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Library of Congress Cataloguing-in-Publication Data. Brown, T.A. (Terence A.) Gene cloning and DNA analysis: an introduction / T.A. Brown.—6th ed. p. cm. Download the App · Facebook footer icon 0ba49efeadcfb19afb3ee1bab88bb04 Twitter footer icon. explored in great depth in my book Men, Women, and Relationships: Making Peace with the. Opposite Sex.) Although the be.

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Read "Gene Cloning and DNA Analysis An Introduction" by T. A. Brown available from Rakuten Kobo. Sign up today and get $5 off your first purchase. Known. Geschenk. Als Download kaufen. Bisher 44,49**. %. 38, Preis in Euro, inkl. MwSt. **Preis der gedruckten Ausgabe (Broschiertes Buch). eBook bestellen. Editorial Reviews. About the Author. Terry Brown Manchester Institute of Biotechnology, Download it once and read it on your Kindle device, PC, phones or tablets. Additional gift options are available when buying one eBook at a time.

Open eBook Preview. Known world-wide as the standard introductory text to this important and exciting area, the sixth edition of Gene Cloning and DNA Analysis addresses new and growing areas of research whilst retaining the philosophy of the previous editions. Assuming the reader has little prior knowledge of the subject, its importance, the principles of the techniques used and their applications are all carefully laid out, with over clearly presented four-colour illustrations. In addition to a number of informative changes to the text throughout the book, the final four chapters have been significantly updated and extended to reflect the striking advances made in recent years in the applications of gene cloning and DNA analysis in biotechnology. Gene Cloning and DNA Analysis remains an essential introductory text to a wide range of biological sciences students; including genetics and genomics, molecular biology, biochemistry, immunology and applied biology.

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Gene Cloning and DNA Analysis (eBook) by T. A. Brown (Author)

The best characterized eukaryotic plasmid is the 2 Fm circle that occurs in many strains of the yeast Saccharomyces cerevisiae. The discovery of the 2 fm plasmid was very fortuitous as it allowed the construction of cloning vectors for this very import- ant industrial organism p.

Like all viruses, phages are very simple in structure, consisting merely of a DNA or occasionally ribonucleic acid RNA molecule carrying a number of genes, including several for replication of the phage, surrounded by a protective coat or capsid made up of protein molecules Figure 2.

With some phage types the entire infection cycle is completed very quickly, possibly in less than 20 minutes. This type of rapid infection is called a lytic cycle, as release of the new phage particles is associated with lysis of the bacterial cell. The characteristic feature of a lytic infection cycle is that phage DNA replication is immediately followed Plasmids and Bacteriophages 19 by synthesis of capsid proteins, and the phage DNA molecule is never maintained in a stable condition in the host cell.

With many lysogenic phages the phage DNA is inserted into the bacterial genome, in a manner similar to episomal insertion see Figure 2. The integrated form of the phage DNA called the prophage is quiescent, and a bacterium referred to as a lysogen that carries a prophage is usually physiologically indistinguishable from an uninfected cell. However, the prophage is eventually released from the host genome and the phage reverts to the lytic mode and lyses the cell.

The infection cycle of lambda E , a typical lysogenic phage of this type, is shown in Figure 2. A limited number of lysogenic phages follow a rather different infection cycle.

When M13 or a related phage infects E. The M13 DNA is not integrated into the bacterial genome and does not become quiescent. With these phages, cell lysis never occurs, and the infected bacterium can continue to grow and divide, albeit at a slower rate than uninfected cells.

Figure 2. Although there are many different varieties of bacteriophage, only e and M13 have found a major role as cloning vectors. We will now consider the properties of these two phages in more detail.

Gene organization in the 5 DNA molecule e is a typical example of a head-and-tail phage see Figure 2.

The DNA is contained in the polyhedral head structure and the tail serves to attach the phage to the bacterial surface and to inject the DNA into the cell see Figure 2. The e DNA molecule is 49 kb in size and has been intensively studied by the tech- niques of gene mapping and DNA sequencing. As a result the positions and identities of all of the genes in the e DNA molecule are known Figure 2. A feature of the e genetic map is that genes related in terms of function are clustered together in the genome.

