AUTHOR’S REPLY

My colleagues and I thank Yurchenko et al. for their constructive and useful letter and in particular for showing that the minicircle sequence that we discovered is similar to a number of other minicircle sequences from L. donovani and L. infantum. This confirms that the sequence that we amplified was indeed of minicircle origin and not an artefact.

Yurchenko et al. ask whether the ability to amplify a single minicircle class is due to the nonrandom distribution of minicircles into the different classes or alternatively to nonrandom amplification of minicircle classes. In principle it seems likely that both these factors are involved; however, we did not have time to investigate these points since our primary objective was to demonstrate the sensitivity of the nested PCR and not to investigate minicircle organization.

Nonrandom distribution of minicircles into the different classes has been demonstrated, as Yurchenko et al. point out. Nonrandom amplification is also likely for two reasons. First, although the amount of variability in the conserved region is small, it is possible that there are sufficient differences between minicircle classes to affect amplification efficiency of some of them. The greater effective primer length in the nested PCR will increase the risk of not amplifying some minicircle classes. Secondly, it seems likely that some minicircle classes will be thermodynamically or kinetically favored in the PCR, particularly given the extensive secondary structure of minicircle DNA. These minicircles will be more efficiently amplified and will be the major products of any PCR; they may be the only product of the nested PCR given the very small quantities of template.

We attempted to minimize the risk of the first problem by designing the primers from an alignment of a wide range of Leishmaniaspecies. Our alignment (Fig. 1-1) shows that we used regions that are conserved across the mainLeishmania species. This strategy was based on the assumption that regions that were conserved across many species would also be conserved between classes in the same species. The inclusion of multiple sequences from L. infantum suggests that this is often the case. The complex fingerprints produced by the digestion of the PCR products from amounts of template greater than 1 fg showed that the nested PCR did indeed amplify multiple minicircle classes. Furthermore, it has been shown that the six 3′ bases of a primer are the most important for specificity and that some mismatches in the 5′ region are tolerated by the Taq enzyme (1-1). Our primers were anchored in regions in which the six 3′ bases were invariable. The alignment also shows that we designed the first set of primers against both CSB2 and CSB3 and not against CSB2 alone, as Yurchenko et al. seem to suggest.

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Fig. 1-1.

Alignment of the conserved region ofLeishmania kDNA minicircles showing the highly conserved sequence blocks (bold). The primers used in the nested PCR are shown. The reverse primers have been reversed and complemented. The alignment shows that the regions selected for the primers are highly conserved across the wide range of species used. It is therefore assumed that they will also be conserved across most minicircle classes in a given strain. The Genbank accession numbers of the strains are as follows:L. braziliensis, M87315; L. guyanensis, M87316;L. infantum 1, Z32847; L. infantum 2, Z32846;L. infantum 3, Z35501; L. peruviana, M87317;L. tropica, Z32843; L. infantum 4, Z35269;L. infantum 5, Z35270; L. infantum 6, Z35272;L. infantum 7, Z35273; L. infantum 8, Z35274;L. infantum 9, Z35292; L. infantum 10, Z35501-2;L. infantum 11, Z35501.

The different efficiencies of amplification of different minicircle classes were largely beyond our control; however, it may be interesting to investigate the effect of different solvent systems, particularly dimethyl sulfoxide, on the amplification.

Consequently, we think it is likely that our primers will not amplify all minicircle classes and will not amplify any minicircle class in proportion to its original abundance. This is a point that merits further attention for the light it might shed on minicircle organization. Nevertheless, we would like to emphasize that our experiments showed that the nested PCR is the most sensitive method available for detecting and identifying kDNA without the use of isotopically labelled probes. This has recently been confirmed by trials in Guatemala, where both L. braziliensis and L. mexicana are endemic. Furthermore, our data showed that the nested PCR reproducibly amplified the same minicircle classes from different members of a presumed clonal population. Consequently, although some selection may be involved it is reproducible and can be managed by using suitable controls.