Towards automatic gene synthesis with bioinformatics software, novel one step real time PCR assembly, and lab chip gene synthesis 1

TOWARDS AUTOMATIC GENE SYNTHESIS WITH BIOINFORMATICS SOFTWARE, NOVEL ONE-STEP REAL-TIME PCR ASSEMBLY, AND LAB-CHIP GENE SYNTHESIS HUANG MO CHAO (B. Eng.), XJTU A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF ELECTRICAL & COMPUTER ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2009 National University of Singapore Department of Electrical and Computer Engineering ACKNOWLEDGEMENTS It is the blessing from my Lord Jesus Christ, who has made this work possible. I would like to express my sincere thanks to my supervisor Dr. Li Mo-Huang, for his patience and unfailing guidance. Without his valuable suggestions and support, this work would not have been successful. Special thanks also go to Associate Professor Adekunle Olusola Adeyeye, my NUS supervisor, for all his kind help and support during my four years PhD study. Many thanks should be addressed to Dr. Yang Yi Yan for the valuable suggestions in developing the hydrogel valve, Dr. Danny Van Noort for the useful comments on the device design, Fan Lee for providing different hydrogel materials and all my friends in the Institute of Bioengineering and Nanotechnology for their endless help. Special thanks go to Professor Jackie Ying and Ms. Noreena AbuBakar for providing me the opportunity to work in IBN and supporting me all the time. I am deeply grateful for the various team mates I encountered during my stay at IBN. I would like to thank Dr. Cheong Wai Chye, Dr. Bode Marcus, Dr Ali Emril Mohamed, Wei Jiashen, Chua Jay, Muller Stefanie, Kuan Yoke Kong, Ye Hongye, Sim Choon Kiat, Khor Samuel, Loh Nicholas and the students at high-throughput gene synthesis group in IBN. In addition to their generous help and strong support, I also enjoyed great companionship. My greatest appreciation should go to my parents and grandmother for their endless love and concern throughout my life. Without them, I would not have made it so far in life. Finally, I would like to give special thanks to my boyfriend, Brian, who has been supportive, loving and encouraging me all the time. This thesis is especially dedicated to all of you. Page I National University of Singapore Department of Electrical and Computer Engineering TABLE OF CONTENTS ACKNOWLEDGEMENTS . I SUMMARY . VI LIST OF FIGURES . VIII LIST OF TABLES . XIV CHAPTER I 1INTRODUCTION 1.1 Overview gene synthesis . 1.2 Challenges in Gene Synthesis . 1.3 Motivation 1.4 Objectives of this PhD thesis 1.5 Thesis Outline 10 CHAPTER II GENE SYNTHESIS METHODS .12 2.1 Introduction 12 2.2 Bioinformatics in Gene Synthesis . 12 2.3 Biochemical method of gene synthesis 16 2.4 2.3.1 LCR based gene assembly 17 2.3.2 PCR based gene assembly 18 2.3.3 Real time PCR 21 2.3.4 DNA extraction and purification . 22 2.3.5 Enzymatic error filtering 23 2.3.6 Cloning and sequencing of synthetic DNA . 25 Fundamentals of lab-on-a-chip 27 2.4.1 Microvalves . 27 2.4.2 Micromixers . 28 2.4.3 On-chip PCR 28 2.4.4 On-chip DNA purification 28 CHAPTER III DESIGN AND OPTIMIZATION OF OLIGONUCLEOTIDES 30 3.1 Introduction 30 3.2 TmPrime oligonucleotide design methods and functional modules 32 Page II National University of Singapore Department of Electrical and Computer Engineering 3.3 3.4 3.2.1 Fast and flexible oligonucleotide design . 32 3.2.2 Multiple-pool assembly 34 3.2.3 Mis-hybridization screening . 34 3.2.4 Codon optimization 35 Experimental evaluation of TmPrime performance . 35 3.3.1 Target Proteins . 35 3.3.2 Real-time gene assembly and amplification 36 3.3.3 LCR assembly 36 Results 37 3.4.1 Designing oligonucleotides for target proteins 37 3.4.2 Oligonucleotide assembly and amplification . 39 3.4.3 Comparison with existing oligonucleotide design programs . 42 3.5 Discussion 43 3.6 Conclusion . 45 CHAPTER IV TOPDOWN ONE-STEP GENE SYNTHESIS 46 4.1 Introduction 46 4.2 Principle of Top-Down PCR based gene synthesis 47 4.3 Experiment verification of TopDown one-step gene synthesis 49 4.4 4.3.1 Design of oligonucleotide for gene synthesis 49 4.3.2 Non-competition one-step real-time gene synthesis 49 4.3.3 One-step and two-step PCR-based gene synthesis 50 4.3.4 Agarose gel electrophoresis 51 Performance of TopDown one-step gene synthesis and its real-time analysis 51 4.4.1 Performance of TD one-step gene synthesis . 51 4.4.2 Analysis of real-time gene synthesis . 54 4.5 Discussion 58 4.6 Conclusion . 61 CHAPTER V AUTOMATIC TOUCHDOWN ONE-STEP GENE SYNTHESIS 63 5.1 5.2 Introduction 63 5.1.1 Principle of Automatic TouchDown one-step gene synthesis 64 5.1.2 Mechanisms of PCR synthesis process . 65 Experiment verification of Automatic TouchDown one-step gene synthesis 66 Page III National University of Singapore Department of Electrical and Computer Engineering 5.2.1 Design of oligonucleotides for gene synthesis 66 5.2.2 Automatic TouchDown one-step real-time gene synthesis 66 5.2.3 Gel electrophoresis . 67 5.3 Theoretical analysis of DNA hybridization kinetics 68 5.4 Real-time performance study of ATD one-step gene synthesis 70 5.4.1 Effect of varying extension time during ATD one-step gene synthesis . 70 5.4.2 Effect of varying initial oligonucleotides concentration 71 5.4.3 Effect of varying annealing temperature . 73 5.4.4 Synthesis of long gene by ATD process 74 5.4.5 Effect of varying dNTP concentration 75 5.4.6 Effect of melting temperature uniformity of partitioned oligonucleotides . 75 5.5 Discussion 78 CHAPTER VI INTEGRATED TWO-STEP GENE SYNTHESIS ON CHIP .80 6.1 Introduction 80 6.2 Device fabrication and thermal cycling system construction . 81 6.3 6.4 6.5 6.2.1 Microfluidic device fabrication . 