Four-Color DNA Sequencing by Synthesis on a Chip Using
`Cleavable Fluorescent Nucleotide Reversible Terminators
`
`Dae Hyun Kim
`
`Submitted in partial fulfillment of the
`requirements for the degree
`of Doctor of Philosophy
`in the Graduate School of Arts and Sciences
`
`COLUMBIA UNIVERSITY
`
`2008
`
`Page a
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`Illumina Ex. 1088
`IPR Petition - USP 10,435,742
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`

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`UMI Number: 3299275
`
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`©2007
`
`Dae Hyun Kim
`All Rights Reserved
`
`Page c
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`

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`ABSTRACT
`
`Four-Color DNA Sequencing by Synthesis on a Chip Using
`
`Cleavable Fluorescent Nucleotide Reversible Terminators
`
`Dae Hyun Kim
`
`DNA sequencing by synthesis (SBS) on a solid surface during the polymerase reaction
`
`offers a new paradigm for the deciphering of DNA sequences. This thesis focuses on the
`
`construction of such a novel DNA sequencing system using molecular engineering
`
`approaches. In this approach, four nucleotides (A, C, G, T) are modified as reversible
`
`terminators by attaching a cleavable fluorophore to the base and capping the 3'-OH with
`
`a small chemically reversible moiety so that they are still recognized by DNA polymerase
`
`as substrates. First, we used a 2-nitrobenzyl based photocleavable (PC) linker to attach a
`
`fluorophore to 3'-O-allyl-modified nucleotides, forming photocleavable fluorescent
`
`nucleotide reversible terminators, 3'-0-allyl-dNTPs-PC-fluorophore, for application in
`
`SBS. The fluorophore and the 3'-O-allyl group on a DNA extension product, which is
`
`generated by incorporating the 3'-0-allyl-dNTPs-PC-fluorophore
`
`in a polymerase
`
`reaction, are removed by photocleavage (irradiation at 355 nm) and Pd-catalyzed
`
`deallylation, respectively. This allows the re-initiation of the polymerase reaction and
`
`continuation of SBS. We then found that an allyl moiety can be used successfully as a
`
`linker to tether a fluorophore to 3'-0-allyl-modified nucleotides, forming chemically
`
`cleavable
`
`fluorescent nucleotide
`
`reversible
`
`terminators,
`
`3'-0-allyl-dNTPs-allyl-
`
`fluorophore, for application in SBS. The fluorophore and the 3'-0-allyl group on a DNA
`
`Page d
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`

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`extension product were now able to be removed simultaneously in 30 seconds by the Pd-
`
`catalyzed deallylation reaction in an aqueous buffer solution. This one-step dual-
`
`deallylation reaction thus allowed the re-initiation of the polymerase reaction and
`
`increased the SBS efficiency. We also developed an alternative sequencing method that
`
`is a hybrid between the Sanger dideoxy chain terminating reaction and sequencing by
`
`synthesis (SBS) and delineate the advantages that come with this hybrid sequencing
`
`strategy. In this approach, four nucleotides, modified as reversible terminators (3'-0-PC-
`
`dNTPs) by capping the 3'-OH with a PC reversible 2-nitrobenzyl moiety so that they are
`
`still recognized by DNA polymerase as substrates, are used in combination with four PC
`
`fluorophore labeled dideoxynucleotides (ddNTPs-PC-fluorophore) to generate Sanger
`
`sequencing fragments during SBS. The DNA sequence was determined by the unique
`
`fluorescence emission of each fluorophore on the ddNTPs. Upon removing the 3'-OH
`
`blocking group on the dNTPs and the fluorophore from the ddNTPs, the polymerase
`
`reaction can reinitiate and the DNA sequences can be continuously determined. Four-
`
`color DNA sequencing was performed using these novel fluorescent nucleotide analogues
`
`and a four-color fluorescent scanner to identify sequences of DNA template immobilized
`
`on a chip. The DNA chip was constructed by covalently attaching alkyne-modified self-
`
`priming DNA template onto an azido-PEG functionalized glass slide by using 1,3-dipolar
`
`cycloaddition chemistry. DNA sequences were obtained for various DNA templates,
`
`including DNA templates containing homopolymeric regions in their sequence.
`
`Page e
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`Table of Contents
`
`List of Figures
`
`Acknowledgement
`
`Abbreviations and Symbols
`
`Chapter 1: Introduction to DNA Sequencing and Analysis Technologies
`
`1.1. Introduction
`
`1.2. Background
`1.2.1. Sanger dideoxynucleotide sequencing
`1.2.2. DNA sequencing by MALDI-TOF MS
`1.2.3. DNA pyrosequencing
`1.2.4. DNA sequencing by ligation
`1.2.5. Nanopore-based single molecule sequencing
`
`1.3. Conclusion
`
`1.4. References
`
`Chapter 2: A Focus on DNA Sequencing By Synthesis
`
`2.1. Introduction
`
`2.2. Experimental Rationale and Overview
`
`2.3. Results and Discussion
`
`2.4. Materials and Methods
`
`2.5. Conclusion
`
`2.6. References
`
`l
`
`iv
`
`xv
`
`xvii
`
`1
`
`2
`8
`11
`14
`17
`19
`
`22
`
`24
`
`28
`
`30
`
`36
`
`44
`
`49
`
`51
`
`

