Figure 1, depicts one such position in Sweh212, where double peaks are present in sequences with DNA from crude feces, and single cyst Sweh212_145, but not in single cysts; Sweh212_243 or Sweh212_236 (Figure 1). Sequencing of the
tpi locus generated from trophozoite cultures of the axenic, assemblage B isolate GS/M-H7, generated double peaks in three positions, namely 39, 45 and 264 SCH 900776 in vitro (Table 1) with the start codon set as position one. This sequence, along with sequences from public databases [GenBank: EF688030, EF688028 and FJ560571], were used as baseline for the GS/M-H7 analysis in order to define potential polymorphic subgroups when performing the single cell analyses (Table 1). Bi-directional sequencing of single GS/M-H7 trophozoites, with (n = 9) and without (n = 5) the pre-treatment of DNAreleasy, on a 530 bp region of tpi was performed in order to verify the occurrence of ASH within single Giardia cells. The chromatograms were carefully analyzed with regards to double peaks, and forward and reverse sequences
were subsequently aligned. All single GS/M-H7 trophozoites, which were pre-treated Gefitinib cost with DNAreleasy, displayed distinct double peaks in the same learn more positions as those from the GS/M-H7 crude isolate (Table 1). However, only one (20%) of the single GS/M-H7 trophozoites, that had not been pre-exposed to treatment with DNAreleasy, showed double peaks in all three positions
(Table 1). Thus, DNAreleasy increases the amplification efficiency from single parasites. Figure 1 Sequence chromatograms of nucleotide variations. Chromatogram of a sequence generated from crude DNA from patient Sweh212, where the position indicated with an arrow shows the presence of a double peak (a). Sequencing of single cysts from the same patient indicates the presence of a double peak or ASH at the single cell level Clomifene (b), and importantly, single cyst analyses also show that there are sub-populations present where double peaks do not exist in the same position (c and d). Bi-directional sequencing was also performed on DNA from clinical single cysts and sequences were aligned using variants of sub-assemblages BIII and BIV, as well as sequences from crude DNA from each respective sample as baselines, where possible. Positions that have earlier been suggested as variable between sub-assemblages BIII and BIV, are highlighted by an asterisk in Tables 1 2 3 4 5[10, 25].