Allele-Specific Primer Design for SNP Genotyping

For each polymorphic variant identified in coding regions of the sequenced candidate genes, a set of allele-specific primers, with GC tail of unequal length attached to their 5′ end, were designed using the Primer3 software (Koressaar and Remm, 2007 (link); Untergasser et al., 2012 (link)), according to melting temperature (Tm)-shift SNP genotyping method developed by Wang et al. (2005) (link). Fragments were amplified by PCR on an Eppendorf Mastercycler using a volume of 20-μl master-mix containing 1.5 mM MgCl₂, 0.2 mM of dNTP Mix (Promega), 0.15 μM of each primer, 1X EvaGreen™ (Biotium, Fremont, CA, United States), 20 ng of genomic DNA, 1X Taq buffer, and 0.1 μl Taq1 polymerase (Promega) under the following profile: 94°C for 2 min, then 38 cycles of denaturation at 92°C for 20 s, annealing for 20 s (i.e., the temperature was specific to each primer trio), extension at 72°C for 20 s, and a final extension at 72°C for 5 min. The melting point analysis for the allele determination of the template DNA was performed with a fluorescence-detecting thermocycler (LightCycler™ 4890 Instrument II, Roche, Basel, Switzerland) with EvaGreen™ Fluorescent Dye (Biotium). The fluorescent detection profile was for 1 min at 95°C, and the melting curve step was ramping from 65 to 95°C in increments of 1°C for every 20 s.