Fabrication of DNA Microarrays by Digital Solid-Phase PCR in a Next Generation Sequencing Chip

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

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.