`

`Chapter 3: Design, Synthesis and Analysis of 3'-0-Modified Fluorescent Nucleotide
`Reversible Terminators for DNA Sequencing By Synthesis
`
`3.1. Introduction
`
`3.2. Experimental Rationale and Overview
`
`3.3. Results and Discussion
`
`3.4. Materials and Methods
`
`3.5. Conclusion
`
`3.6. References
`
`Chapter 4: Optimization of Solid Surface Functionalization for DNA
`
`Immobilization and Four-Color DNA Sequencing By Synthesis
`
`4.1. Introduction
`
`4.2. Experimental Rationale and Overview
`
`4.3. Results and Discussion
`
`4.4. Materials and Methods
`
`4.5. Conclusion
`
`4.6. References
`
`53
`
`55
`
`56
`
`88
`
`97
`
`99
`
`101
`
`103
`
`104
`
`119
`
`127
`
`128
`
`Chapter 5: Four-Color DNA Sequencing By Synthesis Using Cleavable Fluorescent
`Nucleotide Reversible Terminators
`
`5.1. Introduction
`
`5.2. Experimental Rationale and Overview
`
`5.3. Results and Discussion
`
`5.4. Materials and Methods
`
`5.5. Conclusion
`
`5.6. References
`
`ii
`
`130
`
`132
`
`133
`
`148
`
`154
`
`156
`
`

`

`Chapter 6: Four-Color DNA Sequencing with 3'-0-Modified Nucleotide Reversible
`Terminators and Photo cleavable Fluorophore-Modified
`Dideoxynucleotides
`
`6.1. Introduction
`
`6.2. Experimental Rationale and Overview
`
`6.3. Results and Discussion
`
`6.4. Materials and Methods
`
`6.5. Conclusion
`
`6.6. References
`
`Chapter 7: Summary and Future Outlook
`
`7.1. Photocleavable fluorescent nucleotide analogues to confirm
`feasibility of four-color DNA sequencing by synthesis
`
`7.2. Novel cleavable nucleotide reversible terminators for
`DNA sequencing by synthesis
`
`7.3. Four-color DNA sequencing using cleavable fluorescent
`nucleotide reversible terminators on a DNA chip
`
`7.4. Four-color DNA sequencing with a hybrid SBS approach
`
`158
`
`161
`
`163
`
`174
`
`181
`
`184
`
`186
`
`187
`
`188
`
`188
`
`7.5. Future outlook for four-color DNA sequencing by synthesis
`using cleavable fluorescent nucleotide reversible terminators ... 189
`
`7.6. References
`
`192
`
`iii
`
`

`

`List of Figures
`
`Chapter 1: Introduction to DNA Sequencing and Analysis Technologies
`
`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 3)
`
`Figure 1.2. (A) 3-D computer rendered model of the DNA double helix. The two
`strands coil around each other to create the double helix. (B) A cartoon depicting
`two DNA strands (green and red) held together by hydrogen bonds between the
`paired bases. Notice that the direction of the DNA strands are anti-parallel, one
`going from 5' to 3' (red), the other from 3' to 5' (green). (C) A zoomed in section
`of the double helix, which shows that the specific chemical structures of the bases
`only allow efficient hydrogen bonding between A and T and between G and C.
`(page 4)
`
`Figure 1.3. DNA synthesis. Addition of a nucleotide to the 3'-OH end of a DNA
`strand is the fundamental reaction by which DNA is synthesized. The base-pairing
`between the incoming nucleotide and the DNA template strand guides the formation
`of a new DNA strand that is complementary to the template strand. DNA
`polymerase catalyzes the addition of nucleotides to the growing DNA strand by
`incorporating
`the
`incoming nucleotide at
`the 3'-OH end by forming a
`phosphodiester bond and releasing a pyrophosphate, (page 6)
`
`Figure 1.4. Comparison of the chemical structure of 2'-deoxyribonucleotides
`(dNTPs) and 2',3'-dideoxyribonucleotides (ddNTPs). The ddNTPs lack the 3'-OH
`group, which is necessary for DNA synthesis. Therefore, once the ddNTPs are
`incorporated, they terminate further synthesis of DNA. (page 8)
`
`Figure 1.5. Sanger dideoxy sequencing strategy. DNA fragments are produced
`when both dNTPs and ddNTPs are included in the polymerase mixture. Once a
`ddNTP is incorporated, that DNA chain now lacks the 3'-OH group and therefore
`the addition of the next nucleotide is blocked and the DNA synthesis terminates.
`This reaction mixture will eventually produce a set of DNA strands of different
`length complementary to the template DNA that is being sequenced. To determine
`the complete sequence, the DNA fragments are separated by electrophoresis, and
`the separated bands of DNA are then detected by their fluorescence as they emerge
`from the gel. (page 9)
`
`Figure 1.6. Matrix-assisted
`time-of-flight mass
`laser desorption/ionization
`spectrometry (MALDI-TOF MS). Analyte molecules (such as DNA sequencing
`fragments) and matrix molecules (typically ultraviolet (UV) or infrared (IR) light-
`
`IV
`
`