For example, all of the genes coding for components of the capsid are grouped together in the left-hand third of the molecule, and genes controlling integration of the prophage into the host genome are clustered in the middle of the molecule. Clustering of related genes is profoundly important for controlling expression of the e genome, as it allows genes to be switched on and off as a group rather than individually. Clustering is also important in the construction of e-based cloning vectors, as we will discover when we return to this topic in Chapter 6.

The linear and circular forms of 5 DNA A second feature of e that turns out to be of importance in the construction of cloning vectors is the conformation of the DNA molecule. The molecule shown in Figure 2. However, at either end of the molecule is a short nucleotide stretch in which the DNA is single-stranded Figure 2.

The two single strands are complementary, and so can base pair with one another to form a circular, completely double-stranded molecule Figure 2. A B Figure 2. The e cohesive ends are called the cos sites and they play two distinct roles during the e infection cycle. First, they allow the linear DNA molecule that is injected into the cell to be circularized, which is a neces- sary prerequisite for insertion into the bacterial genome see Figure 2.

M13 DNA replication Infected cells continue to grow and divide New M13 phages are continuously extruded from an infected cell M13 phage attaches to a pilus on an E. Plasmids and Bacteriophages 21 The second role of the cos sites is rather different, and comes into play after the prophage has excised from the host genome. At this stage a large number of new e DNA molecules are produced by the rolling circle mechanism of replication Figure 2.

The result is a catenane consisting of a series of linear e genomes joined together at the cos sites. The role of the cos sites is now to act as recognition sequences for an endonuclease that cleaves the catenane at the cos sites, producing individual e genomes. This endonu- clease, which is the product of gene A on the e DNA molecule, creates the single- stranded sticky ends, and also acts in conjunction with other proteins to package each e genome into a phage head structure.

The cleavage and packaging processes recognize just the cos sites and the DNA sequences to either side of them, so changing the structure of the internal regions of the e genome, for example by inserting new genes, has no effect on these events so long as the overall length of the e genome is not altered too greatly. Furthermore, the M13 DNA molecule is much smaller than the e genome, being only nucleotides in length.

It is circular and is unusual in that it consists entirely of single-stranded DNA. The smaller size of the M13 DNA molecule means that it has room for fewer genes than the e genome. This is possible because the M13 capsid is constructed from multiple copies of just three proteins requiring only three genes , whereas synthesis of the e Plasmids and Bacteriophages 23 head-and-tail structure involves over 15 different proteins.

In addition, M13 follows a simpler infection cycle than e, and does not need genes for insertion into the host genome. Once inside the cell the single-stranded molecule acts as the template for synthesis of a comple- mentary strand, resulting in normal double-stranded DNA Figure 2. This molecule is not inserted into the bacterial genome, but instead replicates until over copies are present in the cell Figure 2.

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When the bacterium divides, each daughter cell receives copies of the phage genome, which continues to replicate, thereby maintaining its overall numbers per cell. As shown in Figure 2. Several features of M13 make this phage attractive as a cloning vector. The genome is less than 10 kb in size, well within the range desirable for a potential vector. In addi- tion, the double-stranded replicative form RF of the M13 genome behaves very much like a plasmid, and can be treated as such for experimental purposes.

It is easily prepared from a culture of infected E. Most importantly, genes cloned with an Mbased vector can be obtained in the form of single-stranded DNA.

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Single-stranded versions of cloned genes are useful for several techniques, notably DNA sequencing and in vitro mutagenesis pp.

Cloning in an M13 vector is an easy and reliable way of obtaining single-stranded DNA for this type of work. M13 vectors are also used in phage display, a technique for identifying pairs of genes whose protein products interact with one another p.

This is especially important when it is remembered that plasmids are not commonly found in organisms other than bacteria and yeast. Several eukaryotic viruses have been employed as cloning vectors for specialized applications: These vectors are discussed more fully in Chapter 7. Wiley Blackwell, Chichester. First, total cell DNA will often be required as a source of material from which to obtain genes to be cloned.

