PMC:1271393 / 11034-12659 JSONTXT

{"project":"2_test","denotations":[{"id":"16252244-15290772-2049614","span":{"begin":1300,"end":1304},"obj":"15290772"}],"text":"Molecular Analyses\nThe sequenced fragments of the CD209/CD209L genomic region are shown in figure 1. The entire CD209 region—including exons, introns, and ∼1 kb of the 5′ UTR corresponding to the promoter region—was sequenced, for a total of 5.5 kb per individual. For CD209L, we sequenced a total of ∼5.4 kb per individual, following the same approach used for CD209, with the exception of the neck region. That region was genotyped for its number of repeats, since it turned out to be highly polymorphic, which prevented the sequencing process. Genotyping was performed by a single PCR amplification followed by migration in 2% agarose gels. Human primers were used to both amplify and sequence the orthologous regions in chimpanzees. However, because of polymorphisms specific to the chimpanzee lineage, we could not obtain the entirety of the sequence. Thus, 4.9 kb (90% of the total) of the chimpanzee CD209 sequence were obtained, and 5.3 kb (98% of the total) of CD209L. Detailed information on primer sequences and PCR amplification conditions is available on request. All nucleotide sequences were obtained using the Big Dye terminator kit and the 3100 automated sequencer from Applied Biosystems. Sequence files and chromatograms were inspected using the GENALYS software (Takahashi et al. 2003; Centre National de Genotypage). As a measure of quality control, when new mutations were identified in primer binding regions, new primers were designed and sequence reactions were repeated, to avoid allele-specific amplification. All singletons observed in our data set were systematically reamplified and resequenced."}