`

`absorbing small organic molecules) are mixed in solution and then co-crystallized
`on a flat sample plate, which is subsequently loaded into the vacuum chamber of the
`mass spectrometer. DNA molecules are gently desorbed and ionized along with the
`matrix molecules by UV laser irradiation and the resulting charged ions are
`accelerated under a constant electric voltage, which causes them to fly towards the
`ion detector. The charged molecules arrive at the detector at different times on the
`basis of their masses. The masses of the charged ions are determined from their
`time of flight to the detector, which is proportional to their mass per charge ratio
`(m/z). (page 12)
`
`Figure 1.7. DNA sequencing using MALDI-TOF MS. (A) Sanger sequencing
`fragments generated using biotin-labeled ddNTPs and their corresponding mass in
`Daltons. (B) Mass-sequencing spectrum using Biotin-labeled ddNTPs. The inset
`shows a magnification of the lower intensity region. The mass difference (indicated
`above each base) between a peak and the previous one is used to determine the
`identity of the nucleotide. The sequence when read from left to right corresponds
`to the 5' to 3' direction, (page 13)
`
`Figure 1.8. The general principle of the pyrosequencing method. A polymerase
`catalyzes incorporation of nucleotide(s) into a growing DNA strand. As a result of
`the incorporation, PPi molecules are released and subsequently converted to ATP,
`by ATP sulfurylase. Light is produced in the luciferase reaction during which a
`luciferin molecule is oxidized. A photon detector then registers the luminescence,
`(page 16)
`
`Figure 1.9. A scheme outlining sequencing by ligation method using degenerate
`nonamers. (page 18)
`
`Figure 1.10. An a-hemolysin protein self-assembles in a lipid bilayer to form an
`ion channel and a stretch of nucleic acids passes through it (A), generating a
`corresponding electronic signatures (B). (page 20)
`
`Figure 1.11. Hypothesized plot of translocation time versus blockade current from
`DNA molecules (i), (ii), and (iii). The magnitude and duration of the blockade
`signatures between different nucleotides is easily distinguishable, (page 21)
`
`Chapter 2: A Focus on DNA Sequencing by Synthesis
`
`Figure 2.1. DNA sequencing by synthesis based on modified nucleotides. Step 1:
`Addition of four nucleotide analogues, each of which has a unique label and can act
`as a reversible terminator in DNA polymerase reaction. Step 2: Detection of the
`incorporated nucleotide analogue by its unique label. Step 3: Removal of the
`label/protecting group and reinitiation of the DNA polymerase reaction, (page 29)
`
`v
`
`

