PCR amplification and LDR reaction

The PCR reaction to amplify 110 bp fragment and 850 bp fragment was carried out in 20 μl reaction with primers listed in supplementary table S1, using 10 μl of GoTaq Green Master Mix (M7123, Promega, Madison WI) 0.4 μM each of forward and reverse primers and 1 μl of genomic DNA template. Thermocycling conditions were: 94 °C for 4 min; 94 °C for 30 sec, 60 °C (pre-LDR) and 56 °C (pre-T7E1) for 30 sec; 72 °C for 30 sec for 20–35 cycles; and a 5 min final extension at 72 °C. The 15 μl PCR products were treated with 50 μg protease (19155, Qiagen, Germantown, MD) for 20 min at 50 °C and inactivated at 75 °C for 15 min (Supplementary Fig. S4). The LDR was carried out in 20 μl reaction consisting of 10 μl of protease treated PCR product and 10 μl of LDR master mix (0.25 uM of each LDR primers, 20 mM Tris (20 mM MgCl2, 40 mM KCl, 2 mM NAD) pH 8.3, 0.01% of TritonX-100 and 0.1 μl thermostable ligase (Amphiligase (A8101, Epicentre, Madison, WI; Oakland Genetics, Rochester Hills, MI, www.oaklandgenetics.com) or 0.1 μl ligase expressed and isolated in-house). LDR Thermal cycling parameters: 94 °C for 3 min, 20–30 cycles of 94 °C for 15 sec and 61 °C for 1 min. PAGE was performed at 250 volts using a 15 cm long 10% polyacrylamide non-denaturing gel (12.5 mL 40% Acrylamide/Bis-solution 19:1 (Biorad 1610144), 500 μl of fresh 10% APS and 50 μl of TEMED and 1XTBE to 50 mL) in 1XTBE for 50 min, with compatible gel electrophoresis apparatus (Biometra V15.17, Germany).