In this final chapter of Part II, the various techniques that can be used to identify cloned genes will be described. As with previous chapters, the basis of techniques that are perhaps not so widely used today will be included, to illustrate the principles of gene identification and characterisation. This will lead into the final section of the book, where various applications of the technology will be covered, and where we get a look at some of the more advanced developments in gene manipulation.

Success in any cloning experiment depends on being able to identify the desired gene sequence among the many different recombinants that may be produced. Given that a large genomic library may contain a million or more cloned sequences, whic h are not readily distinguishable from each other by simple analytical methods, it is clear that identification of the target gene is potentially the most difficult part of the cloning process. Fortunately there are several selection/identification methods that can be used to overcome most of the problems that arise.

There are two terms that require definition before we proceed, these being selection and screening. Selection is where some sort of pressure (e.g. the presence of an antibiotic) is applied during the growth of host cells containing recombinant DNA. The cells with the desired characteristics are therefore selected by their ability to survive. This approach ranges in sophistication, from simple selection for the presence of a vector, up to direct selection of cloned genes by complementation of defined mutations.

Before examining some of the specific techniques used in gene manipulation, it is useful to consider the basic methods required for handling, quantifying and analysing nucleic acid molecules. It is often difficult to make the link between theoretical and practical aspects of a subject, and an appreciation of the methods used in routine work with nucleic acids may be of help when the more detailed techniques of gene cloning and analysis are described.

Isolation of DNA and RNA

Every gene manipulation experiment requires a source of nucleic acid, in the form of either DNA or RNA. It is therefore important that reliable methods are available for isolating these components from cells. There are three basic requirements: (i) opening the cells in the sample to expose the nucleic acids for further processing,(ii) separation of the nucleic acids from other cell components, and (iii) recovery of the nucleic acid in purified form. A variety of techniques may be used, ranging from simple procedures with few steps, up to more complex purifications involving several different stages. These days, most biological supply companies sell kits that enable purification of nucleic acids from a range of sources.

The first step in any isolation protocol is disruption of the starting material, which may be viral, bacterial, plant or animal. The method used to open cells should be as gentle as possible, preferably utilising enzymatic degradation of cell wall material (if present) and detergent lysis of cell membranes.

On 5th July 1996 a lamb was born at the Roslin Research Institute near Edinburgh. It was an apparently normal event, yet it marked the achievement of a milestone in biological science. The lamb was a clone, and was named Dolly. She was the first organism to be cloned from adult differentiated cells, which is what makes the achievement such a ground-breaking event. In this chapter we will look briefly at this area of genetic technology.

In this book so far, we have been considering the topic of molecular cloning, where the aim of an experimental process is to isolate a gene sequence for further analysis and use. In organismal cloning, the aim is to generate an organism from a cell that carries a complete set of genetic instructions. We have looked at the methods for generating transgenic organisms in Chapter 12, and a discussion of organismal cloning is a natural extension to this, although transgenic organisms are not necessarily (and at present are not usually) clones. In a similar way, a clone need not necessarily be transgenic. Thus, although not strictly part of gene manipulation technology, organismal cloning has become a major part of genetics in a broader sense. The public have latched on to cloning as an issue for concern, and thus a discussion of the topic is essential even in a book where the primary goal is to illustrate the techniques of gene manipulation.

Early thoughts and experiments

The announcement of the birth of Dolly in a paper in the journal Nature in February 1997 rocked the scientific community.

Advances in genetics continue to be made at an ever increasing rate, which makes writing an introductory text somewhat difficult. In the few years since the first edition was published, many new applications of gene manipulation technology have been developed, covering a diverse range of disciplines. The temptation in preparing this second edition was to concentrate on the applications, and ignore the fundamental principles of the technology. However, I wished to retain many of the features of the first edition, in which a basic technical introduction to the subject was the main aim of the text. Thus some of the original methods used in gene manipulation have been kept as examples of how the technology developed, even though some of these have become little used or even obsolete. From the educational point of view, this should help the reader cope with more advanced information about the subject – a sound grasp of the basic principles is an important part of any introduction to genetic engineering. I have been gratified by the many positive comments about the first edition, and I hope that this new edition is as well received.

