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HIGH-THROUGHPUT GENE SYNTHESIS METHOD COMBINING AUTOMATED MOLECULAR CLONE ISOLATION SYSTEM AND DNA BARCODING : 분자 클론 추출과 DNA 바코딩을 통한 초고속 유전자 합성 기술

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Authors

Taehoon Ryu

Advisor
권성훈
Major
공과대학 전기·컴퓨터공학부
Issue Date
2016-08
Publisher
서울대학교 대학원
Keywords
molecular clone isolationautomationDNA barcodinggene synthesiserror-free DNA
Description
학위논문 (박사)-- 서울대학교 대학원 : 전기·컴퓨터공학부, 2016. 8. 권성훈.
Abstract
In this thesis, I introduce the development of a automated high-throughput DNA synthesis platform based on next-generation sequencing and opto-mechnical particle retrieval system. By using this platform, over 5,000 different error-free DNA sequences whose length ranges from 80bp to 120bp are synthesized during one week with only $5,000, thus it greatly reduces the synthesis cost from $0.3 per bp to $0.01 per bp. To realize this platform, several fundamental technologies such as molecular indexing using DNA barocde, automated particle retrieval system with algorithm for calculating location of specific particle and DNA retrieval system from microarray are introduced.
First, I demonstrate automated particle retrieval system. To calculate exact location of particle on sequencing plate with shorter processing time, two improved computational methods are developed. One is image stitching method based on a recognition of feature on sequencing plate and its center coordinates. The value is derived as a decimal and the offset between different images is not approximated to an integer, therefore, it is prevented that the error is accumulated even though hundreds images are stitched along one axis. The other is an analytic diffusion-like mapping that calculates transformation matrix applying a point pattern matching algorithm which is invariant to translations, rotations and scale changes. The matrix is analytically derived from least-square error estimation of multiple two-dimensional points, thus it makes possible to retrieve desired particle with immediate location-calculation. By combining two computational methods, exact location of particle with desired DNA sequence is obtain, therefore, automated machine could rigorously isolate intended particles without human intervention. Equiped with larger reservoir, such as 384 well plate, than previously used 96 well plate, over 2,500 particles are retrieved per one hour.
In order to confirm that retrieved particle has DNA sequence which I want to obtain, 192 different 7-mer DNA barcodes are designed and used to encode 5,280 samples. To encode sample of larger number than barcode set, combinatorial indexing method is adopted. During amplification step of DNA on the particles, differently indexed n-forward primers and m-reverse primers are used, thus nm combinations are generated. After pooling of amplified product and next-generation sequencing (NGS) analysis of pooled samples, each NGS read is sorted with pre-defined barcode sequence and aligned the sequence to design.
In addition, in order to make the platform more universal, I develop isolation method of glass substrate on which DNA oligonucleotides exist. Focusing pulse-laser beam inside the glass chip, such as microarray or sequencing substrate, due to high energy of focused beam, phase transition is locally occurred from solid state to plasma state. The force from plasma expansion pressure pushes above surface of the substrate which connected with target DNA sequence by covalent bond. The small fraction of substrate is isolated to each PCR tube and the amount of DNA is enough to amplify target DNA with no side reaction. Intended DNA sequences attached to substrate are amplified with high-fidelity DNA polymerase which has 3 -> 5 proof-read activity. Sanger sequencing results of amplified product are well matched to designed sequence, therefore, opto-mechanical retrieval of sequence-verified DNA could be applied to wider-spread next-generation sequencing platform, such as Illumina HiSeq or MiSeq.
I demonstrate that the automated high-throughput DNA synthesis platform is applicable to synthetic biology field of which the cost of DNA is bottleneck to accelerating development. At first, green fluorescent protein (GFP) is assembled from retrieved error-free DNAs using assembly PCR, cloned to E. coli expression vector and transformed to E. coli host cell. After IPTG induction, E. coli express GFP, thus it is demonstrated that synthetic gene assembled from retrieved DNA can be functional. In addition, larger genetic circuit, ~4kbp bacteriophage genome, is synthesized using assembly PCR and isothermal assembly. Cloned products are sequenced by Sanger method and it is identified that 1 of 5 clone has no error. This result suggests that retrieved DNAs have enough error rate to be used in gene synthesis process without additional error-elimination step. Finally, I demonstrate that over 5,000 100bp DNAs which encode sgRNAs guiding Cas9 protein to cut specific double-stranded region are generated in single synthesis cycle. I envision that this novel DNA synthesis platform could establish the base for next-generation of gene synthesis field and be used by many researchers who want to modify existing genetic system and/or make a novel creature.
Language
English
URI
https://hdl.handle.net/10371/119238
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