3130xl automated sequencer

MLPA analysis, unlike FISH, recognizes both the deletions of the entire NSD1 gene and microdeletions of one or more exons.
The analysis was carried out on genomic DNA with the MLPA SALSA P026 Kit (MRC-Holland, Amsterdam, The Netherlands). All reactions (denaturation, ligation, and PCR) were performed following the manufacturer’s instructions. PCR products were run on a 3130xl automated sequencer (Applied Biosystems, Foster City, CA, USA) and data were analyzed using Genemapper v 3.2 and Coffalyser v.140721.1958 software (Applied Biosystems, Foster City). In selected cases, Array-CGH analysis was carried out to define the size and the breaking point of the deletions.
Array-CGH was performed using Superprint G3 CGH 8 × 60 K (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturers’ protocol. Data were analyzed by Agilent Cytogenomics 4.0.3.12 software (Agilent Technologies, Santa Clara, CA, USA). All genomic positions were reported according to the human genome assembly (GRCh37/hg19).

A Gentra Puregene Tissue Kit (Qiagen, Valencia, CA, USA) was used for the DNA extraction. For the polymerase chain reaction (PCR), we used the following nine microsatellite loci: Ppu1, Ppu6, Ppu7, Ppu10 [33 ], STN96, STN163, STN173, STN196 [34 ] and Gac1125PBBE [35 (link)]. PCRs were run in two multiplex reactions on a Thermal Cycler 2720 (Applied Biosystems, Foster City, CA, USA): multiplex 1: Ppu1, Ppu6, Gac1125PBBE, STN96, STN163; multiplex 2: Ppu7, Ppu10, STN173, STN196; 95°C for 15 min; 30 cycles on 94°C for 30 s, 53°C for 90 s and 72°C for 60 s; plus a final extension for 10 min at 72°C. The PCR products were diluted 1:5 and run on a 3130xl Automated Sequencer (Applied Biosystems) using a GeneScan-500 LIZ size standard to estimate the allele sizes. The alleles were scored using Peakscanner (Applied Biosystems).