern hybridization used to examine the results of a restriction digest?
Explain why the efficiency of blunt-end ligation is less than that of sticky-end ligation. What steps can be taken to improve the efficiency of blunt-end ligation?
Draw diagrams of (a) pBR322, and (b) pUC8. Explain how the differences between these two vectors influence the ways in which they are used to clone DNA fragments.
Distinguish between the lytic and lysogenic infection cycles for a bacteriophage.
Write a short description of the way in which a bacteriophage λ vector is used to clone DNA. How does a cosmid differ from a standard λ vector?
Draw a diagram showing a typical YAC. Indicate the key features and explain how a YAC is used to clone DNA.
What problems might arise when a YAC is used to clone a large fragment of DNA? To what extent can these problems be solved by the use of other types of high-capacity cloning vector?
How is DNA cloned in organisms other than Escherichia coli?
Describe how a PCR is carried out, paying particular attention to the role of the primers and the temperatures used during the thermal cycling.
Soon after the first gene cloning experiments were carried out in the early 1970s, a number of scientists argued that there should be a temporary moratorium on this type of research. What was the basis of these scientists' fears and to what extent were these fears justified?
What would be the features of an ideal cloning vector? To what extent are these requirements met by any of the existing cloning vectors?
The specificity of the primers is a critical feature of a successful PCR. If the primers anneal at more than one position in the target DNA then products additional to the one being sought will be synthesized. Explore the factors that determine primer specificity and evaluate the influence of the annealing temperature on the outcome of a PCR.
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