Total cell DNA may be DNA from a culture of bacteria, from a plant, from animal cells, or from any other type of organism that is being studied. It consists of the genomic DNA of the organism along with any additional DNA mole- cules, such as plasmids, that are present. Finally, phage DNA will be needed if a phage cloning vector is to be used.

Phage DNA is generally prepared from bacteriophage particles rather than from infected cells, so there is no problem with contaminating bacterial DNA. However, special techniques are needed to remove the phage capsid.

An exception is the double-stranded replicative form of M13, which is prepared from E. Table 3. The procedure for total DNA preparation from a culture of bacterial cells can be divided into four stages Figure 3. Two typical growth media are detailed in Table 3.

This medium contains a mixture of inorganic nutrients to provide essential elements such as The amount of light that passes through the culture is measured and the OD also called the absorbance calculated as: This curve is plotted from the OD values of a series of cultures of known cell density.

For E. In practice, additional growth factors such as trace elements and vitamins must be added to M9 before it will support bacterial growth. Precisely which supplements are needed depends on the species concerned.

The second medium described in Table 3. This is because two of the ingredients, tryptone and yeast extract, are complicated mixtures of unknown chemical compounds. Tryptone in fact supplies amino acids and small peptides, while yeast extract a dried preparation of partially digested yeast cells provides the nitrogen requirements, along with sugars and inorganic and organic nutrients. Complex media such as LB need no further supple- mentation and support the growth of a wide range of bacterial species.

However, this is not necessary when the culture is being grown simply as a source of DNA, and under these circumstances a complex medium is appropriate. The growth of the culture can be mon- itored by reading the optical density OD at nm Figure 3.

In order to prepare a cell extract, the bacteria must be obtained in as small a volume as possible. Harvesting is therefore performed by spinning the culture in a centrifuge Figure 3. Fairly low centrifugation speeds will pellet the bacteria at the bottom of the centrifuge tube, allowing the culture medium to be poured off. Bacteria from a Bacterial culture Centrifuge rotor Spin at rpm for 10 minutes Pellet of bacteria Figure 3. With some species, including E.

All of these barriers have to be disrupted to release the cell components. Techniques for breaking open bacterial cells can be divided into physical methods, in which the cells are disrupted by mechanical forces, and chemical methods, where cell lysis is brought about by exposure to chemical agents that affect the integrity of the cell barriers. Chemical methods are most commonly used with bacterial cells when the object is DNA preparation.

Chemical lysis generally involves one agent attacking the cell wall and another disrupting the cell membrane Figure 3. The chemicals that are used depend on the species of bacterium involved, but with E.

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Lysozyme is an enzyme that is present in egg white and in secretions such as tears and saliva, and which digests the polymeric compounds that give the cell wall its rigidity. EDTA removes magnesium ions that are essential for pre- serving the overall structure of the cell envelope, and also inhibits cellular enzymes that could degrade DNA. Under some conditions, weakening the cell wall with lysozyme or Detergents aid the process of lysis by removing lipid molecules and thereby cause disruption of the cell membranes.

Components such as partially digested cell wall fractions can be pelleted by centrifugation Figure 3. A variety of methods can be used to purify the DNA from this mixture.

One approach is to treat the mixture with reagents which degrade the contaminants, leaving a pure solution of DNA Figure 3. Other methods use ion-exchange chromatography to separate the mixture into its various components, so the DNA is removed from the proteins and RNA in the extract Figure 3. Removing contaminants by organic extraction and enzyme digestion The standard way to deproteinize a cell extract is to add phenol or a 1: The result is that if the cell extract is mixed gently with the solvent, and the layers then separated by centrifugation, precipitated protein molecules are left as a white coagulated mass at the interface between the aqueous and organic layers Figure 3.

The aqueous solution of nucleic acids can then be removed with a pipette. This problem could be solved by carrying out several phenol extractions one after the other, but this is undesirable as each mixing and centrifugation step results in a certain amount of breakage of the DNA molecules.