`

`Figure 2.2. Possible fluorescence labeling sites on a nucleotide: (A) The
`fluorophore is attached on the gamma phosphate group. (B) The fluorophore is
`attached through a cleavable linker on the 3'-OH group. (C) The fluorophore is
`attached through a cleavable linker on the base, while the 3'-OH group is capped
`with a reversible chemical moiety, (page 31)
`
`Figure 2.3. The 3D structure of the ternary complexes of a rat DNA polymerase, a
`DNA template-primer, and dideoxycytidine triphosphate (ddCTP). The left side of
`the illustration shows the mechanism for the addition of ddCTP and the right side of
`the illustration shows the active site of the polymerase. Note 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 33)
`
`Figure 2.4. SBS approach using cleavable fluorescent nucleotide analogues. In
`this approach, a chip is constructed with immobilized DNA templates that are able
`to self-prime for initiating the polymerase reaction. Four nucleotide analogues are
`designed such that each is labeled with a unique fluorescent dye on a specific
`location of the base, and a small chemical group (R) to cap the 3'-OH group, (page
`34)
`
`Figure 2.5. Structures of four nucleotides labeled through a photocleavable linker
`(PC) using four fluorophores with distinct fluorescent emissions, dGTP-PC-Bodipy-
`FL-510 (Xabs (max) = 502 nm; Xem (max) = 510 nm), dUTP-PC-R6G (X^ (max) = 525
`nm; Xem (max) = 550 nm), dATP-PC-ROX (A,abs (max) = 585 nm; ^em (max) = 602 nm),
`and dCTP-PC-Bodipy-650 (^abs(max) = 630 nm; Xem(max) = 650 nm). (page 37)
`
`Figure 2.6. The polymerase extension scheme (left) and MALDI-TOF MS spectra
`of the four consecutive extension products and their photocleavage products (right).
`Primer extended with dUTP-PC-R6G (1) and its photocleavage product 2; product 2
`extended with dGTP-PC-Bodipy-FL-510 (3), and it photocleavage product 4;
`product 4 extended with dATP-PC-ROX (5), and its photocleavage product 6;
`product 6 extended with dCTP-PC-Bodipy-650 (7), and it photocleavage product 8.
`After 10 seconds of irradiation with a laser at 355 nm, photocleavage is complete
`with all of the fluorophores cleaved from the extended DNA products, (page 38)
`
`Figure 2.7.
`Immobilization of an azido-labeled PCR product on an alkynyl-
`functionalized surface, and a ligation reaction between the immobilized single-
`stranded DNA template and a loop primer to form a self-priming DNA moiety on
`the chip. The sequence of the loop primer is shown in (A), (page 41)
`
`Figure 2.8. Schematic representation of SBS on a chip using four PC fluorescent
`nucleotides (Upper panel) and the scanned fluorescence images for each step of
`SBS on a chip (Lower panel). (1) Incorporation of dATP-PC-ROX
`(2)
`Photocleavage of PC-ROX (3) Incorporation of dGTP-PC-Bodipy-FL-510 (4)
`Photocleavage of PC-Bodipy-FL-510 (5) Incorporation of dATP-PC-ROX (6)
`Photocleavage of PC-ROX (7) Incorporation of dCTP-PC-Bodipy-650
`(8)
`
`VI
`
`

`

`Photocleavage of PC-Bodipy-650 (9) Incorporation of dUTP-PC-R6G (10)
`Photocleavage of PC-R6G
`(11)
`Incorporation of dATP-PC-ROX
`(12)
`Photocleavage of PC-ROX
`(13)
`Incorporation of dUTP-PC-R6G
`(14)
`Photocleavage of PC-R6G
`(15)
`Incorporation of dATP-PC-ROX
`(16)
`Photocleavage of PC-ROX (17) Incorporation of dGTP-PC-Bodipy-FL-510 (18)
`Photocleavage of PC-Bodipy-FL-510 (19) Incorporation of dUTP-PC-R6G (20)
`Photocleavage of PC-R6G (21) Incorporation of dCTP-PC-Bodipy-650 (22)
`Photocleavage of PC-Bodipy-650 (23) Incorporation of dATP-PC-ROX (24)
`Photocleavage of PC-ROX. (page 43)
`
`Chapter 3: Design, Synthesis and Analysis of 3'-0-Modified Fluorescent Nucleotide
`Reversible Terminators for DNA Sequencing By Synthesis
`
`Figure 3.1. General design scheme of 3'-0-allyl photocleavable fluorescent NRTs.
`In this design, the fluorescent dye is attached to the base portion of the nucleotide
`through a photocleavable linker, while the 3'-OH is capped with a chemically
`cleavable allyl group, (page 57)
`
`Figure 3.2. A detailed scheme of a DNA polymerase extension reaction using 3'-
`O-allyl-dGTP-PC-Bodipy-FL-510 as a reversible terminator, (page 58)
`
`Figure 3.3. Continuous polymerase DNA extension scheme using 3'-0-allyl-
`dGTP-PC-Bodipy-FL-510
`(left) and MALDI-TOF MS spectra of the resulting
`DNA products (right), (page 60)
`
`Figure 3.4. Cleavage mechanisms. (A) Photolysis mechanism (Norrish-type II
`reaction) of 2-nitrobenzyl group in the photocleavable linker.
`(B) Deallylation
`mechanism for cleaving allyl groups from allyl ethers in aqueous palladium-
`phosphine solution, (page 62)
`
`Figure 3.5. Structures of 3'-0-allyl-dNTPs-PC-fluorophore, with
`the 4
`fluorophores having distinct fluorescent emissions: 3'-0-allyl-dGTP-PC-Bodipy-
`FL-510 (A-abs(max) = 502 nm; A.em(max) = 510 nm), 3'-0-allyl-dUTP-PC-R6G (A,abS(max)
`= 525 nm; A,em(max) = 550 nm), 3'-0-allyl-dATP-PC-ROX (A,abs(max) = 585 nm; Xem
`(max) = 602 nm), and 3'-O-allyl-dCTP-PC-Bodipy-650 (A,abs(max) = 630 nm; ^em(max)
`= 650 nm). (page 64)
`
`Figure 3.6. A detailed scheme (left) of a polymerase reaction using all four
`photocleavable fluorescent NRTs to extend base specifically with an "A", and the
`subsequent photocleavage and deallylation reaction to cleave off the fluorophore
`and regenerate the 3'-OH, respectively. MALDI-TOF MS spectra (right) verifying
`base specific incorporation of 3'-0-allyl-dATP-PC-ROX (7) (peak at m/z 7228)
`among pool of all four photocleavable fluorescent NRTs, and the corresponding
`photocleavage (8) (m/z 6490) and deallylation (9) (m/z 6450) products, (page 65)
`
`vn
`
`