In trying to strike a balance between the methodology and the applications of gene manipulation, I have divided the text into three sections. Part I deals with basic molecular biology, Part II with the methods used to manipulate genes, and Part III with the applications.

This final chapter is short. It does not answer any questions, but simply raises them for consideration. There are no ‘correct’ answers to these questions, as each must be addressed from the perspective of the individual, family, society, race or nation that is facing up to the situation. There are no diagrams or photographs, and very little factual information. However, the topics discussed are probably the most important that a student of genetic engineering can consider. In practical terms, relatively few people will ever go on to work in science and technology, but we will all have to cope with the consequences of gene-based research and its applications. Informed and vigorous debate is the only way that the developments of gene manipulation technology can become accepted and established.

Is science ethically and morally neutral?

It is often said that science per se is neither ‘good’ nor ‘bad’, and that it is therefore ethically and morally neutral. Whilst this may be true of science as a process, it is the developments and applications that arise from the scientific process that pose the ethical questions. The example that is often quoted is the development of the atomic bomb – the science was interesting and novel, and of itself ethically neutral, but the application (i.e. use of the devices in conflict) posed a completely different set of moral and ethical questions. Also, science is, of course, carried out by scientists, who are most definitely not ethically and morally neutral, as they demonstrate the same breadth and range of opinion as the rest of the human race.

Once recombinant DNA molecules have been constructed in vitro, the desired sequence can be isolated. In some experiments hundreds of thousands of different DNA fragments may be produced, and the isolation of a particular sequence would seem to be an almost impossible task. It is a bit like looking for the proverbial needle in a haystack – with the added complication that the needle is made of the same material as the haystack! Fortunately the methods available provide a relatively simple way to isolate specific gene sequences.

Three things have to be done to isolate a gene from a collection of recombinant DNA sequences: (i) the individual recombinant molecules have to be physically separated from each other,(ii) the recombinant sequences have to be amplified to provide enough material for further analysis, and (iii) the specific fragment of interest has to be selected by some sort of sequence-dependent method. In this chapter I consider the first two of these requirements, which in essence represent the systems and techniques involved in genecloning. This is an essential part of most genetic manipulation programmes. Even if the desired result is a transgenic organism, the gene to be used must first be isolated and characterised, and therefore cloning systems are required. Methods for selecting specific sequences are described in Chapter 8.

The biology of gene cloning is concerned with the selection and use of a suitable carrier molecule or vector, and a living system or host in which the vector can be propagated.

Biotechnology is one of those difficult terms that can mean different things to different people. In essence, it is the use of an organism (usually a microorganism) or a biologically derived substance (usually an enzyme) in a production or conversion process. Thus brewing and wine-making, food processing and manufacture, the production of pharmaceuticals and even the treatment of sewage can all be classed as aspects of biotechnology. In many cases the organism or enzyme is used in its natural form, and is not modified apart from perhaps having been subjected to selection methods to enable the best strain or type of enzyme to be used for a particular application. However, despite its traditional roots, modern biotechnology is often associated with the use of genetically modified systems. In this chapter we will consider the impact that gene manipulation technology has had on some biotechnological applications, with particular reference to the production of useful proteins.

The products of biotechnological processes are destined for use in a variety of fields such as medicine, agriculture and scientific research. It is perhaps an arbitrary distinction to separate the production of a therapeutic protein from its clinical application, as both could be considered as ‘biotechnology’ in its broadest sense. In a similar way, the developing area of transgenic plants and animals is also part of biotechnology, and undoubtedly the information provided by genome sequencing will give rise to many more diverse biotechnological applications.

