Exo sap pcr clean up kit

Variants identified by exome sequence were further confirmed in the parents and other available family members using Sanger sequencing. Exons harboring the variants were amplified using specific primers designed by ExonPrimer SOFTWARE. Primers are available upon request from the corresponding author. PCR cycling conditions were: initial denaturation at 96 °C for 5 min; 30 cycles of denaturation at 96 °C for 30 s; annealing at 60 °C for 30 s; extension at 72 °C for 30 min, and a final extension at 72 °C for 5 min. PCR products were purified using Exo-SAP PCR Clean-up kit (Fermentas, Germany) and sequenced in both directions using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and analyzed on the ABI Prism 3500 Genetic Analyzer (Applied Biosystems) according to the manufacturer's instructions.

Segregation analysis of the identified variant in the parents and the healthy sib was conducted by PCR amplification of exon 5 of the EXOSC8 using specific primer designed by Primer3 software followed by purification with Exo-SAP PCR Clean-up kit (Fermentas, Germany) and sequencing using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) on the ABI Prism 3500 Genetic Analyzer (Applied Biosystems) according to manufacturer’s instructions.

Segregation analysis of the identified variants in the parents and other affected family members of Family 1 was conducted by PCR amplification of exon 4 of the DSPP using specific primer designed by Primer3 software followed by purification with Exo-SAP PCR Clean-up kit (Fermentas, Germany) and sequencing using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) on the ABI Prism 3500 Genetic Analyzer (Applied Biosystems) according to manufacturer’s instructions.