Towards automatic gene synthesis with bioinformatics software, novel one-step real-time PCR assembly, and lab-chip gene synthesis

Abstract

This PhD thesis presents the whole process of gene synthesis method development and optimization, including the development of bioinformatics software TmPrime, TopDown and Automatic TouchDown one-step gene synthesis methods; and based on the developed protocols, this thesis also demonstrates an integrated gene synthesis device which is capable to perform two-step gene synthesis as well as purifying the synthesized product for downstream applications. Bioinformatics software TmPrime is developed to optimize oligonucleotide design. It is able to design oligonucleotides with homologous melting temperature for both LCR and gapless PCR assembly of very long gene sequences. The potential mis-hybridization, hetero-dimer, homo-dimer and hairpin formations among oligonucleotides are screened by pair-wise sequence alignment. The utility of TmPrime is demonstrated by synthesizing three genes using gapless one-step or two-step process. TopDown (TD) one-step gene synthesis method combines the advantages of one-step and two-step gene synthesis process. It conducts gene synthesis with TmPrime particularly designed/partitioned outer primers and inner oligonucleotides with distinct melting temperature (¿Tm > 8°C) difference. This particular reaction condition provides several advantages in (i) eliminating potential competition between the assembly and amplification reactions, (ii) minimizing the possibility of truncated oligonucleotides participating in the assembly process and the resulting errors, (iii) providing an stringent annealing condition to reduce the potential of forming secondary structures, and (iv) increasing the specialization of oligonucleotides hybridization as in Touchdown PCR. All of these would prevent the generation of faulty sequence, especially for gene with high GC contents. Automatic TouchDown (ATD) one-step gene synthesis method is developed to further improve TopDown method. It enables the synthesis of long DNA of up to 1.5 kbp with only one polymerase chain reaction (PCR) process. The method involves two key steps: (i) design of outer primers with two melting temperatures, and (ii) utilization of DNA annealing kinetics to selectively control the oligonucleotide assembly and full-length template amplification. With the help of a novel real-time PCR approach to monitor the gene assembly process, the ability of this ATD method has been demonstrated in the design and synthesis of human protein kinase B-2 (PKB2) (1446 bp) and the promoter of human calcium-binding protein A4 (S100A4) (752 bp) with oligonucleotides concentration of as low as 1 nM. The integrated two-step gene synthesis device is established based on the developed protocols. It is capable of performing two-step gene synthesis to assemble a pool of oligonucleotides into genes with the desired coding sequence. The device comprises of two polymerase chain reaction (PCR) modules, temperature-controlled hydrogel valves, electromagnetic micromixer, shuttle micromixer, volume meters, and magnetic beads based solid-phase PCR purification module, fabricated using a fast prototyping method without lithography process. The fabricated device is combined with a miniaturized thermal cycler to perform gene synthesis. This device has been demonstrated to successfully synthesize a green fluorescent protein fragment (GFPuv) (760 bp), and obtained comparable synthesis yield and error rate with experiments conducted in PCR tube within a commercial thermal cycler. To our knowledge, this is the first microfluidic device demonstrating integrated two-step gene synthesis.