Applied biosystem 3500 dna analyzer

The validation of results by Sanger sequencing was performed for selected samples of ESSE-Vologda, ESSE-Ivanovo, RPS, and all samples of BCS with detected deleterious variants. Sanger sequencing was performed on DNA sequencer Applied Biosystem 3500 DNA Analyzer (Thermo Fisher Scientific, Waltham, MA, USA) using the ABI PRISM BigDye Terminator v3.1 reagent kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol.

The validation of NGS results was done by Sanger sequencing. PCRs were performed in 20 μL of a mixture containing 0.2 mM of each nucleotide, 1× PCR buffer, 20 ng of the DNA, 10 ng of each primer, 2.5 U of DNA polymerase. Amplification was performed on a GeneAmp PCR System 9700 thermocycler (Thermo Fisher Scientific, Waltham, MA, USA) with the following parameters: 95 °C—300 s; 30 cycles: 95 °C—30 s, 62 °C—30 s, 72 °C—30 s; 72 °C—600 s. Before the Sanger reaction, the obtained amplicons were purified using ExoSAP-IT (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s protocol. The nucleotide sequence of PCR products was determined using the ABI PRISM^® BigDye™ Terminator reagent kit v. 3.1 followed by analysis of the reaction products on an automated DNA sequencer Applied Biosystem 3500 DNA Analyzer (Thermo Fisher Scientific, Waltham, MA, USA).