Molecular Engineering of Novel Nucleotide
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`Analogues for DNA Sequencing and Analysis
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`Jia Guo
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`Submitted in partial fulfillment of
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`the requirements for the degree of Doctor of Philosophy
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` in the Graduate School of Arts and Sciences
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` COLUMBIA UNIVERSITY
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`2009
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`Illumina Ex. 1092
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` © 2009
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` Jia GUO
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` All Rights Reserved
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` ABSTRACT
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`Molecular Engineering of Novel Nucleotide Analogues for
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`DNA Sequencing and Analysis
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` Jia GUO
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`DNA sequencing by synthesis (SBS) on a solid surface during the polymerase
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`reaction can decipher multiple DNA sequences in parallel. The first part of this thesis
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`presents the development of a DNA sequencing method that is a hybrid between the
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`Sanger dideoxy chain terminating reaction and SBS. In this approach, four nucleotides,
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`modified as reversible terminators by capping the 3’-OH group with a small reversible
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`moiety so that they are still recognized by DNA polymerase as substrates to extend the
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`DNA chain, are used in combination with a small percentage of four cleavable
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`fluorescent dideoxynucleotides to perform SBS. Sequences are determined by the unique
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`fluorescence emission of each fluorophore on the DNA products terminated by ddNTPs.
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`Upon removing the 3’-OH capping group from the DNA products generated by
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`incorporating the 3’-O-modified dNTPs and the fluorophore from the DNA products
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`terminated with the ddNTPs, the polymerase reaction reinitiates to continue the sequence
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`determination. Various DNA templates, including those with homopolymer regions were
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`accurately sequenced with readlengths of over 30 bases using this hybrid SBS method on
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`a chip and a four-color fluorescent scanner. To further extend the read-length of this
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`hybrid sequencing method, a consecutive DNA sequencing by primer reset approach is
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`developed. Upon removing the sequenced DNA strand and reattaching the original
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`primer to allow the extension of this primer with a combination of natural and modified
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`nucleotide analogues to the end of the first round sequence, the hybrid SBS can be carried
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`out from that point to decipher the adjacent cluster of bases on the template. The
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`sequencing read-length of a DNA template immobilized on a chip is almost doubled
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`using this primer reset approach.
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`Single nucleotide polymorphisms (SNPs) are important markers for disease gene
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`identification and for pharmacogenetic studies. The second part of this thesis describes
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`the design, synthesis and evaluation of a chemically cleavable biotinylated nucleotide
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`analogue, ddATP-N3-biotin, for multiplex SNP analysis by MALDI-TOF MS. This
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`nucleotide analogue has a biotin moiety attached
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`to
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`the 7-position of
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`2’,3’-dideoxyadenosine 5'-triphosphate through a chemically cleavable azide-based linker.
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`We have demonstrated that this ddATP-N3-biotin is faithfully incorporated by the DNA
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`polymerase Thermo Sequenase. The generated DNA extension products can be
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`efficiently isolated by a streptavidin-coated surface and recovered under a mild chemical
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`cleavage conditions. Single and mutiple primer extension reactions were performed using
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`ddATP-N3-biotin to generate and isolate DNA extension products for MALDI-TOF MS
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`analysis.
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`DNA microarray technology offers a paradigm for the study of genome-wide
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`patterns of gene expression. The cDNA labeling step plays an important role in the
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`accuracy and reproducibility of a microarray experiment. The third part of this thesis
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`focuses on the development of a click chemistry based cDNA labeling strategy for
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`microarray analysis. In this approach, azide modified nucleotide analogues along with
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`natural nucleotides are incorporated in reverse transcription reactions with RNA samples
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`as templates. The azide groups on the generated cDNAs are coupled with alkyne
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`functionalized fluorophores by click chemistry. Due to the high stability of the azide and
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`alkyne groups in aqeous solution, the cDNAs are labeled efficiently with sufficient
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`amount of the fluorescent molecules for microarray analysis using this approach.
