Applications of Recombinant DNA Technology

POGC14 9/11/2001 11:11 AM Page 274 CHAPTER 14 Applications of recombinant DNA technology Introduction Theme 1: Nucleic acid sequences as diagnostic tools Biotechnology is not new. The making of beer, wine, bread, yoghurt and cheese was practised by ancient Introduction to theme 1 civilizations, such as the Babylonians, the Romans and the Chinese. Much, much later came vaccines, Nucleic acid sequences can be used diagnostically in the production of basic chemicals (e.g. glycerol, two different ways. The first is to determine whether citric acid, lactic acid) and the development of anti- a particular, relatively long sequence is present in or biotics. In each of these examples, existing properties absent from a test sample. A good example of such of microorganisms were exploited. For example, an application is the diagnosis of infectious disease. Penicillium species naturally make penicillin. What By choosing appropriate probes, one can ascertain the scientists have done is to increase the yield of in a single step which, if any, microorganisms are penicillin by repeated rounds of mutation and present in a sample. Alternatively, a search could be selection, coupled with optimization of the growth made for the presence of known antibiotic-resistance medium. Similarly, sexual crosses between related determinants so that an appropriate therapeutic plant species have created high-yielding and disease- regime can be instituted. In the second way in which resistant varieties of cereals. These improved cereals sequences are used diagnostically, the objective is to represent new combinations of genes and alleles determine the similarity of sequences from different already existing in wild strains. individuals. Good examples of this approach are With the development of gene manipulation tech- prenatal diagnosis of genetic disease and forensic niques in the 1970s, there was a major paradigm profiling (‘DNA fingerprinting’). shift. For the first time microorganisms could be made to synthesize compounds that they had never Detection of sequences at the gross level synthesized before, e.g. insulin production in E. coli ( Johnson 1983). Soon all sorts of commercially or Imagine that a seriously ill individual has a disorder therapeutically useful proteins were being made in of the gastrointestinal tract. A likely cause is a bacteria, principally E. coli, and thus the modern microbial infection and there are a number of candid- biotechnology industry was born. As the techniques ate organisms (Table 14.1). The question is, which developed for manipulating genes in bacteria were organism is present and to which antibiotics is it extended to plants and animals there was a con- susceptible? The sooner one has an answer to these comitant expansion of the biotechnology industry questions, the sooner effective therapy can begin. to exploit the new opportunities being provided. Traditionally, in such a case, a stool specimen would Today there are many different facets to the com- be cultured on a variety of different media and would mercial exploitation of gene manipulation tech- be examined microscopically and tested with vari- niques as shown in Fig. 14.1. Rather than discuss ous immunological reagents. A simpler approach all these topics in detail, for that would take a book is to test the sample with a battery of probes and in itself, we have chosen to focus on six inter- determine which, if any, hybridize in a simple dot- disciplinary themes that reflect both the successes blot assay. With such a simple format it is possible to achieved to date and the likely successes in the next vary the stringency of the hybridization reaction to decade. accommodate any sequence differences that might POGC14 9/11/2001 11:11 AM Page 275 Applications of recombinant DNA technology 275 Transgenic Nucleic animals acids Therapeutic proteins Anti-sense Improved farm Gene therapy/repair animals Diagnostic probes Disease models Vaccines Recombinant DNA technology ‘Pharming’ Vaccines Vaccines Small molecules Small molecules Therapeutic proteins Therapeutic proteins Improved plants Enzymes Biopolymers Transgenic Recombinant plants microbes Fig. 14.1 The different ways that recombinant DNA technology has been exploited. exist between the probe and the target. The down- Table 14.1 Pathogens causing infection of the side of this approach is that if one wishes to test the gastrointestinal tract. sample with 10 different probes, then 10 different dot blots are required; otherwise there is no way of Bacteria Salmonella spp. determining which probe has bound to the target. Shigella spp. Another disadvantage of this approach is that Yersinia enterocolitica sufficient target DNA must be present in the sample Enterotoxigenic E. coli to enable a signal to be detected on hybridization. Clostridium difficile Both of these problems can be overcome by using the Clostridium welchii Campylobacter spp. polymerase chain reaction (PCR). Aeromonas spp. In a traditional dot-blot assay, the sample DNA is Vibrio spp. immobilized on a membrane