Beaudet et al.

GR-1725

Supplemental Information

 

Amplification Controls

 

To further improve genotyping accuracy, amplification of an internal control can be integrated to the AlphaScreen ASA assays to detect PCR failures. An additional PCR primer pair, which amplifies a 318 bp fragment from the human BGT-1 gene, was included in the PCR/AlphaScreen mix for the WIAF-896 assay. After the initial reading of the genotypes shown in Figure 1A, specific probes for the BGT-1 PCR product were added to the samples and a second hybridization step was performed. The results obtained from the reading of a second 10 ml aliquot are shown in Figure 1B. High AlphaScreen signal was detected in every well (Fig. 1B), confirming that PCR/AlphaScreen mix and genomic DNA had been properly dispensed. The addition of an internal control to the ASA assays will help identify PCR failures, which were seen in the initial series of ASA, and increase genotyping accuracy.

As with ASA, the most likely source of errors in genotyping will be from the failure of a sample to amplify. Thus, a heterozygote could be mistyped as a homozygote. To eliminate this possible mistyping, two approaches have been tested. First, amplification of an internal control can be integrated into the assays and detected by the addition of control probes after the allele-specific detection step, as for the ASA approach (data not shown). However, a simpler approach has been developed for ASH, which avoids the extra addition of a second set of probes. Specific probe hybridization is first performed at the optimized hybridization temperature. An aliquot of the sample is read and the remaining sample is simply reheated, and the probes re-annealed at 37°C. At this lower temperature, most probes will show reduced discrimination for mismatched templates. A significant increase in signal should be observed for the mismatched allele in homozygous samples if amplification has occurred. An example of this strategy is shown in Figure 2. In the first panel, the hybridization step is performed at the optimal temperature for allele-discrimination (Fig. 2A). Figure 2B shows the results of the non-specific probe hybridization, made at 37°C. A marked increase in AlphaScreen signal is observed in the wells of the mismatched alleles for the homozygous samples, indicating that PCR amplification had indeed occurred in these wells.

 

Genotyping from Complex Mixtures

Multiplex PCR procedures have been developed in several laboratories to save on reagents, time and operating costs. As a preliminary demonstration of the potential of AlphaScreen for the detection of targets from multiplexed samples, PCR products for NCBI SNPs 394, 4568, 4621, 5173 and WIAF-896 were amplified separately from the same DNA sample and mixed after PCR. The hybridization of the allele-specific bridging was performed both on the single samples and on an aliquot of the pooled samples. The results are represented graphically in Figure 3 as a log chart of allelic ratios obtained for each marker. Correct genotypes were obtained for all the SNP markers and equivalent signal ratios were observed in both the single and pooled series. These results clearly indicate that AlphaScreen could be used for the detection of multiplexed PCR products.

 

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FIGURE LEGENDS

 Figure 1  AlphaScreen ASA genotyping of SNP WIAF-896 in 21 genomic DNA samples. (A) AlphaScreen signal obtained after the detection of the allele-specific amplification product. (B) Detection of the BGT-1 PCR product that was used as a control for PCR amplification. Solid bars, samples amplified with the A-specific PCR primer; white bars, samples amplified with the C-specific primer. Ctrl is a no DNA sample.

 

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Figure 2  AlphaScreen ASH genotyping assay of SNP WIAF-896 in 21 genomic DNA samples. (A) AlphaScreen signal obtained after allele-specific hybridization of the detection probes at 54.5°C. (B) Control for PCR amplification by the reading of the AlphaScreen signal after probe hybridization at 37°C. Solid bars, samples amplified with the A-specific PCR primer; white bars, samples amplified with the C-specific primer. Ctrl is a no DNA sample.

 

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Figure 3  Genotyping of a DNA sample using  five different AlphaScreen ASH SNP assays from single or pooled PCR products. PCR was performed for each SNP assay separately. A fraction of the PCR products were pooled together to mimic multiplexed amplification, and split into five for the individual detection of each SNP genotype. The ratio of AlphaScreen signal obtained for the two alleles of each SNP was determined. (¯) AlphaScreen genotyping using the single PCR products; (¿) AlphaScreen genotyping using the pooled PCR products.