A process for the fabrication of DNA microarrays by copying a next-generation sequencing chip is presented in this thesis. DNA molecules of a DNA library are mixed with a biochemical reaction mix and loaded into hundreds of thousands wells of the commercial sequencing chip PicoTiterPlate (PTP, Roche). The DNA randomly distributes onto the wells so that statistically one well contains one single DNA molecule. All wells of the PTP are closed with a microscope slide and the DNA is amplified by polymerase chain reaction (PCR). Each single PCR product is attached as a DNA cluster in the PTP and on the corresponding position on the slide since both surfaces have been coated with PCR primers. The sequence of each DNA cluster can be obtained by a subsequent sequencing reaction in the PTP. This also reveals the sequences of each DNA cluster on the slide thus allowing the slide to be used as a standard DNA microarray.
Table of contents
1 Introduction
1.1 Background
1.2 Aim of thesis
1.3 Structure of thesis
1.4 Short overview over the chapters
2 Theoretical background
2.1 Immobilization of DNA
2.1.1 Functionalization of substrates
2.1.2 Immobilisation of DNA to substrates
2.1.3 Detection of immobilized DNA
2.2 Fabrication of DNA microarrays
2.3 PCR
2.4 Small-volume PCR
2.5 Solid-phase PCR
2.6 Single-molecule PCR
2.6.1 Digital PCR
2.6.2 Digital solid-phase PCR
2.7 Sequencing technologies
2.8 Strategies against DNA contamination
2.8.1 Chemical decontamination
2.8.2 Enzymatic decontamination
3 Materials
3.1 Consumables
3.2 Instrumentation
3.3 Chemicals and biochemicals
3.4 Oligonucleotides
3.4.1 Synthesized oligonucleotides
3.4.2 Template DNA derived from PCR
4 Methods
4.1 Immobilization of DNA
4.1.1 Coupling of PCR primers to substrates
4.1.2 Coupling of PCR primers into a sequencing chip
4.1.3 Coupling of template DNA to beads
4.2 Manufacturing of DNA microarrays
4.3 Contact replication of DNA microarrays
4.4 Biochemical reactions
4.4.1 Liquid-phase PCR
4.4.2 Solid-phase PCR on a DNA microarray
4.4.3 Solid-phase PCR in a sequencing chip
4.5 Fluorescent staining of surface-bound DNA
4.5.1 Staining of DNA microarrays and target arrays
4.5.2 Staining of a sequencing chip
4.6 Post processing of soluble PCR products
4.7 Fluorescence detection and image analysis
5 DNA Microarrays
5.1 Solid-phase PCR on DNA microarrays
5.1.1 Introduction
5.1.2 Experiments
5.2 Contact replication of DNA microarrays
5.2.1 Introduction
5.2.2 Experiments
5.3 Conclusions and outlook
6 Solid-phase PCR in a sequencing chip
6.1 Solid-phase PCR with DNA molecules
6.1.1 Introduction
6.1.2 Experiments
6.1.3 Conclusions and outlook
6.2 Solid-phase PCR with DNA beads
6.2.1 Introduction
6.2.2 Experiments
6.2.3 Conclusions and outlook
6.3 Digital Solid-phase PCR
6.3.1 Introduction
6.3.2 Experiments
6.3.3 Conclusions and outlook
7 Strategies against DNA contaminations
7.1 Chemical decontamination of surfaces
7.1.1 Introduction
7.1.2 Experiments
7.2 Enzymatic decontamination of PCR reaction mixes
7.2.1 Introduction
7.2.2 Experiments
7.3 Conclusions and outlook
8 Overall conclusions and outlook
8.1 Summary and conclusions
8.2 Outlook
8.2.1 Overall vision based on the technological achievements
8.2.2 Transfer of technological achievements into other fields
Objectives & Topics
This thesis aims to develop a novel process for the fabrication of DNA microarrays by directly copying a next-generation sequencing chip. The research focuses on the integration of solid-phase PCR within a sequencing chip to immobilize amplified DNA clusters, which then serve as a master array for further replication into DNA microarrays.
- Development of immobilization protocols for PCR primers on various substrate materials.
- Implementation of massively-parallel solid-phase PCR in commercial sequencing chips.
- Investigation of digital solid-phase PCR for the amplification of single DNA molecules.
- Establishment of contamination control strategies using chemical and enzymatic methods.
- Development of a contact replication process for generating DNA microarray copies.
Excerpt from the book
Digital solid-phase PCR
Several Digital PCR platforms are developed and partially commercialized offering new possibilities in research and diagnostics. Nevertheless downstream applications are not possible since amplification products are discarded after reaction. By combining Digital PCR with SP-PCR, single-molecule derived PCR products get coupled to a solid surface for downstream applications like sequencing. As illustrated in Figure 2-7, three different approaches for Digital SP-PCR exist: (A) the generation of PCR colonies in a gel matrix: “colonies-in-gel”, (B) bridge PCR: “colonies-on-surfaces”, and (C) emulsion PCR: “colonies-on-beads” (emPCR).
