**4.2.5 RAPD (randomly amplified polymorphic DNA)**

The standard RAPD technology (Williams et al., 1990) utilises short synthetic oligonucleotides (10 bases long) of random sequences as primers to amplify nanogram amounts of total genomic DNA under low annealing temperatures by PCR. Amplification products are generally separated on agarose gels and stained with ethidium bromide. Decamer primers are commercially available from various sources (e.g., Operon Technologies Inc., Alameda, California). PCR amplification with primers shorter than 10 nucleotides [DNA amplification fingerprinting (DAF)] has also been used producing more complex DNA fingerprinting profiles (Caetano-Annoles et al., 1991).

Although these approaches are different with respect to the length of the random primers, amplification conditions and visualisation methods, they all differ from the standard PCR

Laboratory Typing Methods for Diagnostic of

amplified fragments.

sized on an automated sequencer.

second one with a higher cutting frequency (like MseI or TaqI).

ligation, while religated fragments are cleaved again.

Salmonella Strains, the "Old" Organism That Continued Challenges 361

For AFLP analysis, only a small amount of purified genomic DNA is needed; this is digested with two restriction enzymes, one with an average cutting frequency (like EcoRI) and a

Double-stranded oligonucleotide adapters are designed in such a way that the initial restriction site is not restored after ligation, which allows simultaneous restriction and

An aliquot is then subjected to two subsequent PCR amplifications under highly stringent conditions with adapter-specific primers that have at their 39 ends an extension of one to

An extension of one selective nucleotide amplifies 1 of 4 of the ligated fragments, whereas three selective nucleotides in both primers amplify 1 of 4,096 of the fragments. The PCR

After polyacrylamide gel electrophoresis a highly informative pattern of 40 to 200 bands is obtained. The patterns obtained from different strains are polymorphic due to (i) mutations in the restriction sites, (ii) mutations in the sequences adjacent to the restriction sites and complementary to the selective primer extensions, and (iii) insertions or deletions within the

Optimization of restriction enzymes and adapter-specific primers is ongoing for the *Salmonella* (Garaizar et al., 2000), but the technique appears more reproducible than ribotyping techniques (Savelkoul et al., 1999). Some of the studies have shown specificity to

Alternative AFLP typing procedures are based on one enzyme with a single adapter and analysis by agarose gel electrophoresis (Gibson et al., 1998). A major improvement has been obtained using a fluorescent amplified fragment length polymorphisms (FAFLP) technique that followed the same principles of AFLP yet the adapter-specific primers were tagged with a fluorescent moiety (Tamada et al., 2001). Fluorescent tagged fragments are then accurately

FAFLP analysis of *S. typhimurium* generated 45-50 fragments ranging in size from 80-430 bp, though only a subset of these fragments were polymorphic among the strains. FAFLP

Sizing was enhanced by incorporation of a fluorescent internal marker (Tamada et al., 2001). This accurate sizing, combined with the ability to acquire and analyze the data as a gel image, electrophorogram or in a tabular data format will allow comparison of patterns

FAFLP appears quite promising. Disadvantages include the need for a greater technical expertise. In fact, despite that AFLP has been considered as a highly discriminative method, it remains a labour- and cost-intensive technique (Riley, 2004). Set up costs may be

A recently developed methodology (Maiden et al., 1998) called multilocus sequence typing (MLST) may provide an ideal balance of high discriminatory power and a powerful data analysis capability requiring minimal human input. Multilocus sequence typing (MLST) is a molecular typing strategy that compares DNA sequences from portions of housekeeping or virulence genes and/or rRNA sequences which varies due to mutation or recombination events (Maiden et al., 1998). Nucleotide differences in the individual genes

the serotype level with occasional subserotype discrimination (Garaizar et al., 2000).

grouped the isolates into four distinct clusters while PFGE generated three clusters.

among different laboratories or within databanks (Savelkoul et al., 1999).

prohibitive until automated sequencers become more affordable.

**4.2.7 MLST (multilocus sequence typing)** 

three nucleotides running into the unknown chromosomal restriction fragment.

primer which spans the average-frequency restriction site is labeled.

condition (Erlich, 1989) in that only a single oligonucleotide of random sequence is employed and no prior knowledge of the genome subjected to analysis is required.

At an appropriate annealing temperature during the thermal cycle, oligonucleotide primers of random sequence bind several priming sites on the complementary sequences in the template genomic DNA and produce discrete DNA products if these priming sites are within an amplifiable distance of each other.

The profile of amplified DNA primarily depends on nucleotide sequence homology between the template DNA and oligonucleotide primer at the end of each amplified product. Nucleotide variation between different sets of template DNAs will result in the presence or absence of bands because of changes in the priming sites. Recently, sequence characterised amplified regions (SCARs) analysis of RAPD polymorphisms (Bardakci & Skibinski, 1999) showed that one cause of RAPD polymorphisms is chromosomal rearrangements such as insertions/deletions. Therefore, amplification products from the same alleles in a heterozygote differ in length and will be detected as presence and absence of bands in the RAPD profile.

Although the RAPD method is relatively fast, cheap and easy to perform in comparison with other methods that have been used as DNA markers, the issue of reproducibility has been of much concern since the publication of the technique. In fact, ordinary PCR is also sensitive to changes in reaction conditions, but the RAPD reaction is far more sensitive than conventional PCR because of the length of a single and arbitrary primer used to amplify anonymous regions of a given genome. This reproducibility problem is usually the case for bands with lower intensity. The most important factor for reproducibility of the RAPD profile has been found to be the result of inadequately prepared template DNA (Welsh & McClelland, 1994). Differences between the template DNA concentration of 2 individuals' DNA samples result in the loss or gain of some bands (Bardakci, 1996).

Since RAPD amplification is directed with a single, arbitrary and short oligonucleotide primer, DNA from virtually from all sources is amenable to amplification. Therefore, DNA from the genome in question may include contaminant DNA from infections and parasites in the material from which the DNA has been isolated. Special care is needed for keeping out the DNA to be amplified from other sources of DNA.

Finally, due to the amplification conditions, RAPD method is sensitive to slight changes within amplification parameters, thus it is hard to achieve reproducibility. However, ribotyping is a supplementary tool in conjunction with other typing methods (Yan et al., 2003).