81 6.2.2 Preparation of hydrogel valves . 82 6.2.3 PCR thermal cycling 85 Experimental verification of the integrated two-step gene synthesis chip 85 6.3.1 Gene assembly and amplification . 85 6.3.2 Solid-phase buffer exchange . 86 6.3.3 Agarose gel electrophoresis 87 6.3.4 DNA sequencing 87 Results and discussion 88 6.4.1 Device operation 88 6.4.2 In situ hydrogel valve . 92 6.4.3 PCR thermal cycling 93 6.4.4 Comparison of one-step and two-step gene syntheses . 95 6.4.5 Thermally enhanced solid-phase PCR purification 99 Conclusion 102 Page IV National University of Singapore Department of Electrical and Computer Engineering CHAPTER VII CONCLUSIONS AND FUTURE PLAN 104 7.1 Summary .104 7.2 Future work .107 7.2.1 Synthesis of long difficult genes 107 7.2.2 Error filter .107 7.2.3 Integration of real-time fluorescence detection with gene synthesis system .109 Author’s Publications 110 References 111 Appendix I 120 Appendix I 120 Appendix II 125 Appendix III .126 Appendix IV .135 Page V National University of Singapore Department of Electrical and Computer Engineering Towards automatic gene synthesis with bioinformatics software, novel one-step real-time PCR assembly, and lab-chip gene synthesis Huang Mo Chao Under the supervision of Associate Professor Adekunle Olusola Adeyeye At National University of Singapore and Dr. Li Mo-Huang At Institute of Bioengineering and Nanotechnology SUMMARY 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 twostep 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, homodimer and hairpin formations among oligonucleotides are screened by pair-wise sequence alignment. The utility of TmPrime is demonstrated by synthesizing three genes using gapless onestep 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 (∆T m > 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 Page VI National University of Singapore Department of Electrical and Computer Engineering 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 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 solidphase 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. Page VII National University of Singapore Department of Electrical and Computer Engineering LIST OF FIGURES Figure 1.1: Generic gene synthesis process. It takes about two weeks to construct and deliver an error free DNA. . Figure 2.1: Flowchart of bioinformatics software. Both protein sequence and DNA sequence are eligible input files. The program generates the optimized partition themes of the input sequences regarding user requirements. . 13 Figure 2.2: Process steps of gene synthesis. Oligonucleotides are synthesized as building blocks for polymerase cycling assembly or ligase chain reaction. Synthesized mismatch DNA is filtered out via enzymatic error filtering. 16 Figure 2.3: LCR based gene synthesis. (a) Oligos phosphorylation by modifying their 5’ ends from hydroxyl group to phosphate group using a kinase; (b) Oligos are linked together gradually to form template DNA using thermostable ligase enzyme. . 17 Figure 2.4: Operation principle of two-step overlapping polymerase cycling assembly. Different pools of oligos with sequences partially overlapped are first assembled to long DNA blocks. Then the outer primers are added to amplify the amount of assembled full length DNA . 19 Figure 2.5: (a) Successive extension polymerase cycling assembly method. DNA is elongated successively from oligo R5 and F5. (b) Thermodynamically balanced inside-out polymerase cycling assembly method. DNA construction starts from inside oligos F1 and R1, and gradually extended using outside oligos. 20 Figure 2.6: Schematic illustrations of non-specific and specific DNA purifications using (a) ChargeSwitch magnetic beads, (b) streptavidin magnetic beads, (c) oligo (dT)25 magnetic beads 23 Figure 2.7: Principle steps of MutS error filtering. After re-anneal of assembled DNA, mismatched heteroduplex DNA are captured by MutS enzyme and separated from the DNA with correct sequence by gel electrophoresis [60]. . 24 Figure 2.8: Working principle of enzymatic cleavage proteins. Endonuclease such as T4E7 and T7E1 recognize and bind to the mutation site of mismatched DNA and cleave the DNA into two segments . 25 Figure 3.1: Scheme of LCR or gapless PCR assembly. The input sequence is the serial connection of overlap regions of oligonucleotides 32 Figure 3.2: An overview of the oligonucleotide design scheme. The software first divides the input sequence into approximately equal-temperature (EquiTm ) or equal-length fragments (Equi-space) using markers based on the user-specified melting temperature. The positions of the markers are iteratively shifted to globally minimize the deviation in melting temperature among the fragments (Tm Equilibrate). Two adjacent fragments are joined together to generate oligonucleotides for gapless assembly. 33 Page VIII National University of Singapore Department of Electrical and Computer Engineering Figure 3.3: Web interface for TmPrime. TmPrime is implemented as functional modules, each module reflecting a different aspect of the oligonucleotide design process with interface elements organized in a coherently grouped fashion. . 38 Figure 3.4: Base composition plot of gene sequence GC content and melting temperature plot of overlap regions of oligonucleotides partitioned using Equi-space approach. (a) PKB2 (1446 bp, G+C: 58.4%). (b) S100A4 (752 bp, G+C: 56%). The GC plot was obtained using Isochore (http://emboss.bioinformatics.nl/cgi-bin/emboss/isochore). The highly similar profiles of GC and melting temperature plots clearly indicated the affects of GC cluster on the Tm homogeneity of oligonucleotides. . 