Sybr premix ex taq 2 pcr reagent kit

To validate variable copy numbers, real-time quantitative PCR (qPCR) were performed using the 7500 Fast Real-Time PCR systems (Applied Biosystems, Foster City, California). Two primer sets were designed within the boundaries of the CNV region. Primer pairs were designed by an online tool (PrimerQuest, IDT) (http://www.idtdna.com/Primerquest/Home/Index). PCR reactions were prepared with the SYBR Premix Ex Taq II PCR reagent kit (TaKaRa Bio, Dalian, China) according to the manufacturer’s protocol. Amplification efficiencies were identical and the relative copy number was calculated with the 2^-ΔΔCt method.

MS-MLPA was performed using SALSA MLPA kits ME028 and ME030 (MRC Holland, Amsterdam, Netherlands) to identify PWS/AS, and Silver–Russell syndrome, separately, according to the manufacturer’s instructions. The data were analyzed using Coffalyser software. qPCR with SYBR Green chemistry were utilized to verify the potentially clinically relevant CNVs in the cases and their parents. The primer sets for qPCR were designed to target different fragments within variant regions using an online primer designing tool—Primer 3 (http://primer3.ut.ee/)—and were synthesized by Shanghai Invitrogen Biotechnology Company (Shanghai, China) (Supplementary Data 5 and 6). Assays were carried out in accordance with manufacturer recommendations on the 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, California). The copy number variations were determined based on the ratio of target region copies to reference gene (GAPDH) copies in samples. Both male and female genomic DNA samples from unaffected pooled samples stored in our laboratory were used simultaneously as male and female control samples. Each qPCR was carried out in triplicate with the SYBR Premix Ex Taq II PCR reagent kit (TakaRa Bio, Dalian, China) following the manufacturer’s protocol.

For eight potentially pathogenic CNVs, at least two primer sets were designed within the boundaries of the CNV region (primers are listed in the Table S1). Real-time quantitative PCR (qPCR) was performed using a 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, California) to validate variable copy numbers. PCR reactions were prepared using the SYBR Premix Ex Taq II PCR reagent kit (TaKaRa Bio, Dalian, China) according to the manufacturer's instructions. Primer pairs were designed using the online PrimerQuest tool from Integrated DNA Technology (http://www.idtdna.com/Primerquest/Home/Index). Amplification levels were calculated using the 2^−ΔΔCT method.