Allele frequencies for the indel were determined by PCR amplification of 30 ng of genomic DNA using HE3026/HE3027 (Table 1; Fig. 1); this yields a 736 bp fragment in the reference genomic sequence (NT_008183.18) or a 1566 bp fragment if the indel is present. HotStar Taq (Qiagen, Valencia, CA, USA) was used to minimize background. The final reaction contained 1× PCR buffer, 200 µM of each dNTP, 3.5 mm Mg2+, 1× Q-solution, 0.4 µM primer, 1.5 unit of HotStarTaq DNA polymerase (Qiagen) in 15 µl total volume. PCR reactions were carried out in GeneAmp PCR system 9700 (Applied Biosystems, CA, USA) using the following conditions: 95°C 15 min to activate the HotStarTaq polymerase, then 35 cycles of 95°C for 20 s, 60°C for 15 s, 72°C for 2 min 45 s, final extension at 72°C for 10 min. Aliquots of the PCR products (6 µl) were separated on 1% agarose gels (E-Gel 96, Invitrogen, CA, USA). Each gel was read independently by two people, and the data were combined by a third individual.
