Browsing by Subject "TaqMan"

Single nucleotide polymorphism (SNP) is DNA variation of a single nucleotide. SNPs are the most common mutations and millions of single nucleotide differences distinguish humans genetically from each other. Different gene variants of a single nucleotide affect nutritional and pharmacological metabolism and gene test results can therefore guide the diet and medication. SNPs are tested either by sequencing, gene arrays or quantitative polymerase chain reaction (qPCR). In this thesis the SNPs were tested by using 132 different TaqMan probes and the SmartChip qPCR platform. 20 participants donated two buccal swab samples and one dried blood spot (DBS) card. Buccal swabs were extracted using two different methods. DNA was also sent to a reference laboratory to be analysed by gene arrays and two participants also sent their DNA samples to two direct-to-consumer (DTC) commercial gene test companies. Low concentration buccal swab samples (<3 ng/µl) produced mismatches whereas DBS samples had high genotyping accuracy even at low DNA concentration. For buccal swabs there was a correlation between the DNA concentration and qPCR call rate (p<0.0001) but not for DBS samples (p>0.05). The allele separation of one TaqMan assay was not sufficient between minor homozygote and heterozygote clusters. For other 131 TaqMan assays excluding the low DNA concentration swab samples, the reproducibility rate was 99.97 %. The reproducibility rate with the DTC companies was 98.36 %. The lower reproducibility rate emphasizes the importance of the manual review of the genotyping raw data, which is not possible with huge sequencing or microarray SNP datasets. SNPs are found on various genetic structures which leads to inconsistent genotyping data that is difficult to analyse with a single algorithm. The SmartChip system combined with the TaqMan assays is a highly reproducible SNP genotyping method when the DNA concentration of the samples is high and the results are manually reviewed.