Genotyping the PPT and FV Genes by ASO Hybridization and Minisequencing on Oligonucleotide Arrays
| Probes and primers | Ratio between signals from normal and mutant alleles[ii] | Power of discrimination between genotypes[iii] | |||||
| homozygous positions PPT (T/T) FV (G/G) | heterozygous positions PPT (T/A) FV (G/A) | ||||||
| ASO[iv] | MS[v] | ASO[iv] | MS[v] | ASO[iv] | MS[v] | ||
| DNA target | |||||||
| PPT | 15-mer | 1.8 | — | 1.6 | — | 1.1 | |
| 20-mer | 1.0 | 157 | 0.84 | 1.1 | 1.2 | 142 | |
| FV | 15-mer | 11 | — | 4.4 | — | 2.5 | |
| 20-mer | 2.4 | 85 | 1.4 | 1.5 | 1.7 | 57 | |
| RNA target | |||||||
| PPT | 15-mer | 7.4 | — | 1.4 | — | 5.3 | |
| 20-mer | 1.0 | 114 | 0.54 | 2.3 | 1.9 | 49 | |
| FV | 15-mer | 8.0 | — | 28 | — | 1.2 | |
| 20-mer | 14 | 49 | 2.3 | 5.1 | 6.1 | 8 | |
[i] The best results obtained by both methods are highlighted by bold numbers; (—) Not done.
[ii] Mean values of four separate experiments.
[iii] Calculated by dividing the ratio between signals from the normal and mutant alleles obtained at a homozygous position with the corresponding ratio obtained at a heterozygous position.
[iv] Washing conditions of increasing stringency (0.75, 0.15, and additionally 0.03 m NaCl for the RNA hybridizations) were employed for the posthybridization washes at 22°C of the ASO probe arrays. In most cases the best result was obtained at 0.15 mNaCl, but the FV 15-mers performed better at 0.03 m with RNA as template. These results are given here.
[v] (MS) minisequencing. The DyNAseq DNA polymerase was used in the reaction with DNA as template, and the RetroTherm reverse transcriptase was used with RNA as template.