Nucleotide extension chain verification

A method of synthesizing an oligonucleotide and monitoring the synthesis of the oligonucleotide in real time comprising:

• selecting a nucleotide monomer for addition to an oligonucleotide;
• feeding an inlet stream to a reaction chamber for oligonucleotide synthesis, wherein the inlet stream comprises the selected nucleotide monomer;
• providing conditions in the reaction chamber for oligonucleotide synthesis;
• diverging an outlet stream from the reaction chamber to an analysis station;
• monitoring the oligonucleotide synthesis in real time using a computing device comprising a processor and a storage medium, wherein the processor is programmed to execute instructions stored on the storage medium to analyze whether the outlet stream comprises the selected nucleotide monomer or a derivative thereof, the monitoring comprises:
• analyzing the outlet stream or a sample thereof by a mass spectrometry (MS) instrument in the analysis station with the computing device communicably connected with the MS instrument;
• and
• determining, based on results from analyzing by MS, whether the outlet stream comprises the selected nucleotide monomer or a derivative thereof thereby confirming the selected nucleotide monomer was fed in the inlet stream to the reaction chamber in a proper order and as intended by a predetermined sequence of the oligonucleotide to be synthesized.