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` TABLE OF CONTENTS
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`Table of Contents………………………………………………………………………….i
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`List of Figures and Tables………………………………………………………………..iv
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`Abrreviations and Symbols…..…………………………………………………...……..xiv
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`Acknowledgements……………………………………………………………………..xvii
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`Dedication…………………………………………………………..…………………...xix
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`Part I. Four-Color DNA Sequencing with 3’-O-modified Nucleotide
`Reversible Terminators and Chemically Cleavable Fluorescent
`Dideoxynucleotides
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`Chapter 1: Introduction to DNA Sequencing and Analysis Technologies
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`1.1 Introduction ………………………………………………………….2
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`1.2 Background and significance ………………………………………..3
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`1.3 Sanger dideoxynucleotide sequencing ………………………………8
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`1.4 DNA sequencing by hybridization ………………………………….11
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`1.5 DNA sequencing by MALDI-TOF MS……………………………...12
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`1.6 Nanopore DNA sequencing …………………………………………16
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`1.7 DNA sequencing by ligation ………………………………………..18
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`1.8 DNA sequencing by synthesis (SBS) ……………………………….23
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`References ………………………………………………………………32
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`Chapter 2: Four-color DNA Sequencing with 3’-O-Modified Nucleotide Reversible
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`Terminators and Chemically Cleavable Fluorescent Dideoxynucleotides
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`2.1 Introduction …………………………………………………………36
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`2.2 Experimental Rationale and Overview ……………………………..38
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`2.3 Results and Discussion ……………………………………………...41
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`2.4 Materials and Methods ……………………………………………...71
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`2.5 Conclusion …………………………………………………………..96
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`References ………………………………………………………………100
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`Chapter 3: Consecutive Rounds of DNA Sequencing by Primer Reset
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`3.1 Introduction …………………………………………………………102
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`3.2 Experimental Rationale and Overview ……………………………..105
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`3.3 Results and Discussion ……………………………………………..109
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`3.4 Materials and Methods ……………………………………………..119
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`3.5 Conclusion ………………………………………………………….128
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`References ………………………………………………………………130
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`Part II. Design and Synthesis of Chemically Cleavable Biotinylated
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`Dideoxynucleotides
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`for DNA Analysis with Mass
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`Spectrometry
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`4.1 Introduction …………………………………………………………133
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`4.2 Experimental Rationale and Overview ……………………………..139
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`4.3 Results and Discussion ……………………………………………..142
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`4.4 Materials and Methods ……………………………………………...150
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`4.5 Conclusion …………………………………………………………..155
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`References ……………………………………………………………….156
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`Part III. Fluorescent Labeling of cDNA Probes with Click Chemistry
`for Microarray Analysis
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`5.1 Introduction …………………………………………………………159
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`5.2 Experimental Rationale and Overview ……………………………..163
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`5.3 Results and Discussion ……………………………………………..165
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`5.4 Materials and Methods ……………………………………………..173
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`5.5 Conclusion ………………………………………………………….179
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`References ………………………………………………………………181
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` List of Figures and Tables
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`Part I. Four-Color DNA Sequencing with 3’-O-modified Nucleotide Reversible
`Terminators and Chemically Cleavable Fluorescent Dideoxynucleotides
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`Chapter 1: Introduction to DNA Sequencing and Analysis Technologies
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`Figure 1.1. Chemical structures of 2’-deoxyribonucleotides. Each nucleotide is
`composed of a base (adenine, guanine, cytosine or thymine), a sugar, and a phosphate
`group. (page 4)
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`Figure 1.2. DNA molecular structures. (A) 3-D computer rendered model of the
`DNA double helix. (B) A cartoon depicting two DNA molecules held together by
`hydrogen bonds between the paired bases. (C) A zoomed in section of the double
`helix, which shows the specific chemical structures of the bases. The efficient
`hydrogen bonding is only allowed between A and T, or between G and C. (page 6)
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`Figure 1.3. DNA replication. The DNA double helix is unwound and each strand acts
`as a template to incorporate complementary bases into new growing DNA strands.