39 Figure 3.5: Agarose gel electrophoresis of assembled products. One-step synthesis of GFPuv (760 bp) from TmPrime: (Lane 1) optimized and (Lane 2) fixedlength control oligonucleotides. (Lane 3) One-step synthesis of PKB2 (1446 bp). Two-step synthesis of PKB2: (Lane 4) assembly and (Lane 5) amplification. (Lane 6) one-step synthesis of S100A4 (752 bp). Two-step synthesis of S100A4: (Lane 7) assembly and (Lane 8) amplification. The annealing temperatures for the PCR process are as follow: GFPuv, 50°C; PKB2, 61°C; S100A4, 58°C (assembly) and 49°C (amplification). 40 Figure 3.6: (a) Melting peak analyses of the assembled products for GFPuv from onestep synthesis: (---) optimized and (—) fixed-length control oligos. Melting peak analyses of the assembled products for (b) PKB2 and (c) S100A4 from one-step and two-step syntheses; two replicas were performed for each set of oligos. The corresponding agarose gel electrophoresis results of the assembled products are shown in Figure 3.5. The measured Tm values are 86.5°C for GFPuv, 91.5°C for PKB2, and 90.5°C for S100A4 41 Figure 3.7: Agarose gel electrophoresis of LCR assembled GFPuv with TmPrimeoptimized oligonucleotides. (a) LCR products (2, and hrs assembly) before second PCR. (b) Second PCR after LCR (2, and hrs). Lane (L): 100 bp DNA ladder. 41 Figure 4.1: Schematic illustration of TopDown one-step gene synthesis combining PCR assembly and amplification into a single stage with different annealing temperatures designed for assembly and amplification. Inner oligonucleotides and outer primers are designed with melting temperature different > 15ºC to minimize potential interference during PCR. 48 Figure 4.2: Agarose gel (1.5 %) electrophoresis results of one-step (30 cycles), TopDown (TD) one-step (40 cycles), and two-step (PCA: 30 cycles; PCR: 30 cycles) gene syntheses. The TD one-step process is conducted with annealing temperature of 67 °C for the first 20 cycles followed by another 20 cycles with annealing temperature of 49 °C. The concentrations of oligonucleotides and outer primers are 10 nM and 400 nM respectively. 51 Figure 4.3: Continuous fluorescence monitoring of real-time gene synthesis with 1X LCGreen I. The first 20 cycles is conducted with annealing temperature of 67 °C followed by another 20 cycles with annealing temperature of 49 °C. The concentrations of oligonucleotides and outer primers are 10 nM and 400 nM respectively. . 52 Page IX National University of Singapore Department of Electrical and Computer Engineering products of higher molecular weights [8, 14, 26, 34, 78, 79, 92, 93] , which degraded the purity of the synthesized products. This abnormal event is usually neglected without explanation by authors. Although PCR-based gene synthesis has been widely implemented, there is a lack of capability in accurately predicting the gene synthesis. The development of an accurate model of gene synthesis would aid in understanding these phenomena and designing optimal reaction conditions. Another issue associated with gene synthesis is the lack of a standard or universal method [94] . Depending on the complexity of target genes, the synthetic genes are often either constructed with one-step or two-step overlapping process. With its simplicity, the one-step process is preferred for comparative short DNA ( 15ºC to minimize potential interference during PCR. In this study, we also combined the TD one-step gene synthesis with real-time PCR to investigate the gene synthesis process. LC Green I and SYBR Green I intercalation dyes were studied for real-time gene synthesis. Gel electrophoresis results were compared with the real-time fluorescent signals to study the effects of oligonucleotide concentration, outer primers concentration, stringency of annealing temperature, and number of PCR cycles. Performing realtime gene synthesis provided a novel approach to explore these factors in a systematic study, which reveal unique information unavailable in gel results [92] . An analytical model was further developed, which uncovered the mystery of coincident optimal conditions, and the formation of Page 48 National University of Singapore Department of Electrical and Computer Engineering undesired, spurious DNA products. This real-time method provided new insights into gene synthesis and a universal method for gene synthesis. 4.3 Experiment verification of TopDown one-step gene synthesis 4.3.1 Design of oligonucleotide for gene synthesis Gene sequence for the promoter of human calcium binding protein A4 (S100A4, 752 bp; chr1:1503312036-1503311284) was selected for synthesis via assembly PCR. Oligonucleotides were derived by using TmPrime with Equi-space approach (refer to section 3.2.1) and corrected with salt and oligonucleotide concentrations. The summary of oligonucleotide set is shown in Table 4.1 with the detail information provided in Appendix II Table S1. Table 4.1: Data of oligonucleotide set. Gene Length Average Tm (bp) (min, max) (°C) S100A4 752 66.0 (61.1, 69.8) Number of oligonucleotides Overlap length (nt) Oligonucleotide length (nt) 30 19–33 19, 41–66 4.3.2 Non-competition one-step real-time gene synthesis Non-competitive one-step process was optimized using real-time PCR conducted with Roche’s LightCycler 1.5 real-time thermal cycling machine with temperature transition of 20°C/s. Realtime gene synthesis was conducted with 20 µl reaction mixture including 1× PCR buffer (Novagen), µl of 0.25× to 5× SYBR Green I (1× = 1/20,000 dilution; Invitrogen) or LCGreen I (Idaho Technology Inc.), mM of MgSO4, mM each of dNTP (Stratagene), 500 µg/ml of bovine serum albumin (BSA), 5–80 nM