Primers and probes used to detect PTPRD CNVs were designed using Primer Express software (Applied Biosystems, Foster City CA, USA). Primers were purchased from Integrated DNA Technologies (Coralville, IA, USA). Primer sequences are as follows: F primer: 5′-ACATTTCAGAATATCCATCCTTTGG-3′ R primer: 5′-TGCTAATTCGTCCCAGAACGA-3′. Probes were purchased from Operon Biotechnologies, Inc. (Huntsville, AL, USA). The probe 5′-TGGCAGCCAAGCTAAAGCAAATCCTTG-3′ was labeled with FAM (6-carboxy-fluorescine) along with a quencher (BHQ 1; Biosearch Technologies, Novato, CA, USA). Dilutions of control gDNA of 40, 20, 10, 5 and 2.5 ng μl–1 were used as comparators to test DNA samples diluted to ∼10 ng μl–1. All DNAs, probes and primers were diluted in low EDTA TE buffer. Reactions were run on the ABI 7500 Fast Real-time PCR system (Applied Biosystems) using the following cycling parameters: one cycle at 95 °C for 20 s, followed by 40 cycles at 95 °C for 3 s and 60 °C for 30 s. Each reaction was carried out in a total volume of 20 μl containing 1 × Taqman Fast Universal PCR Master mix, 900 nM of each primer and 250 nM probe.