`(page 7)
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`Figure 1.4. The mechanism of the DNA replication reaction. Incorporation of a
`nucleotide at the 3’ end of a growing DNA strand is a fundamental biological process,
`in which the base-pairing between the incoming nucleotide and the DNA template
`strand guides the generation of a new DNA strand. DNA polymerase catalyzes the
`extension of the growing DNA strand by incorporating the incoming nucleotide at the
`3’-OH end with the formation of a phosphodiester bond and the release of
`pyrophosphate. (page 9)
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`Figure 1.5. Sanger dideoxy sequencing. To generate the DNA sequencing ladder,
`dNTPs and dye-labled ddNTPs are combined to perform the DNA replication
`reaction. The incorporation of dNTPs generates a free hydroxyl group at the 3’ end of
`the DNA growing strand, which allows this DNA strand to be further extended; while
`the incorporation of dye-labled ddNTPs eliminates the 3’-OH group of the DNA
`strand and terminates the strand extension. DNA fragments of different lengths
`produced by the DNA replication reaction are separated by gel electrophoresis. The
`unique fluorescence emission of each of the four dye-labled ddNTPs indicates the
`sequence of the DNA template. (page 10)
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`Figure 1.6. Sequencing by hybridization. To resequence a specific base on the
`reference genome, four features are present on a chip, each identical except for the
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`base in the middle. The identity of the query base is determined by the differential
`hybridization of the unknown DNA sample to each of the four features, which contain
`bases complementary to the query sequence. (page 13)
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`time-of-flight mass
`laser desorption/ionization
`Figure 1.7. Matrix-assisted
`spectrometry (MALDI-TOF MS). Analyte molecules (such as DNA sequencing
`fragments) and matrix molecules (typically ultraviolet (UV) or infrared (IR)
`light-absorbing small organic molecules) are mixed in solution. After crystallization
`on a sample plate, analyte molecules are gently desorbed and ionized by UV laser
`irradiation. The resulting charged ions are accelerated under a constant electric
`voltage, which causes them to fly towards the ion detector. The time of the flight can
`be used to determine the masses of the charged ions. (page 15)
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`Figure 1.8. The –hemolysin protein self-assembles in a lipid bilayer to form an ion
`channel and a nucleic acid stretch passes through it (top), generating corresponding
`electronic signatures (bottom). (page 17)
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`Figure 1.9. Hypothesized plot of translocation time versus blockade current when
`DNA molecules (A), (B), (C) are passing through the nanopore. (page 19)
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`Figure 1.10. Exonuclease-assisted nanopore DNA sequencing. An engineered
` -hemolysin protein with an internal cyclodextrin adapter is placed in a well that
`contains two electrodes on either side of the pore. As the exonuclease directs
`individual DNA bases, in sequence, through the nanopore, each base transiently binds
`to the cyclodextrin and generates distinct changes in the electronic current, which
`indicate the sequence of the original DNA strand. (page 19)
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`Figure 1.11. Detector oligonucleotides for sequencing by ligation. (A) 8-mers
`detector oligonucleotides consist of five normal bases at the 3’ end, three universal
`bases at the 5’ end, and a unique fluorophore attached to the last base. Although each
`of the four fluorophores corresponds to four types of dinucleotides (B), with one base
`already known from the previous round in these dinucleotides, the other base can be
`unambiguously identified with the fluorescence signal. (page 21)
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`Figure 1.12. DNA sequencing by ligation. Each sequencing cycle consists of ligation,
`imaging, capping, and cleavage (Step 1-4). After repeating this process for 5-7 cycles
`(Step 5), a new primer with one base shorter length is introduced (Step 6) to perform
`a second round of sequencing (Step 7). Once five such sequencing rounds are
`completed, every base on the DNA strand except the last one has been interrogated
`twice, and the fluorescence signal indicates the sequence of the DNA template. (page
`22)
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`Figure 1.13. DNA pyrosequencing. DNA polymerase catalyzes the incorporation of
`the complementary nucleotide into a growing DNA strand, which leads to the release
`of a pyrophosphate (PPi) molecule. This PPi molecule is converted to ATP by
`sulfurylase, and visible light is subsequently produced by luciferase. The excess
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`nucleotides and ATP are then digested by apyrase. (page 24)
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`Figure 1.14. DNA sequencing by synthesis with chemically modified nucleotides.