of oligonucleotides, 60 nM–1 µM of forward and reverse primers, and U of KOD Hot Start (Novagen). The PCR were conducted under the following conditions: of initial denaturation at 95°C; 20 cycles of 95°C for s, 58–70°C for 10 s, 72°C for 30 s; followed by 20 cycles of 95°C for s, 49°C for 10 s, 72°C for 30 s; and final extension at 72°C for 10 min. Desalted oligonucleotides were purchased from Research Biolabs (Singapore) and Proligo (Singapore) without additional purification. Page 49 National University of Singapore Department of Electrical and Computer Engineering 4.3.3 One-step and two-step PCR-based gene synthesis Conventional gene synthesis via PCR was performed either as a one-step process, combining PCR assembly and amplification into a single stage, or as a two-step process with separate stages for assembly and amplification. All PCR reactions, whether for assembly or amplification, were run in standard 0.2-ml PCR tubes with a commercial thermal cycler (DNA Engine PTC-200, Bio-Rad) using the same oligonucleotides set and out primers as in the non-competitive one-step PCR. The one-step process was performed with 50 µl of reaction mixture including 1× PCR buffer (Novagen), mM of MgSO4, mM each of dNTP (Stratagene), 500 µg/ml of BSA, 10 nM of oligonucleotides, 400 nM of forward and reverse primers, and U of KOD Hot Start (Novagen). The one-step PCR was conducted under the following conditions: initial denaturation at 95°C; 30 cycles of 95°C for s, 58°C for 10 s, 72°C for 30 s; and final extension at 72°C for 10 min. The PCR protocol of the two-step process was essentially the same as that for one-step process except for the concentration of oligonucleotides and annealing temperature. For PCR assembly, 10 nM of oligonucleotides were used without the forward and reverse primers. For gene amplification, µl of the assembled product was diluted in 25 µl of amplification reaction mixture with primers concentration of 400 nM each, and an annealing temperature of 49°C was employed. The PCR conditions of these three gene syntheses are summarized in Table 4.2. Table 4.2: PCR conditions for one-step, non-competition (NC) one-step and two-step gene synthesis. PCA* PCR Method Annealing temperature # of cycles Annealing temperature # of cycles One-step 58°C† 30 – – Two-step NC one-step 58°C 58–70°C 30 20 49°C ‡ 30 49°C ‡ 20 *Average melting temperature of oligonucleotide set = 66.0°C. † Melting temperature of outer primers = 59.4°C and 63.4°C. ‡ Melting temperature of outer primers = 49.4°C and 50.9°C. Page 50 National University of Singapore Department of Electrical and Computer Engineering 4.3.4 Agarose gel electrophoresis Synthesized products were analyzed by 1.5% agarose gel (NuSieve® GTG®, Cambrex Corporation), stained with GelRed (Bio-Rad Laboratories) or SYBR Green (Invitrogen) and visualized using Typhoon 9410 variable imager (Amersham Biosciences). Gel electrophoreses were performed at 100 Volts for 45 mins with 100 bp ladder (New England) and μL of DNA samples. 4.4 Performance of TopDown one-step gene synthesis and its real-time analysis 4.4.1 Performance of TD one-step gene synthesis Successful gene synthesis was achieved using TD one-step and two-step processes, while no obvious full-length gene product was obtained in one-step PCR process as shown by gel electrophoresis (Figure 4.2). The TD one-step process was conducted with an annealing temperature (Tah) of 67°C (average T m of oligonucleotides = 66°C) for the first 20 cycles, followed by an annealing temperature of 49°C (average T m of primers = 50.1°C) for another 20 cycles. The continuous fluorescence monitoring revealed the efficiency of the gene synthesis process (Figure 4.3). Unlike the exponential nature of PCR amplification, the assembly efficiency was more likely linear in nature. Figure 4.2: Agarose gel (1.5 %) electrophoresis results of one-step (30 cycles), TopDown (TD) one-step (40 cycles), and two-step (PCA: 30 cycles; PCR: 30 cycles) gene syntheses. The TD one-step process is conducted with annealing temperature of 67 °C for the first 20 cycles followed by another 20 cycles with annealing temperature of 49 °C. The concentrations of oligonucleotides and outer primers are 10 nM and 400 nM respectively. Page 51 National University of Singapore Department of Electrical and Computer Engineering Two intercalating fluorescent dyes (SYBR Green I and LCGreen I) were investigated for real-time gene synthesis (Figure 4.4). The LCGreen I [96] was more suitable for studying the realtime gene synthesis, which has a fluorescence spectrum similar to the commonly adopted SYBR Green I in real-time PCR. The SYBR Green I would bind preferentially to long DNA fragments [97, 98] , and could redistribute from short DNA fragments to long DNA fragments during thermal cycling [99]. This would make it difficult to analyze the fluorescence signal since the PCA mixture would contain various lengths of dsDNA. Figure 4.3: Continuous fluorescence monitoring of real-time gene synthesis with 1X LCGreen I. The first 20 cycles is conducted with annealing temperature of 67 °C followed by another 20 cycles with annealing temperature of 49 °C. The concentrations of oligonucleotides and outer primers are 10 nM and 400 nM respectively. The initial DNA quantity (~ pmol; 10 nM × 20 µl × 30 oligonucleotides) in PCA mixture was much larger (by > orders of magnitude) than that in standard PCR amplification (< 106 copies of template DNA) [88]. The real-time PCR conditions were adjusted for this factor. The optimal concentration of LCGreen I was studied and increased from 1× for standard PCR to 2× (Figure 4.4). The dNTPs concentration was