Use of a novel DNA melting profile assay for the identification of PCR-amplified genomic sequences encoding for variant-specific surface proteins from the clonal GS/M-83-H7 line of Giardia lamblia

During infections, Giardia lamblia undergoes a continuous change of its maj or surface antigens, the variant-specific surface proteins (VSPs). Many stu dies on antigenic variation have been performed using G. lamblia clone GS/M -83-H7, which expresses surface antigen VSP H7. The present study was focus ed on the identification and characterization of vsp gene sequences within the genome of the clonal G. lamblia GS/M-83-H7 line. For this purpose, we a pplied a PCR which specifically amplified truncated sequences from the 3 ' -terminal region of the vsp genes. Upon cloning, most of the vsp gene ampli fication products were shown to be approximately identical in size and thus could not be distinguished from each other by conventional gel electrophor esis. In order to pre-estimate the sequence complexity within the large pan el of vsp clones isolated, we elaborated a novel concept which facilitated our large-scale genetic screening approach: PCR products from cloned DNA mo lecules were generated and then subjected to a DNA melting profile assay ba sed on the use of the LightCycler Instrument. This high-throughput assay sy stem proved to be well suited to monitor sequence differences between the a mplification products from closely related vsp genes and thus could be used for the primary, sequence-related discrimination of the corresponding clon es. After testing 50 candidates, vsp clones could be divided into five grou ps, each characterized by an individual DNA melting profile of the correspo nding amplification products. Sequence analysis of some of these 50 candida tes confirmed data from the aforementioned assay in that clones were demons trated to be identical within, but different between, the distinct groups. The nucleotide and deduced amino acid sequences of five representative vsp clones showed high similarities both among each other and also with the cor responding gene segment of the variant-specific surface antigen (VSP H7) ex pressed by the original GS/M-83-H7 variant type. Furthermore, three of the genomic vsp sequences turned out to be identical to vsp sequences that repr esented previously characterized transcription products from in vivo- or in vitro-switched GS/M-83-H7 trophozoites. In conclusion, the DNA melting pro file assay seems to be a versatile toot for the PCR-based genotyping of mod erately or highly diversified sequence orthologues.