The production of a transgenic organism involves altering the genome so that a permanent change is effected. This is different from somatic cell gene therapy, in which the effects of the transgene are restricted to the individual who receives the treatment. In fact, the whole point of generating a transgenic organism is to alter the germ line, so that the genetic change is inherited in a stable pattern following reproduction. This is one area of genetic engineering that has caused great public concern, and there are many complex issues surrounding the development and use of transgenic organisms. In addition, the scientific and technical problems associated with genetic engineering in higher organisms are often difficult to overcome. This is partly due to the size and complexity of the genome, and partly due to the fact that the development of plants and animals is an extremely complex process that is still not yet fully understood at the molecular level. Despite these difficulties, methods for the generation of transgenic plants and animals are now well established, and the use of transgenic organisms has already had a major impact in a range of different disciplines. In this chapter we will consider the development and use of transgenic plants and animals.

Transgenic plants

All life on earth is dependent on the photosynthetic fixation of carbon dioxide by plants. We sometimes lose sight of this fact, as most people are removed from the actual process ogfenerating our food, and the supemrarket shelves have all sorts of exotic processed foods and pre-prepared meals that somehow swamp the vegetable section.

The genetic engineer needs to be able to cut and join DNA from different sources. In addition, cer tain modifications may have to be carried out to the DNA during the various steps required to produce, clone and identify recombinant DNA molecules. The tools that enable these manipulations to be performed are enzymes, which are purified from a wide range of organisms and can be bought from various suppliers. In this chapter I examine some of the important classes of enzymes that make up the genetic engineer's toolkit.

Restriction enzymes – cutting DNA

The restriction enzymes, which cut DNA at fidened sites, represent one of the most important groups of enzymes for the manipulation of DNA. These enzymes are found in bacterial cells, where they function as part of a protective mechanism called the restriction–modification system. In this system the restriction enzyme hydrolyses any exogenous DNA that appears in the cell. To prevent the enzyme acting on the host cell DNA, the modification enzyme of the system (a methylase) modifies the host DNA by methylation of particular bases in the recognition sequence, which prevents the restriction enzyme from cutting the DNA.

Restriction enzymes are of three types (I, II or III). Most of the enzymes used today are type II enzymes, which have the simplest mode of action. These enzymes are nucleases (see Section 4.2.1), and as they cut at an internal position in a DNA strand (as opposed to beginning degradation at one end) they are known as endonucleases.

What is genetic engineering?

Progress in any scientific discipline is dependent on the availability of techniques and methods that extend the range and sophistication of experiments which may be performed. Over the last 30 years or so this has been demonstrated in a spectacular way by the emergence of genetic engineering. This field has grown rapidly to the point where, in many laboratories around the world, it is now routine practice to isolate a specific DNA fragment from the genome of an organism, determine its base sequence, and assess its function. The technology is also now used in many other applications, including forensic analysis of scene-of-crime samples, pater nity disputes, medical diagnosis, genome mapping and sequencing, and the biotechnology industry. What is particularly striking about the technology of gene manipulation is that it is readily accessible by individual scientists, without the need for large-scale equipment or resources outside the scope of a reasonably well-found research laboratory.

The term genetic engineering is often thought to be rather emotive or even trivial, yet it is probably the label that most people would recognise. However, there are several other terms that can be used to describe the tecnology, including gene manipulation, gene cloning, recombinant DNA technology, genetic modification, and the new genetics. There are also legal definitions used in administering regulatory mechanisms in countries where genetic engineering is practised.

Although there are many diverse and complex techniques involved, the basic principles of genetic manipulation are reasonably simple.

Now and again a scientific discovery is made that changes the whole course of the development of a subject. In the field of molecular biology we can identify several major milestones – the emergence of bacterial genetics, the discovery of the mechanism of DNA replication, the double helix and the genetic code, restriction enzymes, and finally the techniques of recombinant DNA. Many of these areas of molecular biology have been recognised by the award of the Nobel prize in either Chemistry or in Medicine and Physiology. Some of these key discoveries are listed in Table 7.1.