adjusted from 0.2 mM each for standard PCR to mM each to prevent the depletion of dNTPs. The Mg2+ ion (MgSO4) concentration has been empirically optimized (at mM) based on the concentration of dNTP, which could chelate with Mg2+ and affect the polymerase activity [100, 101] . (Figure 4.5). The manufacturer’s recommended Mg2+ ion concentration was 1.5 mM for standard PCR with 0.2 mM of dNTPs each. Page 52 National University of Singapore Department of Electrical and Computer Engineering (a) (b) Figure 4.4: Concentration effects of SYBR Green I and LCGreen I for TD one-step real-time gene synthesis of S100A4. (a) 0.25× to 5× SYBR Green I. The fluorescence intensity of 1× LCGreen I is also included in this plot for comparison. The fluorescence curves of SYBR Green I are insensitive to the number of PCR cycles, and fail to indicate the DNA length extension during gene synthesis. (b) 0.25× to 5× LCGreen I. The annealing temperatures for assembly and amplification are 58°C and 49°C, respectively. The concentrations of oligonucleotide and outer primer are 64 nM and 400 nM, respectively. (a) (b) Figure 4.5: The MgSO4 concentration is critical for successful gene synthesis. (a) Fluorescence of 1× LCGreen I as a function of PCR cycle number for various concentrations of MgSO4: 1.5 mM (◊), 2.5 mM (□), 3.0 mM (∆), 3.5 mM (×), 4.0 mM (●), and 5.0 mM (○). (b) The corresponding agarose gel electrophoresis results. The TD one-step gene synthesis is conducted with annealing temperatures of 58°C and 49°C for assembly and amplification, respectively, mM each of dNTP, 10 nM of oligonucleotides, and 400 nM of forward and reverse primers. Gene synthesis with mM of MgSO4 provides the best yield of full-length product. Page 53 National University of Singapore Department of Electrical and Computer Engineering 4.4.2 Analysis of real-time gene synthesis Mechanistically, gene synthesis took place in several phases, as revealed by the variation in slopes with the number of PCR cycles (Figure 4.6). This phenomenon was remarkable with an oligonucleotide concentration of 10–20 nM. In the early cycles of PCA, most annealing between paired oligonucleotides formed an extendable duplex, which could undergo extension by polymerase (phase 1; cycles < 7). The fluorescence signal revealed a linear increment of DNA length extension with each cycle. In contrast to that reported by Wu et al. [92] and Lee et al. [95], the assembly efficiency increased with further PCR cycles (phase 2; cycles ~ 7–14). Our hypothesis was that the assembly process switched in favor of full-length template amplification as the fulllength fragments emerged, and was promoted by the excess outer primers. The PCA reaction then reached the first plateau (phase 3; cycles 15–20) whereby the outer primers priming was limited by the elevated annealing conditions (Tah – Tm = 15°C). At cycle #21, the annealing temperature was reduced to 49°C to match with the Tm of primers (phase 4; cycles ~ 21–29). The exponential amplification was boosted, and caused a sudden jump in fluorescence signals. Finally, the process reached the second plateau, presumably due to the depletion of outer primers or non-specific products annealing (phase 5). The plateau stages were delayed or completely missing for low oligonucleotide concentration (< nM) due to its low assembly efficiency. For gene synthesis with > 64 nM of oligonucleotides, the PCR process reached the plateau within 15 cycles. Additional cycles would most likely favor non-specific PCR, and lead to the generation of spurious bands and the buildup of high molecular weight products in gel electrophoresis (Figure 4.6b), as observed in most reported gene synthesis results 93] [8, 14, 26, 34, 78, 79, 92, . The consistent gel results and real-time PCR curves suggested that the optimal oligonucleotide concentration was 10–20 nM for TD gene synthesis, which coincided with that of both the onestep [34, 92] and two-step [93] processes. Page 54 National University of Singapore Department of Electrical and Computer Engineering (a) (b) Figure 4.6: The oligonucleotide concentration is critical in the successful gene synthesis. S100A4 (752 bp) is synthesized with various oligonucleotide concentrations ranging from nM to 80 nM, and annealing temperatures of 67°C for the first 20 cycles and 49°C for the next 20 cycles. (a) Fluorescence as a function of PCR cycle number for oligonucleotide concentrations of nM (◊), nM (□), 10 nM (∆), 13 nM (+), 17 nM (×), 20 nM (○), 40 nM (●), 64 nM (▲), and 80 nM (♦). The slopes of fluorescence increment in the early cycles and cycles #21 indicate the efficiencies of the assembly and amplification processes. (b) The corresponding agarose gel electrophoresis results. We further investigated the effect of primer by varying primer concentration from 60 nM to µM while keeping the oligonucleotide concentration at 10 nM. The highest full-length quantity was obtained with 400 nM of primers (Figure 4.7), which was consistent with observations in one-step [92] and two step [93] processes. Assembly efficiencies, depicted by the slopes of fluorescence increment, were indifferent in the early cycles (< cycle 7), even though the primer concentration was varied by 16-fold (inset in Figure 4.7a). This demonstrated the noninterference feature of the TD process, wherein the outer primers did not intervene with the assembly process. The assembly efficiencies started to deviate at around cycle as the full-length products emerged, in favor of full-length template amplification. Unlike the oligonucleotide Page 55 National University of Singapore Department of Electrical and Computer Engineering