A 50 bp or 200 bp DNA ladder marker (TaKaRa) was included in all gels to determine the size of the amplified DNA fragments. The selected VNTR loci and their characteristics are shown in Table 2. The forward primers for the PCR were labeled at the 5′ end with either FAM or HEX or TAMRA. The reverse primers were synthesized unlabelled (Table 2) (20–22). The final protocol selleck compound consisted of three multiplex PCR. M1 contained 10 pmol TR1, 8 pmol TR3, 6 pmol TR5, and 8 pmol TR6 of the primer sets;
M2 contained 2.5 pmol TR2, 10 pmol TR7 and 15 pmol TR9 of the primer sets. M3 contained 10 pmol TR4 and 10 pmol TR8 of the primer-sets. M1 and M3 were performed according to standard PCR cycling as above. For M2, the initial denaturation at 95°C for 10 min was followed by 35 cycles: denaturation at 95°C for 1 min, 58°C for 40 s, and 72°C for 2 min; and a final extension of 10 min at 72°C. PCR fragments from M1 and M2 were analyzed using multicolored capillary electrophoresis (20–22). The amplicons of M1 and M2 were diluted in water to 1:120. After denaturation by heating, the amplicons were separated by capillary electrophoresis on an ABI 3730xl genetic analyzer with a GeneScan 500 LIZ size standard (Applied Biosystems, Tokyo,
Japan). Data were collected and the lengths of the amplicons determined according to color and size using GeneMapper software v. 4.0 (Applied Biosystems). Because the fragments from M3 PCR amplification are larger learn more than 500 bp (at the upper limitation for the GeneScan 500 LIZ size marker), the PCR fragments from M3 were resolved using horizontal agarose gel electrophoresis; and the sizes of the PCR amplification were deduced by visual inspection using a flanking reference DNA ladder. Whereas, because the unit sizes of the repeat TR8 and TR4 were 231 bp and 90 bp, respectively, they were determined directly. All of the tandem repeat loci patterns generated from TRF and the repeat copy numbers (alleles) of GZ1, P1/7, SC84 and 89/1591 were rounded to the nearest whole numbers. The number of repeat units for the nine VNTR loci and the calculated sizes of amplicons for S.
suis strains P1/7, SC84 and GZ1 were used as the standards to infer the number of repeat units HSP90 for each locus in the isolates tested. All amplicons of different lengths in each locus were subjected to nucleotide sequence determination to verify the repeat sequence and the number of repeat units (20, 23). The primers (without the dye label) used for nucleotide sequence determination were the same as the primer sets used for PCR amplification. In those instances where no amplification was observed at a particular locus despite multiple attempts, the allele was denoted as “0”, whereas a decimal allele was designated to describe a locus allele that contained both flanking sequences but non-whole number repeat units. In each strain, the sequence of TR9 was also determined in both directions to confirm the results of the capillary electrophoresis.