`

`Figure 3.7. A detailed scheme (left) of a polymerase reaction using all four
`photocleavable fluorescent NRTs to extend base specifically with a "C", and the
`subsequent photocleavage and deallylation reaction to cleave off the fluorophore
`and regenerate the 3'-OH, respectively. MALDI-TOF MS spectra (right) verifying
`base specific incorporation of 3'-O-allyl-dCTP-PC-Bodipy-650 (10) (peak at m/z
`8525) among pool of all four photocleavable fluorescent NRTs, and the
`corresponding photocleavage (11) (m/z 7758) and deallylation (12) (m/z 7718)
`products, (page 67)
`
`Figure 3.8. A detailed scheme (left) of a polymerase reaction using all four
`photocleavable fluorescent NRTs to extend base specifically with a "G", and the
`subsequent photocleavage and deallylation reaction to cleave off the fluorophore
`and regenerate the 3'-OH, respectively. MALDI-TOF MS spectra (right) verifying
`base specific incorporation of 3'-O-allyl-dGTP-PC-Bodipy-FL-510 (13) (peak at
`m/z 7052) among pool of all four photocleavable fluorescent NRTs, and the
`corresponding photocleavage (14) {m/z 6556) and deallylation (15) (m/z 6516)
`products, (page 68)
`
`Figure 3.9. A detailed scheme (left) of a polymerase reaction using all four
`photocleavable fluorescent NRTs to extend base specifically with a "T", and the
`subsequent photocleavage and deallylation reaction to cleave off the fluorophore
`and regenerate the 3'-OH, respectively. MALDI-TOF MS spectra (right) verifying
`base specific incorporation of 3'-0-allyl-dUTP-PC-R6G (16) (peak at m/z 6210)
`among pool of all four photocleavable fluorescent NRTs, and the corresponding
`photocleavage (17) (m/z 5548) and deallylation (18) (m/z 5508) products, (page 68)
`
`Figure 3.10. Synthesis of a fluorophore-labeled DNA with a novel allyl based
`linker and a model deallylation reaction experiment to cleave the fluorophore from
`the DNA. (page 70)
`
`Figure 3.11. Synthesis of a 5'-Bodipy-FL-510-allyl-22 mer DNA 24. (page 71)
`
`Figure 3.12. A synthesis scheme of fluorophore-labeled DNA via a novel allyl
`based linker and the subsequent deallylation reaction experiment to cleave the
`fluorophore from the DNA (left) and MALDI-TOF MS spectra (right) of 5'-
`Bodipy-FL-510-allyl-22 mer DNA 24 and deallylation product 23. (page 72)
`
`Figure 3.13. General design scheme of 3'-0-allyl chemically cleavable fluorescent
`NRTs. In this design, the fluorescent dye is attached to the base portion of the
`nucleotide through a chemically cleavable allyl-based linker, and the 3'-OH is also
`capped with a chemically cleavable allyl group, (page 74)
`
`Figure 3.14. A detailed scheme of polymerase reaction using 3'-0-allyl-dGTP-
`allyl-Bodipy-FL-510 as a reversible terminator, (page 75)
`
`Vlll
`
`