The following enumeration describes the different approaches, which are visualized in Figure 2-7, with respect to sequencing applications.
Colonies-in-gel. In the patent from Chetverin et al., colonies of immobilized PCR products are generated in different gel matrices. DNA molecules and PCR reagents are mixed with the components of a gel, and applied in a thin film to a substrate. After polymerization, the randomly distributed DNA molecules and PCR reagents are constrained within the three-dimensional network for subsequent PCR thermocycling. The spatial extend of the growing DNA colonies is limited by diffusion. Mitra and Church described a similar system, which covalently immobilize DNA polonies (PCR colonies = polonies) into a gel. A polyacrylamide gel is spiked with one PCR primer and co-polymerized via its 5’-end acrydite label into the gel. A film of this gel is applied to a silanized glass slide and polymerized thereto. Size of the polonies is dependent on the length of the template DNA and on the pore size of the gel. Several applications are examined, such as copying polonies from one slide to another, genotyping and haplotyping, and sequencing.
Summary of the Chapters
1 Introduction: Provides the background on genomics and DNA microarrays, defines the aim of the thesis, and outlines the structure of the work.
2 Theoretical background: Covers the fundamentals of DNA immobilization, DNA microarray fabrication, PCR, and sequencing technologies.
3 Materials: Lists all consumables, instrumentation, chemicals, and oligonucleotides used for the experimental parts.
4 Methods: Details the protocols for DNA immobilization, PCR setup, staining, and data analysis.
5 DNA Microarrays: Describes experiments regarding solid-phase PCR on microarrays and the process of contact replication for microarray manufacturing.
6 Solid-phase PCR in a sequencing chip: Focuses on the configuration of solid-phase PCR within a sequencing chip using DNA molecules, beads, and digital PCR.
7 Strategies against DNA contaminations: Examines chemical and enzymatic approaches to mitigate DNA contamination in sensitive PCR systems.
8 Overall conclusions and outlook: Summarizes the major technological achievements and provides an outlook on future potential applications.
Keywords
DNA Microarrays, Solid-phase PCR, Digital PCR, Sequencing chip, PicoTiterPlate, Immobilization, Contact replication, DNA contamination, Decontamination, Next-generation sequencing, Microfluidics, Polymerase chain reaction, Surface chemistry, DNA replication, Molecular cloning.
Frequently Asked Questions
What is the core objective of this doctoral thesis?
The primary goal is to establish a novel process chain for the fabrication of DNA microarrays by directly copying a commercial next-generation sequencing chip (PicoTiterPlate, Roche).
What are the main thematic pillars of the research?
The research focuses on three major areas: optimizing solid-phase PCR for sequencing chips, developing surface chemistry for reliable DNA immobilization, and creating a surface transfer process for contact replication of DNA microarrays.
Which central research question is addressed?
The work investigates whether it is possible to perform massively-parallel solid-phase PCR within the small-volume wells of a sequencing chip and subsequently use these immobilized DNA products to fabricate molecularly identical microarray copies.
What scientific methods were primarily employed?
The methodology includes surface functionalization via organosilanes, chemical immobilization using PDITC, various PCR configurations (liquid-phase, solid-phase, and digital PCR), fluorescent labeling/staining, and quantitative data analysis via real-time PCR and fluorescence scanning.
What is covered in the main experimental section?
The main part details the successful immobilization of PCR primers on different polymers (COP, COC, PP, PDMS) and glass, the execution of solid-phase PCR within picoliter-scale wells of a sequencing chip, and the verification of DNA transfer from a master array to a target array via PDMS polymerization.
Which specific terms characterize this work?
Key terms include solid-phase PCR, digital PCR, DNA microarrays, PTP (PicoTiterPlate), surface modification, and DNA replication via contact transfer.
Why are decontamination strategies particularly relevant for this research?
Since the project involves digital PCR (dPCR), which operates at the single-molecule level, even minor carry-over DNA contamination from previous experiments can lead to false-positive results, necessitating robust chemical and enzymatic decontamination protocols.
What role does the sequencing chip play as a master array?
The sequencing chip, after successfully performing digital solid-phase PCR within its wells, acts as a master array, allowing the contained DNA information to be replicated onto target substrates using the developed contact replication method.
- Arbeit zitieren
- Dipl.- Ing. Jochen Hoffmann (Autor:in), 2013, Fabrication of DNA Microarrays by Digital Solid-Phase PCR in a Next Generation Sequencing Chip, München, GRIN Verlag, https://www.grin.com/document/300928
