Development Of A High-throughput Solid Phase De Novo DNA Synthesizer

There is no doubt that DNA acts as the carrier of life information.Since the application of chemical synthesis methods of oligonucleotides,synthetic oligonucleotides are widely used and play a vital role in various research fields,including drug development(such as artificial antibodies and biological bulk drugs),precision medical treatment(such as gene diagnosis and editing),industrial biology(such as biofuel and biosynthesis of important chemicals),agriculture(such as genetically modified food,bio-derived food and crop disease resistance),nanotechnology(such as DNA assembly materials and bio-based materials),information storage and calculation,etc.In recent years,the increasing demands for large-scale oligonucleotide synthesis caused by the rapid development of the above-mentioned fields,poses new challenges to the DNA synthesizers,including related throughput,as well as efficiency and cost.Considering that hundreds of thousands of different oligonucleotides are required by the genome-wide researches,synthesis throughput of DNA synthesizers available on the market is absolutely far from enough;In addition,the cost of materials is also another huge obstacle to genome-wide research,because the synthesis cost of the current oligonucleotide synthesis method is too high.As a consequence,it is urgent to create special oligonucleotide synthesis instruments with high throughput and low cost,which can affect the genome researches and even the general development of synthetic biology.Based on it,the non-contact piezoelectric valve is applied to replace the traditional solenoid valve in DNA synthesis reagent dosing.Although the liquid outlet diameter of this method is much smaller than that of the solenoid valve,its speed can still reach10m/s.Due to the characteristics of small diameter and high speed,the reagent can fall to a certain position on the synthetic plate more easily.The accuracy of the piezoelectric valve is not affected by the moving speed of the platform.By offering an extremely optimistic imagination space,these characteristics will contribute greatly to breaking through the shackles of traditional DNA synthesizers.Based on such high precision piezoelectric valve,the instrument of DNA chemical synthesis in situ is developed in this paper,which is characterized by higher throughput,faster speed and lower cost.1.To satisfy the functional demands of the new DNA synthesizer,the structure of the instrument is successfully designed by modularization,including three main parts of liquid adding module,as well as motion module and core reaction unit.The core reagent flows through the piezoelectric valve and the general reagent flows through the solenoid valve.This focuses more on the synthesis efficiency and cost.The purpose of high-speed X-Y axis of motion module is to greatly shorten the synthesis reaction time.The use of four-axis manipulator focuses on solving the needs of instrument automation and unmanned.Through precision design and processing,the core reaction unit has reached the highest throughput of 6144 holes,which can easily exceed 10000 or even100000 by array stacking.At the same time,it can meet the requirements of high throughput and high capacity characteristics.2.Perform finite element analysis on the main stressed components of the instrument,including structural mechanics analysis and modal simulation.The weak parts in the process of instrument force and vibration is calculated,which effectively verifies the rationality and safety of the structure design,and puts forward the improvement plan for the unreasonable structure.3.Independently carry out control circuit design and software programming.The control part adopts Ether CAT bus to dynamically connect the motion control card,PC,piezoelectric valve,solenoid valve,linear motor,mechanical arm,etc.and has realized the functions of multi-axis point motion,interpolation motion,encoder position detection,IO control,position comparison,position lock and so on.The software development of upper-end is carried out by C # mainly including the internal logic of control process,interface UI design,communication protocol,etc.4.After systematic assembly debugging and performance verification,a complete round of experiments was designed to complete the DNA synthesis and detection process.The fragment which coincides with the designed sequence was obtained by liquid chromatography and mass spectrometry,which further verified the rationality and feasibility of using piezoelectric valve for high-throughput DNA synthesis.The experimental prototype and theoretical basis are provided for the next step of comprehensive realization of high-throughput and large-scale column synthesis.