`

`Figure 3.15. Continuous polymerase extension scheme using 3'-0-allyl-dGTP-
`allyl-Bodipy-FL-510 (left) and MALDI-TOF MS spectra of the resulting DNA
`products (right), (page 76)
`
`Figure 3.16. Cleavage mechanism of an allyl linker: Deallylation of allyl groups
`from allyl carbamates in aqueous palladium-phosphine solution, (page 77)
`
`Figure 3.17.
`Structures of 3'-0-allyl-dNTPs-allyl-fluorophore, with the 4
`fluorophores having distinct fluorescent emissions: 3'-0-allyl-dCTP-allyl-Bodipy-
`FL-510 (Xabs(max) = 502 iim; tam(max) = 510 nm), 3'-0-allyl-dUTP-allyl-R6G (Xabs
`(max) = 525 nm; X,em(max) = 550 nm), 3'-0-allyl-dATP-allyl-ROX (tabs (max) = 585 nm;
`tam(max) = 602 nm), and 3'-O-allyl-dGTP-allyl-Bodipy-650 (tabs (max) = 630 nm; Xem
`(max) = 650 nm). (page 79)
`
`Figure 3.18. A detailed scheme (left) of a polymerase reaction using all four
`chemically cleavable fluorescent NRTs to extend base specifically with an "A", and
`the subsequent deallylation reaction to cleave off the fluorophore and regenerate the
`3'-OH, simultaneously. MALDI-TOF MS spectra (right) verifying base specific
`incorporation of 3'-0-allyl-dATP-allyl-ROX (29) (peak at m/z 7136) among pool of
`all four chemically cleavable fluorescent NRTs, and the corresponding deallylation
`(30) (m/z 6452) product, (page 81)
`
`Figure 3.19. A detailed scheme (left) of a polymerase reaction using all four
`chemically cleavable fluorescent NRTs to extend base specifically with a "C", and
`the subsequent deallylation reaction to cleave off the fluorophore and regenerate the
`3'-OH, simultaneously. MALDI-TOF MS spectra (right) verifying base specific
`incorporation of 3'-O-allyl-dCTP-allyl-Bodipy-FL-510 (31) (peak at m/z 5930)
`among pool of all four chemically cleavable fluorescent NRTs, and
`the
`corresponding deallylation (32) (m/z 5489) product, (page 81)
`
`Figure 3.20. A detailed scheme (left) of a polymerase reaction using all four
`chemically cleavable fluorescent NRTs to extend base specifically with a "G", and
`the subsequent deallylation reaction to cleave off the fluorophore and regenerate the
`3'-OH, simultaneously. MALDI-TOF MS spectra (right) verifying base specific
`incorporation of 3'-O-allyl-dGTP-allyl-Bodipy-650 (33) (peak at m/z 7224) among
`pool of all four chemically cleavable fluorescent NRTs, and the corresponding
`deallylation (34) (m/z 6512) product, (page 82)
`
`Figure 3.21. A detailed scheme (left) of a polymerase reaction using all four
`chemically cleavable fluorescent NRTs to extend base specifically with a "T", and
`the subsequent deallylation reaction to cleave off the fluorophore and regenerate the
`3'-OH, simultaneously. MALDI-TOF MS spectra (right) verifying base specific
`incorporation of 3'-0-allyl-dUTP-allyl-R6G (35) (peak at m/z 6116) among pool of
`all four chemically cleavable fluorescent NRTs, and the corresponding deallylation
`(36) (m/z 5509) product, (page 82)
`
`IX
`
`

`

`Figure 3.22. A general design scheme for 3'-0-(2-nitrobenzyl) photocleavable
`fluorescent NRTs. In this design, the fluorescent dye is attached to the base portion
`of the nucleotide through a photocleavable linker, and the 3'-OH is also capped
`with a photocleavable 2-nitrobenzyl group, (page 84)
`
`Figure 3.23. A detailed scheme of polymerase extension reaction using 3'-0-PC-
`dCTP-PC-Bodipy-FL-510 as a reversible terminator, (page 86)
`
`Figure 3.24. Continuous polymerase extension scheme using 3'-0-PC-dCTP-PC-
`Bodipy-FL-510 (left) and MALDI-TOF MS spectra of the resulting DNA products
`(right), (page 87)
`
`Optimization of Solid Surface Functionalization
`Chapter 4:
`Immobilization and Four-Color DNA Sequencing By Synthesis
`
`for DNA
`
`Figure 4.1. Immobilization of an azido-labeled PCR product on an alkynyl -
`functionalized surface, and a ligation reaction between the immobilized single-
`stranded DNA template and a loop primer to form a self-priming DNA moiety on
`the chip. The sequence of the loop primer is shown in (A), (page 105)
`
`Figure 4.2. Functionalization of a glass surface with different terminal functional
`groups.
`(A) An alkyne functionalized glass slide: a commercially available
`heterobifunctional crosslinker 6-heptynoic acid is used to alkyne functionalized the
`glass surface.
`(B) An azido functionalized glass slide: heterobifunctional
`crosslinker, 5-azidopentanoic acid is used to azido functionalize the glass surface.
`(C) Comparison of non-specific absorption of different fluorophores on the alkyne
`and azido functionalized glass slides, (page 108)
`
`Figure 4.3.
`Synthesis of an alternative heterobifunctional crosslinker, 5-
`azidopentanoic acid, (page 109)
`
`Figure 4.4. Construction of an azido-PEG functionalized glass slide: PEG derived
`heterobifunctional
`crosslinker,
`0-(2-azidoethyl)-0'-[2-(diglycolyl-amino)-
`ethyljheptaethylene glycol (azido-PEG-linker) is used to functionalize the glass
`surface with azido-PEG groups, (page 110)
`
`Figure 4.5. Comparison of non-specific absorption of different
`fluorophores
`between an azido and azido-PEG functionalized glass slides, (page 111)
`
`Figure 4.6. Synthesis and verification of a 3'-FAM, 5'-alkynyl 18-mer DNA. (A)
`UV-Vis absorption spectrum of FAM-labeled alkynyl 18-mer DNA. (B) MALDI-
`TOF MS spectrum of FAM-labeled alkynyl 18-mer DNA. (page 113)
`
`x
`
`