`DNA polymerase catalyzes the incorporation of a fluorescently labeled nucleotide
`reversible terminator, which is complementary to the next base on the unknown DNA
`template. After removing unincorporated nucleotide analogues and other excess
`reagents, a fluorescence imager is used to identify the base just incorporated. Then
`the fluorophore and the 3’ capping moiety are chemically cleaved to reinitiate the
`DNA polymerase reaction. (page 26)
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`Figure 1.15. The 3D structure of a ternary complex of a rat DNA polymerase, a DNA
`template-primer, and a dideoxycytidine triphosphate (ddCTP). This figure, which
`shows the active site of the polymerase in the context of the polymerase DNA
`complex, illustrates that the 3' position of the dideoxyribose ring is very crowded,
`while ample space is available at the 5 position of the cytidine base. (page 28)
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`Figure 1.16. SBS approach conducted in a high-throughput manner. Different DNA
`templates are immobilized on a chip to initiate the sequencing reaction. DNA
`polymerase catalyzes the incorporation of the complementary nucleotide analogues
`(step 1), which generate unique fluorescence emissions on the spot (step 2). This
`unique fluorescence signal indicates the identity of the incorporated nucleotide. After
`capping the unextended primers with ddNTPs (step 3), the fluorophore moiety and
`the 3’ capping group are chemically removed (step 4). With a reconstituted free
`3’-OH group, the sequencing primer is ready for the next sequencing cycle (step 5).
`(page 29)
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`Chapter 2: Four-color DNA Sequencing with 3’-O-Modified Nucleotide Reversible
`
`Terminators and Chemically Cleavable Fluorescent Dideoxynucleotides
`
`
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`Figure 2.1. The hybrid DNA sequencing approach between the Sanger dideoxy chain
`terminating reaction and sequencing by synthesis. In this approach, four nucleotides
`(3’-O-R1-dNTPs) modified as reversible terminators by capping the 3’-OH with a
`small reversible moiety R1 so that they are still recognized by DNA polymerase as
`substrates, are used in combination with a small percentage of four cleavable
`fluorescent dideoxynucleotides (ddNTP-R2-fluorophores) to perform SBS. DNA
`sequences are determined by the unique fluorescence emission of each fluorophore on
`the DNA products terminated by ddNTPs. Upon removing the 3’-OH capping group
`R1 from the DNA products generated by incorporating the 3’-O-R1-dNTPs, and the
`cleavage of the R2 linker to remove the fluorophore from the DNA products
`terminated with the ddNTPs, the polymerase reaction reinitiates to continue the
`sequence determination. (page 40)
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`Figure 2.2. Structures of the nucleotide reversible terminators, 3’-O-N3-dATP,
`3’-O-N3-dCTP, 3’-O-N3-dGTP, and 3’-O-N3-dTTP. (page 42)
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`Figure 2.3. Synthesis of 3’-O-azidomethyl-dATP. (page 44)
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`Figure 2.4. Synthesis of 3’-O-azidomethyl-dTTP. (page 44)
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`Figure 2.5. Synthesis of 3’-O-azidomethyl-dCTP. (page 45)
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`Figure 2.6. Synthesis of 3’-O-azidomethyl-dGTP. (page 45)
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`Figure 2.7. Staudinger reaction with TCEP to regenerate the 3’-OH group of the
`DNA extension product. (page 47)
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`Figure 2.8. The polymerase extension scheme (left) and MALDI-TOF MS spectra of
`the four consecutive extension products and their cleaveage products (right) using the
`nucleotide
`reversible
`terminators
`(3’-O-N3-dNTPs). Primer extended with
`3’-O-N3-dCTP (1) (A), and its cleavage product 2 (B); Product 2 extended with
`3’-O-N3-dGTP (3) (C), and its cleavage product 4 (D); Product 4 extended with
`3’-O-N3-dATP (5) (E), and its cleavage product 6 (F); Product 6 extended with
`3’-O-N3-dTTP (7) (G), and its cleavage product 8 (H). After brief incubation in a
`TCEP aqueous solution the azidomethyl moiety capping the 3’-OH group of the DNA
`extension products is completely removed to continue the polymerase reaction. (page
`48)
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`Figure 2.9. Structures of cleavable fluorescent dideoxynucleotide terminators (A)
`ddNTP-N3 (version I)-fluorophores and (B) ddNTP-N3 (version II)-fluorophores, with
`the 4 fluorophores having distinct fluorescent emissions: Bodipy-FL-510 ( abs (max) =
`502 nm; em (max) = 510 nm), R6G ( abs (max) = 525 nm; em (max) = 550 nm), ROX ( abs
`(max) = 585 nm; em (max) = 602 nm), Cy5 ( abs (max) = 649 nm; em (max) = 670 nm).