The topic of this chapter is the polymerase chain reaction (PCR), whic h was discovered by Kary Mullis and for which he was awarded the Nobel prize in Chemistry in 1993. The PCR technique produces a similar result to DNA cloning – the selective amplification of a DNA sequence – and has become such an important part of the genetic engineer's toolkit that in many situations it has essentially replaced traditional cloning methodology. In this chapter we will look at some of the techniques and applications of PCR technology.

The (short) history of the PCR

The essentials for PCR were in place by the late 1970s. In 1979 Kary Mullis joined the Cetus Corporation, based in Emeryville, Califor nia. He was working on oligonucleotide synthesis, which by the early 1980s had become an automated and somewhat tedious process. Thus, his mind was free to investigate other avenues.

The diagnosis and treatment of human disease is one area in which genetic manipulation is beginning to have a considerable effect. As outlined in Chapter 10, many therapeutic proteins are now made by recombinant DNA methods, and the number available is increasing steadily. Thus the treatment of conditions by recombinant-derived products is already well established. In this chapter we will look at how the techniques of gene manipulation impact more directly on medical diagnosis and treatment, and will also examine the use of rDNA technology in forensic science. Progress in both of these areas is of course closely linked to our increasing knowledge of the human genome, and thus many new developments in medical and forensic applications will appear as we decipher the genome.

Diagnosis and characterisation of medical conditions

Genetically based diseases (often called simply ‘genetic diseases’) represent one of the most important classes of disease, particularly in children. A disorder present at birth is termed a congenital abnormality, and around 5% of newborn babies will suffer from a serious medical problem of this type. In most of these cases there will be a significant genetic component in the aetiology (cause) of the disease state. It is estimated that about a third of primary admissions to paediatric hospitals are due to genetically based problems, whilst some 70% of cases presenting more than once are due to genetic defects. In addition to genetic problems appearing at birth or in childhood, it seems that a large proportion of diseases presenting in later life also have a genetic cause or predisposition.

In Part II we have examined some of the basic techniques of gene manipulation. These techniques, and many more sophisticated variations of them, give the scientist the tools that enable genes to be isolated and characterised. In this final section of the book we will consider some of the applications of gene manipulation. Of necessity, this will be a highly selective treatment, the aim being to give some idea of the immense scope of the subject whilst trying to include some detail in certain key areas. We will also look more broadly at some of the ethical problems that gene manipulation poses, and at the topic of organismal cloning.

In many ways genetic engineering has undergone a shift in emphasis over the past few years, a way from the technical problems that had to be solved before the technology became ‘user friendly’ enough for widespread use. Gene manipulation is now used as a tool to address many diverse biological problems that were previously intractable, and the applications of the subject appear at times to be limited only by the imagination of the scientists who use the technology in basic research, medicine, biotechnology and other related disciplines.

Analysis of gene structure and function

In terms of ‘pure’ science, the major impact of gene manipulation has been in the study of gene structure and expression. The organisation of genes within genomes is a fast-developing area that is essentially an extension of the early work on gene structure.

In this chapter I present a brief overview of the structure and function of DNA, and its organisation within the genome (the total genetic complement of an organism). This provides the non-specialist reader with an introduction to the topic, and may also act as a useful refresher for those who have some background knowledge of DNA. More extensive accounts of the topics presented here may be found in the textbooks listed in Suggestions for further reading.

The flow of genetic information

It is a remarkable fact that an organism's characteristics are encoded by a fourletter alphabet, de fining a language of three-letter words. The letters of this alphabet are the bases adenine (A), guanine (G), cytosine (C) and thymine (T), with triplet combinations of these bases making up the ‘dictionary’ that is the genetic code.

The expression of genetic information is achieved ultimately via proteins, particularly the enzymes that catalyse the reactions of metabolism. Proteins are condensation heteropolymers synthesised from amino acids, of which 20 are used in natural proteins. Given that a protein may consist of several hundred amino acid residues, the number of different proteins that may be made is essentially unlimited; thus great diversity of protein form and function can be achieved using an elegantly simple coding system. The genetic code is shown in Table 2.1.

The flow of genetic information is unidirectional, from DNA to protein, with messenger RNA (mRNA) as an intermediate.