The answer is to treat the cell extract with a protease such as pronase or These enzymes break polypeptides down into smaller units, which are more easily removed by phenol. The only effective way to remove the RNA is with the enzyme ribonuclease, which rapidly degrades these molecules into ribonucleotide subunits. Using ion-exchange chromatography to purify DNA from a cell extract Biochemists have devised various methods for using differences in electrical charge to separate mixtures of chemicals into their individual components.

One of these methods is ion-exchange chromatography, which separates molecules according to how tightly they bind to electrically charged particles present in a chromatographic matrix or resin. DNA and RNA are both negatively charged, as are some proteins, and so bind to a positively charged resin.

The electrical attachment is disrupted by salt Figure 3. By gradually increasing the salt concentration, different types of molecule can be detached from the resin one after another. The simplest way to carry out ion-exchange chromatography is to place the resin in a glass or plastic column and then add the cell extract to the top Figure 3. The extract passes through the column, and because this extract contains very little salt all the negatively charged molecules bind to the resin and are retained in the column.

If a salt solution of gradually increasing concentration is now passed through the column, the different types of molecule will elute i. The most frequently used method of concentration is ethanol precipitation. With a thick solution of DNA the ethanol can be layered on top of the sample, causing molecules to precipitate at the interface.

A spectacular trick is to push a glass rod through the ethanol into the DNA solution. When the rod is removed, The solutions passing through the column can be collected by gravity flow or by the spin column method, in which the column is placed in a low-speed centrifuge.

Alternatively, if ethanol is mixed with a dilute DNA solution, the precipit- ate can be collected by centrifugation Figure 3. Ethanol precipitation has the added advantage of leaving short-chain and monomeric nucleic acid components in solution. Ribonucleotides produced by ribonuclease treatment are therefore lost at this stage.

Fortunately DNA concentrations can be accurately measured by ultraviolet UV absorbance spectrophotometry. Fibers of DNA can be withdrawn with a glass rod. Usually absorbance is measured at nm, at which wavelength an absorbance A of 1.

Measurements of as little as 1 fl of a DNA solution can be carried out in spectrophotometers designed especially for this purpose. Ultraviolet absorbance can also be used to check the purity of a DNA preparation. Ratios of less than 1. Total cell DNA from, for example, plants or animals will be needed if the aim of the genetic engineering project is to clone genes from these organisms. Obviously growth of cells in liquid medium is appropriate only for bacteria, other microorganisms, and plant and animal cell cultures.

The chemicals used for disrupting bacterial cells do not usually work with other organisms: On the other hand, most animal cells have no cell wall at all, and can be lysed simply by treating with detergent. Another important consideration is the biochemical content of the cells from which DNA is being extracted.

Instead a different approach must be used. One method makes use of a detergent called cetyltrimethylammonium bromide CTAB , which forms an insoluble complex with nucleic acids. When CTAB is added to a plant cell extract the The precipitate is then collected by centrifugation and resuspended in 1 M sodium chloride, which causes the complex to break down. The nucleic acids can now be concentrated by ethanol precipitation and the RNA removed by ribonuclease treatment. This is one of the reasons why ion-exchange chromatography has become so popular.

First, it denatures and dissolves all biochemicals other than nucleic acids and can therefore be used to release DNA from virtually any type of cell or tissue. Second, in the presence of guanidinium thiocyanate, DNA binds tightly to silica particles Figure 3. This provides an easy way of recovering the DNA from the denatured mix of biochemicals. One possibility is to add the silica directly to the cell extract but, as with the ion-exchange methods, it is more convenient to use a chromatography column.

The silica is placed in the column and the cell extract added Figure 3. DNA binds to the silica and is retained in the column, whereas the denatured biochemicals pass straight through. After washing away the last contaminants with guanidinium thiocyanate solution, the DNA is recovered by adding water, which destabilizes the interactions between the DNA molecules and the silica.

A culture of cells, containing plasmids, is grown in liquid medium, harvested, and a cell extract prepared. In a plasmid preparation it is always necessary to separate the plasmid DNA from the large amount of bacterial chromosomal DNA that is also present in the cells.