`

`Figure 4.7. Immobilization of the 3'-FAM, 5'-alkynyl 18-mer DNA on an azido-
`PEG functionalized glass slide.
`(A) Cu(I) catalyzed cycloaddition coupling
`chemistry using the 3'-FAM, 5'-alkynyl 18-mer DNA. (B) Coupling mechanism of
`Cu(I) catalyzed coupling reaction of azides and terminal alkynes. (page 114)
`
`Figure 4.8. Confirmation of site-specific cycloaddition coupling reaction using the
`3'-FAM, 5'-alkynyl 18-mer DNA. (A) Comparison of fluorescent intensity of the
`glass surface spotted with the 3'-FAM, 5'-alkynyl 18-mer DNA and a 3'-FAM, 5'-
`amino 18-mer DNA (negative control), (page 115)
`
`Figure 4.9. Various concentrations of 3'-FAM, 5'-alkynyl 18-mer DNA spotted on
`a glass surface functionalized with azido-PEG groups. Analysis of the fluorescence
`intensities at these various concentrations showed
`that
`the optimal DNA
`concentration for maximal DNA immobilization is estimated to be around 50 uM.
`(page 116)
`
`Figure 4.10. Synthesis scheme of an alkyne-labeled self-priming DNA template,
`(page 118)
`
`Figure 4.11. Construction of a DNA chip with self-priming DNA templates
`immobilized on an azido-PEG functionalized glass slide, (page 119)
`
`Chapter 5: Four-Color DNA Sequencing By Synthesis Using Cleavable Fluorescent
`Nucleotide Reversible Terminators
`
`Figure 5.1. The polymerase extension scheme (left) and MALDI-TOF MS spectra
`of the four consecutive extension products and their deallylated products (right).
`Primer extended with 3'-0-allyl-dUTP-allyl-R6G (1), and its deallylated product 2;
`Product 2 extended with 3'-O-allyl-dGTP-allyl-Bodipy-650 (3), and its deallylated
`product 4; Product 4 extended with 3'-0-allyl-dATP-allyl-ROX (5), and its
`deallylated product 6; Product 6 extended with 3'-0-allyl-dCTP-allyl-Bodipy-FL-
`510 (7), and its deallylated product 8. After 30 seconds of incubation with the
`palladium/TPPTS cocktail at 70°C, deallylation
`is complete with both the
`fluorophores and the 3'-0-allyl groups cleaved from the extended DNA products,
`(page 135)
`
`Figure 5.2. DNA extension reaction performed in solution phase to characterize
`the four different chemically cleavable fluorescent nucleotide analogues (3'-0-
`allyl-dUTP-allyl-R6G, 3'-O-allyl-dGTP-allyl-Bodipy-650, 3'-0-allyl-dATP-allyl-
`ROX and 3'-O-allyl-dCTP-allyl-Bodipy-FL-510). After each extension reaction,
`the DNA extension product is purified by HPLC for MALDI-TOF MS analysis to
`verify that it is the correct extension product. A Pd-catalyzed deallylation reaction
`is performed to produce a DNA product that is used as a primer for the next DNA
`extension reaction, (page 137)
`
`XI
`
`