`(page 50)
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`Figure 2.10. Synthesis of the cleavable linker (version I). (page 52)
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`Figure 2.11. Synthesis of ddCTP-N3 (version I)-Bodipy-FL-510. (page 53)
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`Figure 2.12. Synthesis of ddUTP-N3 (version I)-R6G. (page 55)
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`Figure 2.13. Synthesis of ddATP-N3 (version I)-ROX. (page 56)
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`Figure 2.14. Synthesis of ddGTP-N3 (version I)-Cy5. (page 57)
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`Figure 2.15. Synthesis of the cleavable linker (version II). (page 58)
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`Figure 2.16. Synthesis of ddCTP-N3 (version II)-Bodipy-FL-510. (page 58)
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`Figure 2.17. Synthesis of ddUTP-N3 (version II)-R6G. (page 59)
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`Figure 2.18. Synthesis of ddATP-N3 (version II)-ROX. (page 60)
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`Figure 2.19. Synthesis of ddGTP-N3 (version II)-Cy5. (page 61)
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`Figure 2.20. Staudinger reaction with TCEP to cleave the N3-fluorophore from the
`dideoxynucleotide. (page 63)
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`Figure 2.21. A polymerase reaction scheme (top) to yield DNA extension products
`by incorporating each of the four ddNTP-N3 (version I)-fluorophores and the
`subsequent cleavage reaction to remove the fluorophores from the DNA extension
`products. MALDI-TOF MS spectra (bottom) showing efficient base specific
`incorporation of the ddNTP-N3-fluorophores and the subsequent cleavage of the
`fluorophores from the DNA extension products: (A) primer extended with
`ddATP-N3(version I)-ROX (1) (peak at 9,180 m/z), (B) its cleavage product 2 (8,417
`m/z); (C) primer extended with ddCTP-N3(version I)-Bodipy-FL-510 (3) (peak at
`8,915 m/z), (D) its cleavage product 4 (8,394 m/z); (E) primer extended with
`ddGTP-N3(version I)-Cy5 (5) (peak at 9,261 m/z), (F) its cleavage product 6 (8,432
`m/z); (G) primer extended with ddUTP-N3(version I)-R6G (7) (peak at 9,082 m/z) and
`(H) its cleavage product 8 (8,395 m/z). (page 64)
`
`Figure 2.22. A polymerase reaction scheme (top) to yield DNA extension products
`by incorporating each of the four ddNTP-N3 (version II)-fluorophores and the
`subsequent cleavage reaction to remove the fluorophores from the DNA extension
`products. MALDI-TOF MS spectra (bottom) showing efficient base specific
`incorporation of the ddNTP-N3-fluorophores and the subsequent cleavage of the
`fluorophores from the DNA extension products: (A) primer extended with
`ddATP-N3(version II)-ROX (1) (peak at 9,003 m/z), (B) its cleavage product 2 (8,403
`m/z); (C) primer extended with ddCTP-N3(version II)-Bodipy-FL-510 (3) (peak at
`8,738 m/z), (D) its cleavage product 4 (8,380 m/z); (E) primer extended with
`ddGTP-N3(version II)-Cy5 (5) (peak at 9,140 m/z), (F) its cleavage product 6 (8,419
`m/z); (G) primer extended with ddUTP-N3(version II)-R6G (7) (peak at 8,905 m/z)
`and (H) its cleavage product 8 (8,381 m/z). (page 66)
`
`Figure 2.23. (A) A hybrid SBS scheme for 4-color sequencing on a chip using the
`nucleotide reversible
`terminators (3’-O-N3-dNTPs) and cleavable fluorescent
`dideoxynucleotide terminators (ddNTP-N3 (version I)-fluorophores). (B) The 4-color