The presence of the smallest amount of contaminating bacterial DNA in a gene cloning experiment can easily lead to undesirable results. Fortunately several methods are available for removal of bacterial DNA during The methods are based on the several physical differences between plasmid DNA and bacterial DNA, the most obvious of which is size. In addition to size, plasmids and bacterial DNA differ in conformation.

Plasmids and the bacterial chromosome are circular, but during preparation of the cell extract the chromosome is always broken to give linear fragments.

A method for separating circular from linear molecules will therefore result in pure plasmids. If the cells are lysed under very carefully controlled conditions, only a minimal amount of chromosomal DNA breakage occurs. The resulting DNA fragments are still very large—much larger than the plasmids—and can be removed with the cell debris by centrifugation.

This process is aided by the fact that the bacterial chromosome is physically attached to the cell envelope, so fragments of the chromosome sediment with the cell debris if these attachments are not broken. Cell disruption must therefore be carried out very gently to prevent wholesale break- age of the bacterial DNA.

Treatment with EDTA and lysozyme is carried out in the presence of sucrose, which prevents the cells from bursting immediately. Instead, sphaeroplasts are formed, cells with partially degraded cell walls that retain an intact cytoplasmic membrane. Cell lysis is now induced by adding a non-ionic detergent such as Triton X ionic detergents, such as SDS, cause chromosomal breakage.

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This method causes very little breakage of the bacterial DNA, so centrifugation leaves a cleared lysate, consisting almost entirely of plasmid DNA. A cleared lysate will, however, invariably retain some chromosomal DNA. Further- more, if the plasmids themselves are large molecules, they may also sediment with the Most plasmids exist in the cell as supercoiled molecules Figure 3.

Supercoiling occurs because the double helix of the plasmid DNA is partially unwound during the plasmid replication process by enzymes called topoisomerases p. The supercoiled conformation can be maintained only if both polynucleotide strands are intact, hence the more technical name of covalently closed- circular ccc DNA. If one of the polynucleotide strands is broken the double helix reverts to its normal relaxed state, and the plasmid takes on the alternative conforma- tion, called open-circular oc Figure 3.

Supercoiling is important in plasmid preparation because supercoiled molecules can be fairly easily separated from non-supercoiled DNA. Two different methods are commonly used. Alkaline denaturation The basis of this technique is that there is a narrow pH range at which non-supercoiled DNA is denatured, whereas supercoiled plasmids are not. If sodium hydroxide is added to a cell extract or cleared lysate, so that the pH is adjusted to If acid is now added, these denatured bacterial DNA strands reaggregate into a tangled mass.

The insoluble network can be pelleted by centrifugation, leaving plasmid DNA in the supernatant. Ethidium bromide—caesium chloride density gradient centrifugation This is a specialized version of the more general technique of equilibrium or density gradient centrifugation.

A density gradient is produced by centrifuging a solution of Macromolecules present in the CsCl solution when it is centrifuged form bands at distinct points in the gradient Figure 3. Exactly where a particular molecule bands depends on its buoyant density: DNA has a buoyant density of about 1. More importantly, density gradient centrifugation in the presence of ethidium bro- mide EtBr can be used to separate supercoiled DNA from non-supercoiled molecules.

Ethidium bromide binds to DNA molecules by intercalating between adjacent base pairs, causing partial unwinding of the double helix Figure 3. This unwinding results in a decrease in the buoyant density, by as much as 0.

However, supercoiled DNA, with no free ends, has very little freedom to unwind, and can only bind a limited amount of EtBr. The decrease in buoyant density of a supercoiled molecule is therefore much less, only about 0.

When a cleared lysate is subjected to this procedure, plasmids band at a distinct point, separated from the linear bacterial DNA, The pure plasmid DNA is removed by puncturing the side of the tube and withdrawing a sample with a syringe Figure 3. The yield of DNA from a bacterial culture may therefore be disappointingly low.

Some multi- copy plasmids those with copy numbers of 20 or more have the useful property of being able to replicate in the absence of protein synthesis. This contrasts with the main bacterial chromosome, which cannot replicate under these conditions.