Dye terminator cycle sequencing

We confirmed the presence of the two c-terminus RUNX2 mutations, initially identified in the CCD patients by “Casa del sollievo e della sofferenza” U.O.C. Genetica Medica—S. Giovanni Rotondo (FG)-Italy, by targeted sanger sequencing of a 470 base-pair (bp) PCR product corresponding to exon 7 and adjacent intron regions of RUNX2 gene (NM_001024630.3). Specific primers (purchased from Thermo Fisher Scientific, Waltham, MA, USA) were designed using Primer3 and BLAST (NCBI, https:www.ncbi.nlm.nih.gov/tools/primer-blast/, accessed on 3 February 2019) and were the following: Forward 5′-TAAGGCCTGAAAGGATGGGGT-3′ and Reverse 5′-ATGTGGGCAAGGGAATGACAA-3′. PCR was conducted with Mastercycler^® ep Gradient S^® (Eppendorf, Milan, Italy) and GoTaq^® Hot Start Polymerase kit (Promega, Madison, WI, USA) by 2 min at 96 °C followed by 35 cycles of 96 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s. A final step of 72 °C for 5 min concluded the PCR program. Purity and identity (by means of length in bp) of PCR product was checked by 1.5% agarose and gel electrophoresis. After purification with FastGene™ kit (Nippon Genetics, Mariaweilerstraße, Düren, Germania), 1.5 µL of the PCR product was used with Dye Terminator Cycle Sequencing (DTCS), Beckman Coulter; Fullerton, CA, USA) Quick Start Kit (Sciex, Milan, Italy) following the manufacturer’s instructions.

To determine the peptide-encoding nucleotide sequence contained in each phage clone, sequence analysis was performed using the ssDNA of the phage as template. E. coli TG1 infected with phages were plated on medium with X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside)-isopropyl β-D-1-thiogalactopyranoside (IPTG) to obtain phage plaques. Thirty plaques were randomly selected.
Sequencing reactions were performed as described in the Beckman Coulter Dye Terminator Cycle Sequencing (DTCS) protocol. The purified sequencing reactions were run on a CEQ 8000 DNA Sequencer (Beckman, Fullerton, CA, USA).