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Temperature (°C)

Fig. 3. SYBR Green I melting curve analysis of Cp492 amplicons from one C. parvum type 1 isolate (TU502) and three type 2 isolates (TAMU, 7C, CISD). Because the differences in the melting temperature are determined by the length of the amplicon and by the base composition, melting temperatures do not always correlate with amplicon length or gel mobility. In the example shown, isolate TU502 has a lower melting temperature than isolate CISD in spite of the fact that TU502 has the highest number of repeats. F1, SYBR Green fluorescence measured at 530 nm. The negative value of the first derivative of F1 over the temperature is plotted on the y-axis.

pipet and repeating the aspiration. As with all procedures involving ether, buildup of pressure should be prevented by periodically venting the tubes while performing the extraction.

5. Because of the relatively high cost of the immunomagnetic products, it is often possible to reduce the volume of immunomagnetic beads and adjust the buffer volume proportionally. Unless we expect to extract a large number of oocysts, say more than 107, the volume of beads and buffers can be reduced to one-half (50-^L bead suspension) or one-third (30-^L bead suspension) of the recommended volumes. For routine genetic analyses, this method will recover sufficient oocysts for extracting DNA and performing multiple PCR assays.

6. Several modifications to the standard amplification program were attempted, but the amplification curves obtained with (AT) microsatellites remained flat in comparison with those amplified from nonrepeat sequences or repeats containing C or G residues. This observation indicates suboptimal amplification. In addition, melting curves obtained from (AT) microsatellites frequently display multiple peaks. We assume that this is caused by partial melting of the AT-rich region at a lower temperature than that of the sequences flanking the repeat. If this assumption is confirmed, (AT) repeats may not be suitable for real-time PCR typing. Additional microsatellites containing repeats of G and/or C residues are being evaluated as alternative markers.

7. The general applicability of SYBR Green I real-time PCR for allele discrimination is still being investigated. Preliminary results indicate that AT microsatellites are not efficiently amplified. Repeats containing G and C nucleotides appear to amplify better, but rela tively large differences in repeat length (6 bp or more) may only result in relatively small differences in melting temperature. This observation contrasts with those from real-time PCR methods based on internal fluorescent probes, which can generate measurable and reproducible melting profiles on the basis of single-nucleotide polymorphisms (30,31).

References

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