Dale_Molecular Genetics of Bacteria 4th ed

genes generated did not correspond to known organisms and were clearly derived from hitherto unknown and uncultured bacterial species.

9.5.2 Diagnostic use of PCR

Traditional methods for the detection and identification of bacteria rely on growing the organism in pure culture and identifying it by a combination of staining methods, biochemical reactions and other tests. This applies equally to detection of environmental organisms (in soil or water), bacteria in food (including milk and drinking water) or pathogens in samples from patients with an infectious disease. However these methods are slow, requiring at least 24 h or several weeks for slow-growing organisms such as Mycobacterium tuberculosis. In addition, there are some bacteria, such as Mycobacterium leprae (the causative agent of leprosy) that still cannot be grown in the laboratory.

In principle, gene probes could be used to provide quicker results by directly detecting the presence of specific DNA in the specimen. However, this only works if the bacteria present are plentiful. Gene probes are not sensitive enough to detect the small numbers of organisms that may be present, and significant, in such specimens. The technique that is needed is the polymerase chain reaction (PCR) as described in Chapter 2. This provides greatly enhanced sensitivity, being capable (in theory) of detecting a single organism. In order to apply this to the detection of a specific species, it is necessary to know the sequence of a gene that is characteristic of that species – that is, it is always present (and the sequence is conserved) in that species, but is absent or significantly different in other bacteria. A pair of PCR primers can then be designed which will anneal to this target sequence so that PCR will amplify a DNA fragment that can be easily detected. Other bacteria, lacking the specific binding sites for those primers, will not give an amplified product. In a research laboratory the amplified product (amplicon) would commonly be detected by gel electrophoresis, sometimes combined with Southern blotting and hybridization with specific gene probes (see Chapter 2) to increase the sensitivity and specificity of the procedure. The commercial kits that are now available for detection of some bacterial pathogens (some using forms of gene amplification that are distinct from PCR) use other, quicker, ways of detecting product amplification. A technique known as real-time PCR which produces results more rapidly than gel electrophoresis and has the additional advantage of quantifying the target present in the sample, will be discussed in Chapter 10.

9.5.3 Molecular epidemiology

Epidemiology is the study of the occurrence and distribution of diseases. By identifying the source of infection, the measures necessary to control an outbreak

c

Figure 9.13 Restriction fragment length polymorphism (RFLP). The diagram shows a region of the chromosome that hybridizes to a specific probe and some of the possible reasons for polymorphism in the Southern blot pattern: (1) The ‘original’ sequence, in which the probe detects three DNA fragments labelled a,b and c. (2) Loss of a restriction site by mutation results in fragments b and c being replaced by a single larger fragment.

(3) An insertion within fragment b changes its size, without altering the number of fragments. (4) A deletion within fragment a reduces its size

10

Gene Mapping to Genomics

In this chapter, the methods available for studying the location, structure and expression of genes will be examined. This includes in vivo methods of gene mapping as well as genome sequencing and methods for studying the expression of genes individually and genome-wide. At the level of this book it is possible to provide only an introduction to some of the most exciting aspects of modern molecular biology and genetics.

10.1 Gene mapping

One of the main objectives in genetic analysis is the determination of the position of genes on the chromosome. In isolation, this may seem a rather arcane occupation, but knowledge of the organization of the chromosome does play a major role in understanding gene function and has contributed extensively to the advances described in Chapter 9.

In bacteria, the classical methods of gene mapping depend on the production of recombinants by gene transfer using conjugation, transformation and transduction. These methods have now been supplemented, although not entirely supplanted, by methods based on in vitro gene technology. Nevertheless, a basic understanding of these methods is valuable for an appreciation of the development of our knowledge of bacterial genetics.

10.1.1 Conjugational analysis

In an earlier chapter it was shown that integration of the F plasmid into the E. coli chromosome produces an Hfr strain which is capable of transferring a copy of the chromosome to a suitable recipient. Transfer of the whole chromosome would

with a multiply auxotrophic recipient (e.g. thr leu trp his arg), the number of recipients that have received each of the markers can be determined by plating aliquots of the mixture on a minimal medium supplemented with four out of the five amino acids. For example, the number of thr recombinants is measured using a medium that contains leucine, tryptophan, histidine and arginine, but not threonine. It is of course necessary to prevent growth of the prototrophic donor, for example by using a streptomycin-resistant recipient and including streptomycin in the medium. Streptomycin in this instance is used as a counterselecting agent. On this medium, the donor will be unable to grow (because of the streptomycin) and the parental recipient will not grow (because of the absence of threonine). The only cells that can grow will be the recombinant recipients that have received the thr gene.

The result is illustrated by Figure 10.2. In this instance, the HfrH donor has been used, from which the genes are transferred in a clockwise direction starting very close to the thr locus. There is a linear relationship between the logarithm of the number of recombinants and the map position of the genes concerned. If it is assumed that the position of the trp gene is not known, determining the number of Trpþ recombinants will allow the gene to be mapped as shown in Figure 10.2.

An alternative method for more accurate mapping of genes that are transferred relatively early in mating involves deliberately separating the mating pairs (by violent agitation) in samples of the mixture at different times after the start of mating (interrupted mating). Recombinants that have received a specific gene start

Figure 10.2 Gene mapping using the gradient of transfer by conjugation. Determination of the position of the trp gene

to appear at a certain time after the start of mating (the time of entry), which is a measure of the distance of that gene from the origin of transfer.

10.1.2 Co-transformation and co-transduction

Transformation can be used for mapping the relative positions of genes, by selecting recombinants in which one marker has been transferred and then determining the frequency of recombination for a second marker. If the two are close together, they will tend to be inherited together.

If the two markers are labelled A and B: the donor is wild type for both genes (AþBþ), while the recipient is the double mutant (A B ). After transformation of the recipient with chromosomal DNA extracted from the donor, the cells are plated on a medium that only allows Aþ cells to grow. The colonies that result from this (transformants) can then be tested for the presence of the Bþ gene (the unselected marker). Two recombination events (crossovers) are needed to incorporate a piece of linear DNA into the chromosome. Since transformants that have received gene A have been selected, one crossover must be to the left of A in Figure 10.3 and the second will be to the right of A – either between A and B or

Figure 10.3 Determination of the relative position of two genes by co-transformation frequency