J. Chartier and H.C. Birnboim
DNA Genotek Inc., Ottawa, ON, Canada
DNA collected with Oragene works well with TaqMan SNP Genotyping Assays. SNPs in the thymidylate synthetase and apolipoprotein E genes were reliably detected.
Single-nucleotide polymorphisms (SNPs) are highly abundant, and are estimated to occur at 1 out of every 1,000 bases in the human genome (ref. 1). In addition to diagnostic applications, SNPs are useful as markers in population genetics and evolutionary studies (ref. 2). The TaqMan 5 nuclease assay is a widely-used SNP genotyping technology from Applied Biosystems. The purpose of this study was to evaluate the compatibility of Oragene DNA Self-Collection Kits with TaqMan SNP Genotyping Assays.
Materials and Methods
Saliva was collected from 25 donors using Oragene kits. DNA was purified from 200 L aliquots of Oragene/saliva samples using the ethanol precipitation protocol supplied with the kits. Purified DNA was redissolved in 200 L of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). DNA was quantified using a fluorimeter and SYBR Green I dye (Molecular Probes) according to the F/D Protocol (ref. 3).
Validated TaqMan SNP Genotyping Assays were obtained from Applied Biosystems. The assays are described in Table 1. The probes were labeled with FAM or VIC dye at the 5 end and a minor-groove binder and non-fluorescent quencher at the 3 end. The reaction components for the allelic discrimination reactions were set up according to Table 2. Although it is recommended to use 1 to 20 ng of DNA per reaction (ref. 4), 1 L of DNA was added to each 25 L PCR reaction without adjusting the concentrations. SNP genotyping reactions were performed on a Rotor-Gene 3000 real-time quantitative thermal cycler (Corbett Research) using the cycling conditions in Table 3.
The purifi ed DNA from 25 samples had a median concentration of 19.1 ng/L and ranged from 1.2 to 53.4 ng/L. Consequently, a number of the genotyping reactions used more than the 20 ng of DNA template recommended by the manufacturer.
Allelic discrimination plots
Figure 1 shows the allelic discrimination plot for the thymidylate synthetase assay and Figure 2 shows the plot for the apolipoprotein E assay. The plots are presented as the signal (average fluorescence between cycles 41 and 45) minus the background (average fluorescence between cycles 21 and 25).
Discussion and Conclusions
TaqMan assays are widely used for SNP genotyping. As with other molecular genetic techniques, the quality and purity of DNA is important for reliable results. Our findings indicate that DNA samples collected and purified using Oragene kits are suitable for allelic discrimination using TaqMan probes. Clear discrimination between different genotypes is evident in Figures 1 and 2.
Although it is recommended to use 1 to 20 ng of DNA per reaction, this study used DNA amounts ranging from 1.2 to 53.4 ng and all 25 samples gave clear, interpretable results. It was possible to perform the reactions without first determining the amount of DNA. Thus, the step of adjusting the DNA concentrations co uld be avoided. In summary, DNA collected with Oragene is suitable for SNP genotyping with TaqMan assays.
1. Sachidanandam, R. et al. (2001). A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms. Nature. 409, 928933.
2. Schork, N. J., Cardon, L. R. & Xu, X. (1998). The future of genetic epidemiology. Trends Genet. 14, 266272.
3. Technical Bulletin: DNA Quantification Using the Fluorescence/DNase (F/D) Assay, v1.2. DNA Genotek. Nov 29, 2004.
4. TaqMan SNP Genotyping Assays Protocol. Part Number 4332856 Rev. B. Applied Biosystems. June, 2004.
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