Fig 1.
Genotyping of rs1057910 (left) and rs4986893 using cassette with Cal Red 610 and Quasar 670 in routine qPCR master mix following KASP protocol. Allele specific primers were added with tail for Cal Red 610 (G for rs1057910, and C for rs4986893) or Quasar 670 (T for rs1057910, and T for rs4986893). Synthetic positive control of each gene was genotyped individually (homozygotes being approximately 300,000 copies/ reactions, and heterozygotes being 150,000 copies of each allele per reaction). One None-Template Control (NTC) was included in each run.
Table 1.
Genotyping result reported by hospital and by KASP using new cassettes.
Fig 2.
Limit of detection of 4-fluorescent KASP.
Allele specific primers were added with tails for Cal Red 610 (G for rs1057910), Quasar 670 (T for rs1057910), FAM (A for rs9923231) and Hex (G for rs9923231). Positive control of rs1057910 and rs9923231 were diluted by 10-fold from 6.23 x 1010 copies/ μl (100 nM stock solution) to 6.2 copies/ μl. The two synthetic genes were diluted together to make duplex-SNP controls, where allele-G for rs1057910 and allele-A for rs9923231 were put in one solution and the other two alleles were put in another solution. The heterozygote controls were made by mixing homozygote solution of both alleles with 1:1 ratio at each concentration. 5 μl of each concentration was used in each reaction. For each concentration, each genotype was run in duplicates, with a total of 12 NTCs included. The data above showed the concentrations from 62 copies/ μl to 6.23 x 106 copies/ μl (6 concentrations in total). The 6.2 copies/ μl samples were not clustered correctly and therefore not included.