concentration, which dominated the assembly reaction and critically influenced the success of gene synthesis, the primer concentration was less critical. It presumably controlled the late amplification process and the quantity of desired DNA. The optimal PCR cycles depended on the initial oligonucleotide concentration and target gene length. This was clearly demonstrated by the experiment on oligonucleotide concentration (Figure 4.6). As oligonucleotide concentration increased from 20 nM to 80 nM, the full-length band gradually disappeared and became widened. (a) (b) Figure 4.7: S100A4 (752 bp) is successfully synthesized with various primer concentrations ranging from 60 nM to µM, as indicated by the sharp, narrow gel band of the desired length. (a) Fluorescence as a function of PCR cycle number for outer primer concentrations of 60 nM (◊), 120 nM (□), 200 nM (∆), 300 nM (×), 400 nM (+), and µM (○). The inset shows the fluorescence signal of the first 20 cycles. (b) The corresponding agarose gel electrophoresis results. The overlapping assembly was a parallel process. Relatively few PCR cycles were needed to complete the assembly. The theoretical minimum number of cycles (x) in order to construct a dsDNA molecule of length (L) from uniform oligonucleotide length (n) and overlapping size (s) is given by: x n − (2 x − 1) s > L Theoretically, six PCA cycles were sufficient for assembling S100A4 (752 bp) from a pool of 40nt oligonucleotides with an overlap of 20nt. To determine whether excess cycling was necessary for gene assembly, we used the optimal condition determined in previous experiments Page 56 National University of Singapore Department of Electrical and Computer Engineering with various PCA cycles of 6–20, followed by 20 amplification cycles. Gene synthesis was fairly efficient. Indeed, full-length assembly was achieved within 11 PCA cycles (Figure 4.8). Figure 4.8: S100A4 is synthesized with various assembly cycles (6-20 cycles), followed by another 20 cycles for amplification. Agarose gel (1.5%) electrophoresis results indicate fulllength assembly is achieved within 11 cycles. The gene synthesis was insensitive to the variation in assembly annealing temperature (Tah) from 58°C to 70°C, as visualized in both gel results and fluorescence signals (Figure 4.9). The fluorescence intensity curves were indiscriminant to the annealing temperatures during the assembly phase (first 13 cycles), and began to deviate only after the first phase (see inset in Figure 4.9a). The indifference in fluorescence intensity during the first 13 cycles implied that the primers did not intervene with the assembly reaction. The primers were designed with an average Tm of 50.9°C, which meant that the primers encountered an annealing stringent of 7.1–19.9°C (Tah – Tm) during the PCA process. This suggested that we could potentially reduce the melting temperature window (∆Tm of primers and oligonucleotides) to 7.1°C, and ensure the non-competitive feature of TD gene synthesis method. Interestingly, a higher yield of the desired DNA was obtained with a stringent annealing temperature (> 67°C) higher than the average Tm of oligonucleotides (66°C). Page 57 National University of Singapore Department of Electrical and Computer Engineering (b) (a) Figure 4.9: S100A4 (752 bp) synthesized with various assembly annealing temperatures ranging from 58°C to 70°C for the first 20 cycles, followed by an annealing temperature of 49°C for the next 20 cycles. (a) Fluorescence as a function of PRC cycle number for annealing temperatures of 58°C (◊), 60°C (□), 62°C (∆), 65°C (×), 67°C (+), and 70°C (○). The inset shows the middle 15 cycles (#13–27). (b) The corresponding agarose gel electrophoresis results. Higher synthesis yield was obtained with a stringent assembly annealing temperature (> 67°C). 4.5 Discussion As presented above, TD gene synthesis method offered a simple, rapid and low-cost method for synthesizing fairly long DNA (752 bp) with only one PCR step. The primer interference problem present in one-step process has been eliminated by designing primers and assembly oligonucleotide set with melting temperature variation (∆Tm) of > 15°C, and performing Tmmatched PCR to selectively control the efficiencies of oligonucleotide assembly and full-length template amplification. Experimental data also suggested the TD process might also work well with a relaxed ∆Tm of 7.1°C. The TD synthesis conditions have been empirically optimized using real-time PCR with LCGreen I dye. It was noted that the popular SYBR Green I dye was not suitable for real-time gene synthesis. Other intercalating dyes such as LCGreen [102] [97] and SYTO9 might also work, but required further optimization in dye concentration. Based on the data presented herein, an analytical model has also been developed, which described TD one-step gene synthesis process clearly. TD synthesis occurred in five phases in term of PCR cycles: (i) linear assembly, (ii) emerging amplification, (iii) first plateau, (iv) Page 58 National University of Singapore Department of Electrical and Computer Engineering exponential amplification, and (v) non-specific amplification. The experiments have demonstrated that TD one-step gene synthesis was fairly efficient, as compared to the conventional one-step and two-step processes. Also, the assembly process automatically switched to preferential full-length amplification as the full-length template emerged. This greatly improved the assembly efficiency of the PCA process as compared to the conventional one-step and two-step processes. It was found that the quality and quantity of PCR-based gene synthesis were