HIGH-THROUGHPUT RETRIEVAL OF MOLECULAR CLONES FOR POLYNUCLEOTIDE SYNTHESIS

Abstract

Molecular clone is a large population of DNA replicated from a single DNA molecule. Production, analysis and retrieval of the molecular clones are the key techniques in current biotechnology. Here, high-throughput method for the manipulation of molecular clones is presented using highly parallel sequencing and radiation-based non-contact molecular clone retrieval system. Specifically, production and analysis methods of the molecular clones are investigated using a highly parallel sequencing platform. In emulsion polymerase chain reaction (PCR) at the sample preparation of parallel pyrosequencing, large amount of molecular clones are created. After the production of large amount of molecular clones, individual molecular clone is sequenced so that sequence and position information of each molecular clone are identified. Molecular clone retrieval system based on non-contact energy transmission is developed for rapid and precise retrieval of each molecular clone. Pulse laser, optical component, vision module, and motorized stage are integrated in the system. Also, system control software is developed combining functions such as: (i) extraction of sequence and positional information of each molecular clone from output data of highly parallel sequencing, (ii) scanning sequencing plate and high-resolution image reconstruction, (iii) algorithmic identification of molecular clone in the reconstructed image, (iv) synchronization of sequencing plate movement, pulse-laser radiation, and carrier plate movement. Furthermore, retrieval method of molecular clones produced by bacterial cell is presented. Mixture of genetically diverse bacterial cell stock is spread and grown on a solid growth medium. After the short period of growth, position of each cell clone is identified using microscope vision scanning system, and then each cell clone is retrieved using focused pulse laser. Retrieved individual cell clone is contained in carrier micro well plate. DNA of retrieved individual cell clone is tagged with barcode DNA, and analyzed and identified by highly parallel sequencing. Method and system developed here may be applicable in a wide range of biotechnology area such as synthetic DNA production, antibody drug discovery, and functional genome study with exceptional cost reduction and increased throughput due to highly parallel nature of the method.