`

`Figure 5.3. Fluorescence absorption (dark solid line) and emission spectra of 3'-0-
`allyl-dCTP-allyl-Bodipy-FL-510, 3' -0-allyl-dUTP-allyl-R6G, 3 '-O-allyl-dATP-
`allyl-ROX and 3'-0-allyl-dGTP-allyl-Cy5. (page 139)
`
`Figure 5.4. Fluorescence imaging of 3'-O-allyl-dCTP-allyl-Bodipy-FL-510, 3'-0-
`allyl-dUTP-allyl-R6G, 3'-0-allyl-dATP-allyl-ROX and 3'-0-allyl-dGTP-allyl-Cy5
`using a fluorescent laser scanner, (page 140)
`
`Figure 5.5. (A) Reaction scheme of SBS on a chip using four chemically cleavable
`fluorescent NRTs. (B) The scanned 4-color fluorescence images for each step of
`SBS on a chip: (1) incorporation of 3'-0-allyl-dGTP-allyl-Cy5; (2) cleavage of
`allyl-Cy5 and 3'-allyl group; (3) incorporation of 3'-0-allyl-dATP-allyl-ROX; (4)
`cleavage of allyl-ROX and 3'-allyl group; (5) incorporation of 3'-0-allyl-dUTP-
`allyl-R6G; (6) cleavage of allyl-R6G and 3'-allyl group; (7) incorporation of 3'-0-
`allyl-dCTP-allyl-Bodipy-FL-510; (8) cleavage of allyl-Bodipy-FL-510 and 3'-allyl
`group; images (9) to (25) are similarly produced. (C) A plot (4-color sequencing
`data) of raw fluorescence emission intensity at the four designated emission
`wavelengths of the four chemically cleavable fluorescent NRTs. (page 142)
`
`Figure 5.6. Structures of 3'-0-allyl-dATP, 3'-0-allyl-dCTP, 3'-0-allyl-dGTP, and
`3'-0-allyl-dTTP. (page 144)
`
`Figure 5.7. (A) Four-color DNA sequencing raw data with our sequencing by
`synthesis chemistry using a template containing two homopolymeric regions. The
`individual base (A, T, C, G), the 10 repeated A's and the 5 repeated A's are clearly
`identified. The small groups of peaks between the identified bases are fluorescent
`background from the DNA chip, which does not build up as the cycle continues.
`(B) The pyrosequencing data for the same DNA template containing the
`homopolymeric regions (10 T's and 5 T's). The first 4 individual bases are clearly
`identified. The two homopolymeric regions (10 A's) and (5 A's) produce two large
`peaks, from which it is very difficult to identify the exact sequence, (page 145)
`
`Figure 5.8. The scanned 4-color fluorescence images for each step of SBS on a
`chip using 3'-0-allyl-dNTPs-PC-fluorophore:
`(1) incorporation of 3'-0-allyl-
`dGTP-PC-Bodipy-FL-510; (2) cleavage of PC-Bodipy-FL-510 by photolysis and
`3'-allyl group by deallylation; (3) incorporation of 3'-0-allyl-dGTP-PC-Bodipy-
`FL-510; (4) cleavage of PC-Bodipy-FL-510 by photolysis and 3'-allyl group by
`deallylation; (5) incorporation of 3'-0-allyl-dUTP-PC-R6G; (6) cleavage of PC-
`R6G by photolysis and 3'-allyl group by deallylation; (7) incorporation of 3'-0-
`allyl-dUTP-PC-R6G; (8) cleavage of PC-R6G by photolysis and 3'-allyl group by
`deallylation; (9) incorporation of 3'-0-allyl-dATP-PC-ROX; (10) cleavage of PC-
`ROX by photolysis and 3'-allyl group by deallylation; (11) incorporation of 3'-0-
`allyl-dATP-PC-ROX; (12) cleavage of PC-ROX by photolysis and 3'-allyl group
`by deallylation; (13) incorporation of 3'-O-allyl-dCTP-PC-Bodipy-650. (page 147)
`
`xn
`
`

`

`Chapter 6: Four-Color DNA Sequencing With 3'-0-Modified Nucleotide Reversible
`Terminators and Photocleavable Fluorophore-Modified Dideoxynucleotides
`
`Figure 6.1. Reaction scheme of sequencing on a chip using combination of 3'-0-
`modified NRTs
`(3'-0-Ri-dNTPs)
`and
`cleavable
`fluorophore modified
`dideoxynucleotide
`terminators
`(ddNTPs-R2-fluorophore).
`In
`this sequencing
`approach, a chip is constructed with immobilized DNA templates that are able to
`self-prime for initiating the polymerase reaction. The four 3'-0-modified NRTs
`have a small chemically reversible group (Ri) to cap the 3'-OH moiety. Four
`cleavable fluorophore modified dideoxynucleotides are designed such that each is
`attached with a unique fluorophore on the base through a cleavable linker (R2).
`Upon adding the mixture of 3'-0-Ri-dNTPs and ddNTPs-R2-fluorophore with the
`DNA polymerase, only the dideoxynucleotide/3'-0-modified NRTs complementary
`to the next nucleotide