and hybridized with a Viruses selection of labelled probes. An alternative is a Rotaviruses ‘reverse dot blot’, where the probes are immobilized Enteroviruses on the membrane and hybridized with the sample. Enteric adenoviruses In this way, only one hybridization step is required, Protozoa because each probe occupies a unique position on Entamoeba histolytica the membrane (Fig. 14.2). For this approach to Giardia Iamblia work, the sample DNA needs to be labelled and this can be achieved using the PCR. This has the added POGC14 9/11/2001 11:11 AM Page 276 276 CHAPTER 14 Conventional dot blot Membrane Test DNA Test DNA Test DNA Test DNA Test DNA Probe Probe Probe Probe Probe A B C D E Reverse dot blot Probe AProbe B Probe C Probe D Probe E Labelled Fig. 14.2 Comparison of conventional test DNA dot blot assay with reverse dot blot method. Note that in the latter there is only a single hybridization reaction, regardless of the number of probes used, whereas in the former each probe has to be tested in a separate hybridization step. benefit of amplifying the sample, thereby minimizing Despite the difficulties noted above, multiplex PCR the amount of target DNA required. The downside has been used successfully in the diagnosis of infec- of this approach is that the amplification step needs tious diseases. For example, Heredia et al. (1996) to be done with multiple pairs of primers (multiplex have used the method to simultaneously examine PCR), one for each target sequence. Although blood for the presence of HIV-1, HIV-2, human multiplex PCR is now an established method, con- T-cell lymphotropic virus (HTLV)-I and HTLV-II. siderable optimization is needed for each application Similarly, Grondahl et al. (1999) have used the (Elnifro et al. 2000). The major problems encoun- method to identify which of nine different organisms tered are poor sensitivity or specificity and/or pre- is responsible for respiratory infections. Once the ferential amplification of certain specific targets. clinical microbiologist knows the identity of a micro- The presence of more than one primer pair in the organism in a specimen, he/she can select those reaction increases the chance of obtaining spurious antibiotics that might be effective, provided that amplification products because of the formation of the organism in question does not carry multiple primer dimers. Such non-specific products may be drug-resistance determinants. The presence of these amplified more efficiently than the desired target. determinants can also be established using multi- Clearly, the design of each primer pair is crucial to plex PCR. Alternatively, if a virus causes the infec- avoiding this problem. Another important feature is tion, one can monitor the progress of the infection by that all primer pairs should enable similar amplifica- using quantitative PCR (see p. 23). This approach tion efficiencies for their respective targets. has been used to monitor cytomegalovirus infec- POGC14 9/11/2001 11:11 AM Page 277 Applications of recombinant DNA technology 277 tions in kidney-transplant patients (Caballero et al. two 19-mer oligonucleotides, one of which was 1997). It is worth noting that the development complementary to the amino-terminal region of the of multiplex PCR technology is being facilitated by normal β-globin (βA) gene and the other of which the rapid progress in the sequencing of microbial was complementary to the sickle-cell β-globin gene genomes (for an up-to-date list, see http//igweb.integ- (βS). These oligonucleotides were radiolabelled and ratedgenomics.com/GOLD/), since the data gener- used to probe Southern blots. Under appropriate ated enable species-specific genes or sequences to be conditions, the probes could distinguish the normal identified. and mutant alleles. The DNA from normal homozygotes only hybridized with the βA probe and DNA from sickle-cell homozygotes only hybridized with Comparative sequence analysis: single- the βS probe. DNA of heterozygotes hybridized with nucleotide polymorphisms (SNPs) both probes. These experiments, therefore, showed In prenatal diagnosis of genetic disorders, there is that oligonucleotide hybridization probes can dis- a need to determine which alleles of a particular criminate between a fully complementary DNA and locus are being carried by the fetus, i.e. is the fetus one containing a single mismatched base. Similar homozygous for the normal or the deleterious allele results have been obtained (Fig. 14.3) with a point or is it heterozygous? In forensic DNA profiling the mutation in the α-antitrypsin gene, which is implic- requirement is to match DNA from the perpetrator ated in pulmonary emphysema (Cox et al. 1985). of a crime with that of a suspect. In each case, a The single-base changes that occur in the two definitive answer could be obtained by sequencing clinical examples just quoted are examples of single- the relevant samples of DNA. While this is possible, it nucleotide polymorphisms (SNPs, pronounced is not practicable for mass screening.

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