`fluorescence images for each step of SBS: (1) incorporation of 3’-O-N3-dCTP and
`ddCTP-N3 (version I)-Bodipy-FL-510; (2) cleavage of N3 (version I)-Bodipy-FL-510
`and 3’-CH2N3 group; (3) incorporation of 3’-O-N3-dATP and ddATP-N3 (version
`I)-Rox; (4) cleavage of N3 (version I)-ROX and 3’-CH2N3 group; (5) incorporation of
`3’-O-N3-dTTP and ddTTP-N3 (version I)-R6G; (6) cleavage of N3 (version I)-R6G
`and 3’-CH2N3 group; (7) incorporation of 3’-O-N3-dGTP and ddGTP-N3 (version
`I)-Cy5; (8) cleavage of N3 (version I)-Cy5 and 3’-CH2N3 group; images 9–63 are
`similarly produced. (C) A plot (4-color sequencing data) of raw fluorescence
`intensity obtained using 3’-O-N3-dNTPs and ddNTP-N3 (version
`emission
`I)-fluorophores at the four designated emission wavelengths. (page 69)
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`Figure 2.24. A plot (4-color sequencing data) of raw fluorescence emission intensity
`obtained using 3’-O-N3-dNTPs and ddNTP-N3 (version I)-fluorophores at the four
`designated
`emission wavelengths
`of
`the
`four
`cleavable
`fluorescent
`dideoxynucleotides. (page 72)
`
`Table 2.1 Volumes of solution A and B in each SBS cycle during de novo DNA
`sequencing. (page 97)
`
`
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`Chapter 3: Consecutive Rounds of DNA Sequencing by Primer Reset
`
`terminators, 3’-O-R1-dATP,
`Figure 3.1. Structures of nucleotide reversible
`3’-O-R1-dCTP, 3’-O-R1-dGTP and 3’-O-R1-dTTP. With the 3’-OH capped by a small
`reversible moiety R1, these nucleotide analogues are still recognized as good
`substrates by DNA polymerase. Upon removing the 3’-OH capping group R1 from
`the DNA extension product generated by incorporation of 3’-O-R1-dNTPs, the
`polymerase reaction reinitiates to continue the sequence determination. (page 103)
`
`Figure 3.2. Structures of cleavable fluorescent dideoxynucleotide terminators,
`ddCTP-R2-Bodipy-FL 510, ddUTP-R2-R6G, ddATP-R2-ROX and ddGTP-R2-Cy5.
`Each of the four fluorophores is attached to the 5-position of pyrimidines and the
`7-position of purines through a chemically cleavable linker R2. After incorporation of
`these dideoxynucleotide analogues, the fluorophores can be removed from the DNA
`extension products. (page 104)
`
`Figure 3.3. The hybrid DNA sequencing approach between the Sanger dideoxy chain
`terminating reaction and sequencing by synthesis. In this approach, four nucleotides
`(3’-O-R1-dNTPs) modified as reversible terminators by capping the 3’-OH with a
`small reversible moiety R1 so that they are still recognized by DNA polymerase as
`substrates, are used in combination with a small percentage of four cleavable
`fluorescent dideoxynucleotides (ddNTP-R2-fluorophores) to perform SBS. DNA
`sequences are determined by the unique fluorescence emission of each fluorophore on
`the DNA products terminated by ddNTPs. Upon removing the 3’-OH capping group
`R1 from the DNA products generated by incorporating the 3’-O-R1-dNTPs, and the
`cleavage of the R2 linker to remove the fluorophore from the DNA products
`terminated with the ddNTPs, the polymerase reaction reinitiates to continue the
`sequence determination. (page 106)
`
`Figure 3.4. Consecutive rounds of DNA sequencing by template “walking”. Upon
`ligation of universal primers A and B to both ends of each DNA template, a DNA
`library is prepared (1). Different DNA templates are immobilized on a PEG
`(polyethylene glycol) functionalized surface to initiate the sequencing reaction (2).
