There are a number of PCR-based typing systems which are in use, but for purposes of this discussion only two will be considered: amplified fragment length polymorphism (AFLP) and multilocus variable number tandem (MLVA) repeat analysis. AFLP was first described in 1995.33 It is a powerful DNA fingerprinting technique that incorporates aspects of PCR and RFLP In AFLP, restriction fragments from a total genomic digest (the enzymes employed are determined by the user) have short double-stranded oligonucleotides (adaptors) of defined sequence joined (ligated is the correct term) to both ends of the restriction fragment.33 The adaptors can then serve as complimentary sequences for a defined set of PCR primers that are complimentary to the adaptor sequence and part of the restriction site sequence with some extension into the sequence flanking the restriction site. The fragments are then selectively amplified by PCR to produce a pattern of fragments of various sizes similar to the patterns generated by RFLP analysis. Incorporation of labeled primers which carry a fluorescent molecule (a "fluorescent tag" ) has adapted the AFLP technique to run on automated DNA sequencing machines. Some examples of applications for the AFLP technique include typing geographically diverse collections of Bacillus anthracis isolates, studying the diversity of Bacillus cereus and Bacillus thurengensis isolates from Norwegian soil, and studying the genetic diversity of Vibrio cholerae (causative agent of cholera) isolates from the Chesapeake Bay.34-38 AFLP is also being explored as a tool in bacterial taxonomy investigations, because of the power shown by AFLP to discriminate between highly related strains of the same species.39 AFLP is advantageous because it can be used to type organisms for which very little or even no sequence data are available.
AFLP generates data that are, in a sense, binary, meaning the fragments are either present or they are not. Such "two-form" data in the lexicon of the geneticist is termed "bi-allelic" where alleles can be thought of as different forms of a genetic site or a gene. For pathogens that show very little genetic diversity, bi-allelic typing markers may be insufficient to discriminate between closely related isolates. More complex discriminatory markers with more than two forms may be required. VNTR elements are such markers, and they can have many different forms. The number of forms will vary according to the number of repeats that are found in the element. In VNTR analyses, sequences flanking the VNTR element are amplified using PCR primers, and the size of the fragments generated is then scored. Fragment sizes will vary depending upon how many repeats are present in the element.40 Fluorescent-tagged PCR primers are used to facilitate automated analysis. By using different colored fluorescent tags and primers, the analyses can be multiplexed to score several VNTR elements at once, hence the name multilocus VNTR analysis (also known as MLVA).40 This approach has been shown to be very powerful for discrimination between closely related isolates of organisms such as Bacillus anthracis, Yersinia pestis, and Francisella tularensis.40-42 The power of the MLVA approach lies in the multi-allelic nature of the data produced compared to the bi-allelic nature of AFLP or RFLP data and the high mutation rate of VNTR elements. The drawback to the MLVA approach is that it cannot be used on an organism for which no sequence information is available. DNA sequencing (or hybridization assays) is required to identify possible VNTR locations (loci) in the genome of an organism.
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