Towards automatic gene synthesis with bioinformatics software, novel one-step real-time PCR assembly, and lab-chip gene synthesis
type: CreativeWork
description: 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 cha…
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 cha…
author: HUANG MOCHAO
keywords: Gene synthesis, Bioinformatics, TmPrime, TopDown PCR, Automatic-TouchDown PCR, Microfluidic system
date published: 2009-08-14
date modified: 2026-07-24T03:32:30Z
provider: National University of Singapore
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: National University of Singapore