`The sequencing primer is then annealed to the DNA template (3). A first round of
`SBS extends the sequencing primer to produce the maximal read-length (4). After
`denaturing the sequencing primers, including those terminated with ddNTP analogues,
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`the original sequencing primer is reattached (5). Then unmodified or 3’-O-modified
`nucleotides are used to extend the primer approximately to the end of the first round
`sequence (6). Following that, the second round of SBS is performed to further extend
`the read-length. (page 108)
`
`Figure 3.5 Consecutive rounds of DNA sequencing by template “walking”. After the
`first sequencing round with the hybrid SBS approach, the extended primer is
`denatured. Upon reattachment of the original primer, three normal nucleotides are
`combined with another 3’-O-modified nucleotide to extend the primer to the end of
`the first round sequence. Then the second sequencing round is performed to further
`extend the read-length. (page 110)
`
`Figure 3.6 The polymerase extension scheme (left) and MALDI-TOF MS spectra of
`the three consecutive extension products and their cleaveage products (right) using
`the combination of dATP, dCTP, dTTP and 3’-O-N3-dGTP. Primer extended with
`3’-O-N3-dGTP (1) (A), and its cleavage product 2 (B); product 2 extended with
`3’-O-N3-dGTP (3) (C), and its cleavage product 4 (D); product 4 extended with dATP,
`dCTP and 3’-O-N3-dGTP (5) (E), and its cleavage product 6 (F). (page 112)
`
`Figure 3.7. (A) A general “template walking” scheme followed by hybrid SBS. The
`polymerase reaction with dATP, dCTP, dTTP and 3’-O-N3-dGTP extends the primer
`until it reaches the next C on the DNA template. After regenerating the 3’-OH group
`by using TCEP, the second and the third extension and cleavage reactions are
`performed in a similar manner. Then a subsequent round of hybrid SBS is carried out
`to generate the four-color DNA sequencing raw data (B). (page 115)
`
`Figure 3.8. Consecutive rounds of DNA sequencing by primer reset on a chip. (A)
`Consecutive DNA sequencing scheme. The first round sequencing is carried out with
`the hybrid SBS approach. Upon the removal of the extended primer and the
`subsequent annealing of the original primer, the polymerase reaction with dATP,
`dCTP, dTTP and 3’-O-N3-dGTP extends the primer until the incorporation of
`3’-O-N3-dGTP. After regenerating the 3’-OH group by using TCEP, the second
`extension and cleavage reactions are performed in a similar manner. Then a second
`round of sequencing is performed with the hybrid SBS approach to interrogate the
`identity of the consecutive bases. (B) A plot (four-color sequencing data) of raw
`fluorescence emission intensity vs. the progress of sequencing extension during the
`first and the second rounds of SBS. (page 117)
`
`Table 3.1 Volumes of solution A and B in each SBS cycle during de novo DNA
`sequencing. (page 126)
`
`Table 3.2 Volumes of solution A and B in each SBS cycle during the first round (A)
`and the second round (B) of de novo DNA sequencing. (page 126)
`
`
`
`Part
`
`II. Design and Synthesis of Chemically Cleavable Biotinylated
`
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`Dideoxynucleotides for DNA Analysis with Mass Spectrometry
`
`
`Figure 4.1. The structures of biotinylated dideoxynucleotides, biotin-11-ddATP,
`biotin-11-ddCTP, biotin-11-ddGTP and biotin-16-ddUTP. (page 135)
`
`Figure 4.2. DNA sequencing by MALDI-TOF MS. (A) The DNA sequencing
`fragments are generated by the polymerase reaction by using the combination of
`dNTPs and biotion-ddNTPs. Then the pure DNA extension products are isolated with
`solid phase capture for MALDI-TOF MS analysis. (B) Illustration of the DNA
`sequencing data generated from the DNA fragments. (page 136)
`
`Figure 4.3. Multiplex genotyping using molecular affinity and mass spectrometry.