influenced by several factors, including annealing temperature, concentration of oligonucleotides, concentration of monomers, and number of PCR cycles. The fluorescence curve (10 nM curve in Figure 4.6) suggested the assembly and amplification processes reached the plateau at around cycle #15, and cycle #35, respectively. This implied that the optimal PCR cycles were 30 cycles: 15 cycles each for assembly and amplification reactions. Furthermore, after the PCR reached the plateau, the amplification efficiency of PCR decreased. Additional PCR cycling would favor the non-specific annealing of the full-length product to assembled random-fragments or the fulllength themselves. Both extendable and unextendable pairings could occur. Products annealed in the 3’ recessed configuration could be extended to higher molecular weight DNA and randomly terminated during the later cycles (extenable). In contrast, products annealed with 3’ ends protruded would reduce the amount of full-length DNA, and generate DNA with lower molecular weight. Both of these types of non-specific annealing would result in diminished full-length gel band [103] , as observed in gel electrophoresis. These abnormal products with incorrect DNA sequences would potentially complicate the enzymatic cleavage or consensus shuffling error correction process [60, 104] when consecutive PCRs were adopted for gene re-assembly or DNA re- amplification. Although the gel results (Figure 4.2) indicated that the two-step process was superior to the TD one-step process, it could be argued that the optimized TD approach could eventually outperform the two-step process in providing better purity of synthetic products, with the understanding of gene synthesis mechanism. The two-step method suffered the same rapid decrease on extension efficiency as the one-step process during the assembly process (PCA), which created a lot of intermediate DNAs with lower molecular weight besides the full-length Page 59 National University of Singapore Department of Electrical and Computer Engineering products. The non-specific annealing and extension would most likely also occur during PCA, considering the observed fairly efficient full-length assembly (< 10 cycles) (Figure 4.7). These higher molecular weight products could also contain primers’ binding sites, and be exponentially amplified at the next amplification step (PCR) as the full-length products products would complicate the error filtering process [104, 105] [86, 103] . These incorrect , requiring excess MutS enzyme. TD process could be improved by having the PCR amplification tailored after the emergence of fulllength DNA to avoid excess PCR. With the help of the developed model, insights into the coincidental optimal conditions reported for various PCR-based gene synthesis processes were attained (Table 4.3). The optimal conditions were universal with an oligonucleotide concentration of 10–60 nM and a primer concentration of 200–800 nM, as reported by a distinct gel band of full-length products. It has been demonstrated that the success of gene synthesis relied on the assembly efficiency of PCA, which was in turn dominated by the oligonucleotide concentration and number of PCR cycles (Figures 4.6 and 4.8). The fluorescence signals indicated that an oligonucleotide concentration of 10–60 nM provided optimal assembly efficiency with full-length products. It was noteworthy that the optimal number of PCR cycles should be adjusted according to the oligonucleotide concentration and gene length to avoid excessive PCR. Successful gene synthesis (752 bp) was also achieved with an oligonucleotide concentration of < nM in 40 PCR cycles. However, it might not be reliable for genes with a high GC content or a high length due to the comparatively low assembly efficiency. The primer concentration was less critical; it controlled the late amplification process and the quantity of desired DNA (Figure 4.7). The optimal primer concentration balanced the quantity and purity of the desired DNA, avoiding the formation of spurious DNA products. As the synthesis yield was insensitive to the assembly annealing temperature (Figure 4.7), the assembly could be performed with an annealing temperature slightly higher than the average Tm of oligonucleotides. This provided several advantages in (i) eliminating potential competition between the assembly and amplification reactions, (ii) minimizing the possibility of truncated oligonucleotides (n–1) participating in the assembly process and the resulting errors, (iii) Page 60 National University of Singapore Department of Electrical and Computer Engineering 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 [106]. All of these would prevent the generation of faulty sequence, especially for gene with high GC contents. Table 4.3: Some reported optimal gene synthesis conditions. Length (bp) [Oligos] (nM) [Primer] (nM) # of PCR cycles Method PDK1 gene 1712 20 200 25–35 / 25–35 2-Step PCR [26] PDK1 gene 1712 40–200 200 25 / 25 TBIO [26] 470–1200 40 200 20 / 15 2-Step PCR [79] 2370 30 600 25 / 25 2-Step PCR [8] 209–936 10–25 400–800 25 1-Step PCR [92] 12000 60 600 25 / 25 PCR-based [91] Various DNAs 327–993 10–25 500 35–45 1-Step PCR [34] GFP segment 752 5–15 400 30 / 30 2-Step PCR [93] Name of synthesized DNA Various DNAs vip3aI gene Various DNAs Pur operon Another important factor for successful gene synthesis was the polymerase enzyme. The performance of various polymerases has been studied and compared for the gene synthesis process [1, 37, 107-109]. The KOD series of polymerases were suggested for gene synthesis [37, 92, 107]. It was observed that the KOD Hot Start outperformed the Taq and Pfu polymerases (data not shown). No obvious full-length gene product was obtained with Taq or Pfu for the TD method. 