For detection of the E2 and e2 allele variants, a SimpleProbe assay was created.
The PCR primers E2for (5′-TGCAACCCCACTACAGCCT-3′) and E2Rev (5′-GAGGCAGAGCCAAAGCCTAT-3′) were designed from the Williams 82 sequence as described by Watanabe et al. 2011 [12 (link)]. This 222-bp amplicon includes the A/T SNP (Gm10:44,732,850, W82 Glyma v1.0) at base 1561 in exon 10 where a nonsense mutation occurs in the e2 allele [12 (link)]. The SimpleProbe oligonucleotide (Fluorescein-SPC-GGCATGTCTTATGAAAATATTTGCTGC-Phosphate) was designed to the E2 sequence on the sense strand using the LightCycler Probe Design software (Roche Applied Science, Indianapolis, IN). PCR reactions and melting curve parameters were identical to the E1 genotyping assay except that the forward E2for and reverse E2Rev primers had a concentration of 0.2 μM and 0.5 μM, respectively. The melting curve from 55 °C to 77 °C distinguished the E2 peak at 64 °C and the e2 peak at 60 °C.
The PCR primers E2for (5′-TGCAACCCCACTACAGCCT-3′) and E2Rev (5′-GAGGCAGAGCCAAAGCCTAT-3′) were designed from the Williams 82 sequence as described by Watanabe et al. 2011 [12 (link)]. This 222-bp amplicon includes the A/T SNP (Gm10:44,732,850, W82 Glyma v1.0) at base 1561 in exon 10 where a nonsense mutation occurs in the e2 allele [12 (link)]. The SimpleProbe oligonucleotide (Fluorescein-SPC-GGCATGTCTTATGAAAATATTTGCTGC-Phosphate) was designed to the E2 sequence on the sense strand using the LightCycler Probe Design software (Roche Applied Science, Indianapolis, IN). PCR reactions and melting curve parameters were identical to the E1 genotyping assay except that the forward E2for and reverse E2Rev primers had a concentration of 0.2 μM and 0.5 μM, respectively. The melting curve from 55 °C to 77 °C distinguished the E2 peak at 64 °C and the e2 peak at 60 °C.
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