`The biotinylated DNA extension products generated by single base extension using
`four biotin-ddNTPs are isolated on a streptavidin-coated surface. After removing the
`unextended primers and other reaction components, the pure DNA fragments are
`released for subsequent MALDI-TOF MS analysis. (page 138)
`
`Figure 4.4. Affinity capture and chemical cleavage for isolating biotinylated DNA
`fragments on a streptavidin-coated surface. The DNA extension products with a
`cleavable biotin moiety at 3’ end are captured and then cleaved chemically to release
`the DNA fragments for analysis by mass spectrometry, leaving the biotin moiety still
`bound to the surface. (page 141)
`
`Figure 4.5. Synthesis of ddATP-N3-biotin. (page 143)
`
`Figure 4.6. Staudinger reaction with TCEP to cleave the biotin moiety and release the
`DNA extension products from the streptavidin-coated surface. (page 145)
`
`Figure 4.7. DNA extension reaction using ddATP-N3-biotin and cleavage of the
`generated DNA fragment captured on a solid surface. DNA polymerase incorporates
`ddATP-N3-biotin into a growing DNA stand, generating the cleavable biotinylated
`DNA fragment. Chemical cleavage using TCEP of this DNA fragment captured on
`the streptavidin-coated surface releases the DNA fragment, with the boitin moiety
`remaining on the surface. (page 146)
`
`Figure 4.8. MALDI-TOF mass spectra of the DNA extension product generated from
`ddATP-N3-biotin and the subsequent cleavage of the generated DNA fragment
`captured on a solid surface. (A) The DNA polymerase incorporated ddATP-N3-biotin,
`yielding DNA extension product 6. (B) This generated DNA fragment was captured
`on a streptavidin-coated surface and a chemical cleavage reaction using TCEP was
`carried out to release the DNA fragment 7. (page 148)
`
`Figure 4.9. (A) Multiplex DNA analysis scheme. Four DNA primers with different
`molecular weights were extended with ddATP-N3-biotin by DNA polymerase. After
`solid phase capture and elimination of the unextended primers, a chemical cleavage
`reaction was carried out under mild conditions to release the DNA extension products.
`
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`(B) MALDI-TOF mass spectra of the generated DNA fragments. (page 149)
`
`Table 4.1 Sequences of oligonucleotide primers and the synthetic DNA template for
`the multiplex DNA extension reaction. (page 154)
`
`
`Part III. Fluorescent Labeling of cDNA Probes with Click Chemistry for
`Microarray Analysis
`
`
`Figure 5.1. Chemical structures of Cy3-dCTP and Cy5-dCTP. (page 161)
`
`labeling for microarray experiments.
`Figure 5.2. Direct cDNA fluorescent
`Cy3-labeled dCTP and Cy5-labeled dCTP are incorporated during cDNA synthesis
`from control and test mRNA samples respectively. After degrading the mRNAs, the
`Cy3-labeled cDNA and Cy5-labeled cDNA are purified, mixed and hybridized on a
`DNA microarray chip. (page 162)
`
`Figure 5.3. Chemical structure of aminoallyl-dUTP. (page 164)
`
`Figure 5.4. Indirect cDNA fluorescent labeling for microarray experiments. The
`control and test mRNA samples are reverse-transcribed into cDNA using reverse
`transcriptase, dNTPs and aminoallyl-dUTP. Then the amino modified cDNA from the
`two samples are labeled with Cy3 and Cy5 respectively, followed by mixing and
`hybridization on the microarray. (page 164)
`
`Figure 5.5. Indirect cDNA fluorescent labeling with click chemistry for microarray
`analysis. Taking the control and test mRNAs as templates, their corresponding
`cDNAs are synthesized using reverse transcriptase, dNTPs and azide-modified dUTP
`(N3-dUTP). After degrading the mRNAs, the azide labeled cDNA from the two
`samples are coupled with alkyne modified Cy3 and Cy5 respectively. Subsequently,
`the two fluorescently labeled cDNA samples are mixed and hybridized on the
`microarray. (page 166)
`
`Figure 5.6. Synthesis of N3-dUTP. (page 168)
`
`Figure 5.7. Synthesis of alkyne functionalized Cy3 and Cy5. (page 168)
`
`Figure 5.8. Primer extension using N3-dUTP along with reverse transcriptase and the
`subsequent fluorescent labeling with click chemistry. Taking RNA as the template,
`reverse tra