4.6 Conclusion A simple, efficient and cost-effective TD one-step gene synthesis method has been developed that combines the advantages of one-step and two-step gene synthesis processes. TD synthesis is recommended to be conducted with the following conditions: (i) design primers (Tm = 50–55°C) and inner oligonucleotide (Tm ~ 65°C) with distinct melting temperature (∆Tm > 8°C); (ii) 2× LCGreen I, mM of MgSO4, µM each of dNTP, 500 µg/ml of BSA, 10 nM of oligonucleotides, 400 nM of forward and reverse primers, and U of KOD Hot Start, (iii) of initial denaturation at 95°C; 15 cycles of 95°C for s, 67–70°C (according to the Tm of inner Page 61 National University of Singapore Department of Electrical and Computer Engineering oligonucleotides) for 30 s, 72°C for 30 s; followed by 15 cycles of 95°C for s, 50–55°C (according to the Tm of outer primers) for 30 s, and 72°C for 30 s with 10 final extension. Page 62 [...]... pair of forward and reverse primers Several PCR- based gene synthesis methods have been developed including: 1) Two -step /one- step overlapping synthesis, 2) Two -step /one- step successive synthesis, 3) Thermodynamically balanced inside-out synthesis, 4) TopDown one- step synthesis (Chapter 4), and 5) Automatically TouchDown one- step synthesis (Chapter 5) Two -step /one- step overlapping PCA gene synthesis Figure... following areas: (1) the biochemistry background knowledge of gene synthesis and gene synthesis related lab- on-a -chip technology overview; (2) development of bioinformatics software and its experimental verification; (3) development and the real- time experimental analysis of TopDown one- step gene synthesis protocol; (4) development and realtime experimental analysis of automatic TouchDown gene synthesis protocol;... of S100A4 -1 (lanes 1 and 3) and S100A4-2 (lanes 2 and 4) with oligonucleotide concentrations of 10 nM and 1 nM, and PKB2 (lane 5) with 1 nM oligonucleotides The arrow indicates the full-length DNA Syntheses are performed with 30 and 36 cycles, respectively, for 10 nM and 1 nM oligonucleotides, with 30-s annealing at 70°C and 90-s extension at 72°C 77 Figure 6 .1: Schematic illustration of PCR- based... of TmPrime with other gene synthesis programs for S100A4, PKB2, GFPuv and the whole genome of Poliovirus [1] (Genbank FJ 517 648; 7 418 bp) and øX174 bacteriophage [3] (Genbank J02482; 5386 bp) with oligonucleotide concentration of 10 nM 42 Table 4 .1: Data of oligonucleotide set 49 Table 4.2: PCR conditions for one- step, non-competition (NC) one- step and two -step gene synthesis ... gene synthesis conditions 61 Table 5 .1: Data of oligonucleotide set 66 Table 5.2: Summary of primers for conventional one- step, and ATD one- step gene syntheses All PCR assemblies are performed with an annealing temperature of 70°C 67 Table 6 .1: Errors and efficiencies in the synthesis of GFPuv using one- step and twostep processes in the microfluidic device vs standard PCR. .. limitation of current gene synthesis approaches and then provide our solutions These include new bioinformatics program for gene synthesis, investigation of kinetics and mechanisms of PCR- based gene synthesis, novel gene synthesis approach with ultra-low oligonucleotide concentration, and finally the lab- chip devices to integrate the tedious gene synthesis process into a chip Several bioinformatics programs... PCR Gibson, 08 [5] φX174 bacteriophage 5,386 bp 96 of 42 mers LCR & PCR Smith, 03 [3] 13 9 10 42 bp PCR 1. 8 / kb Hoover, 02 [14 ] 14 76 bp 64 0f 40 mers LCR & PCR Chalmers, 01 [19 ] Phenylalanine ammonia-lyase Plasmodium falciparum Ornithine transcarbamylase 2.2 kbp 10 8 of 40 mers PCR Baedeker, 99 [6] 2 .1 kbp 10 4 of 40 mers PCR 3.5 / kb Martinez, 99 [20] 10 44bp 18 of 70-80 mers PCR 2.7... 5.7: Fluorescent curves of conventional 1 -step (▲,♦) and ATD one- step gene syntheses (Δ,◊) with dNTPs concentration of 4 mM (♦,◊) and 0.8 mM (▲,Δ) for (a) S100A4 -1 (752 bp), (b) S100A4-2 (752 bp), and (c) PKB2 (14 46 bp) All PCRs are conducted with 30-s annealing at 70°C and 90-s extension at 72°C The concentrations of oligonucleotides and outer primers are 10 nM and 400 nM, respectively 77 Figure... LCR based gene synthesis, PCR based gene synthesis, real- time PCR, DNA purification/extraction and enzymatic error filtering Also, fundamentals of gene synthesis related lab- on-a -chip technology including microvalves, micromixers, and PCR on a chip are presented Chapter 3 focuses on the discussion of the development of bioinformatics software and its experimental verifications Especially a novel approach... understanding of genes synthesis, the shortcomings or limitation of various synthesis schemes and appreciations on the complexity of current genes synthesis methods 1. 1 Overview gene synthesis In general, generic gene synthesis often employs a “topdown” approach that involves a series of highly complex processing steps as shown in Figure 1. 1 Basically, it includes sequential activities of (i) pre-synthesis . TOWARDS AUTOMATIC GENE SYNTHESIS WITH BIOINFORMATICS SOFTWARE, NOVEL ONE- STEP REAL- TIME PCR ASSEMBLY, AND LAB- CHIP GENE SYNTHESIS HUANG. Computer Engineering Page VI Towards automatic gene synthesis with bioinformatics software, novel one- step real- time PCR assembly, and lab- chip gene synthesis Huang Mo Chao Under the. 12 2 .1 Introduction 12 2.2 Bioinformatics in Gene Synthesis 12 2.3 Biochemical method of gene synthesis 16 2.3 .1 LCR based gene assembly 17 2.3.2 PCR based gene assembly 18 2.3.3 Real time