Towards low cost, multiplex clinical genotyping: 4-fluorescent Kompetitive Allele-Specific PCR and its application on pharmacogenetics

Abstract

Single-nucleotide polymorphisms (SNPs) is associated with efficacy of specific drugs. Although there are several methods for SNP genotyping in clinical settings, alternative methods with lower cost, higher throughout and less complexity are still needed. In this study, we modified Kompetitive Allele Specific PCR to enable multiplex SNP genotyping by introducing additional fluorescent cassettes that specifically help to differentiate more amplification signals in a single reaction. This new format of assay achieved a limit of detection down to 310 copies/ reactions for simultaneous detection of 2 SNPs with only standard end-point PCR workflow for synthetic controls, and genotyped 117 clinical samples with results that were in 100% agreement with hospital reports. This study presented a simplified, cost-effective high-throughput SNP genotyping alternative for pharmacogenetic variants, and enabled easier access to pharmaceutical guidance when needed.

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.

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.