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


Doctoral Thesis / Dissertation, 2013

124 Pages, Grade: 1,0


Excerpt


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

Glossary

References

1 Introduction

1.1 Background

Higher organisms are made of thousands of trillions of cells, each containing deoxyribonucleic acid (DNA) encoding the basic instructions for cellular function. The genome is defined as the entity of the organism’s DNA and often referred to as its “blueprint”. Genetic information is encoded by the order of the four nucleotides adenine

(A), cytosine (C), guanine (G), and thymine (T) [1]. A human genome comprises 3.2 billion nucleotides and was first decoded in the frame of the Human Genome Project (HUGO). After a duration of 11 years and 3 billion $, the results have been published in 2001 [2]. A human genome is divided into about 23,000 smaller regions called genes [3]. The functioning of an organism is regulated by the expression of genes, where defined sections on the DNA are copied (expressed) into messenger ribonucleic acid (mRNA). These blueprints encode all information for the synthesis of proteins, which are responsible for structure and function of single cells and thus the whole organism.

DNA microarrays are analytical tools for the highly parallel detection of molecules like DNA, mRNA, or proteins. A DNA microarray is a substrate featuring DNA sequences (probes) in a regular pattern on its surface [4-6]. Probes behave like biosensors for the specific and parallel detection of target molecules from a sample solution. At whole-genome DNA microarrays, all probes together target all DNA from an organism. Over the last two decades, microarrays emerged as important analytical tools for the investigation of gene expression [7], the detection of DNA-protein interactions [8], or the identification of single nucleotide polymorphisms [9]. Microarrays evolved from southern blots using cellulose nitrate filters as substrates [10], to dot blots [11], over DNA microarrays [7] to whole-genome DNA microarrays. A commercial example of the latter features 4.2 million DNA probes (Human CGH 4.2M WholeGenome Tiling Arrays, NimbleGen).

DNA sequencing is a process for decoding the sequence of a given DNA molecule [12-16]. Until the arrival of next-generation sequencing systems in 2005 [17], sequencing was done by an electrophoretic method developed by Frederick Sanger [18]. With this method, the first prokaryote was decoded in 1977 [18] and the first eukaryote in 1996 [19]. Over the last two decades, much effort has been put into the investigation and commercialization of new high-throughput sequencing technologies [17,20-23]. Nowadays, these so called next-generation sequencing systems are able to decipher a whole genome in a few days [13,24], opening the door to new applications like de-novo sequencing of unknown genomes [25], RNA sequencing for the identification and quantification of active genes [26], determination of DNA - protein interactions in regulatory networks [27], and in metagenomics for the identification of the microbial composition of a sample [28]. Some authors already distinguish between sequencers of the second generation - sequencing DNA which has been amplified before - and the third generation sequencers decoding one single DNA molecules [29].

1.2 Aim of thesis

This thesis aims at a novel process for the generation of DNA microarrays directly from a next-generation sequencing chip. It starts with the extraction and purification of DNA from a natural organism. The DNA is fragmented, together with a PCR reaction mix introduced into the wells of a sequencing chip (PicoTiterPlate - PTP, GS systems, Roche), and sealed with a planar substrate (target array). By a PCR reaction, amplified DNA fragments get immobilized into the wells and onto corresponding positions on the target array since these surfaces are modified for this purpose. By a subsequent sequencing reaction in the chip, DNA sequences in the chip and on the target array are deciphered. This process also identifies wells that contained initially zero or more than one DNA molecule. The DNA on the target array provides a first DNA microarray wherefrom multiple molecular copies are generated by a biosynthetic process. The whole process chain promises the following:

DNA microarrays derived from natural DNA from any organism.

Low error rates of the sequences due to the biosynthetic process compared to the in-situ synthesis of the state-of-the-art.

Different length of the sequences (e.g. 100 - 1500 nucleotides) by adjusting the degree of fragmentation of the initial DNA.

Additionally, the process of preparing a chip for sequencing is streamlined in terms of reduced time, material, and costs. In case of success, researchers will be able to produce DNA microarrays from an organism of interest on their own at affordable time and costs. The required sequencing reaction, which is necessary for allocating the DNA sequences to each position on the master array, can be conducted by an external provider.

1.3 Structure of thesis

The thesis is composed of two major parts: chapters 1 - 4 presents all necessary information for understanding and performing the experiments described in chapters 5 - 7. The main technological challenges to be solved are:

- Establishment of a protocol to perform massively-parallel PCR within the 18.5 pL wells of a sequencing chip (“Small-volume PCR”).
- Investigation of protocols to immobilize PCR primers to surfaces for binding PCR products thereto (“Solid-phase PCR”).
- Immobilization of PCR products derived from single DNA molecules to surfaces (“Digital solid-phase PCR”).
- Strategies to avoid and reduce DNA contaminations.
- Investigation of a method for the replication of DNA microarrays by means of a surface transfer process (“Contact replication”).

1.4 Short overview over the chapters

Chapter 2 presents the state-of-the-art and theoretical background. Section 2.1 gives an overview on different chemical immobilization methods to attach DNA molecules to surfaces. Section 2.2 presents the state-of-the-art for the fabrication of DNA microarrays. Section 2.3 describes PCR in general; section 2.4 presents measures to successfully perform small-volume PCR. Section 2.5 explains the concept of solidphase PCR, where PCR products are bound to surfaces via an immobilized PCR primer. Section 2.6 introduces digital PCR, where a PCR product is generated based on single DNA molecules which can be additionally immobilized to surfaces by the concept of solid-phase PCR. Section 2.7 deals with different next-generation sequencing strategies and describes the sequencing system (GS, Roche) this thesis is based on in more detail. Section 2.8 summarizes chemical and enzymatic strategies to prevent and fight DNA contaminations, which is especially important for digital PCR.

Chapter 3 lists the used materials like consumables (3.1), instrumentation (3.2), chemicals and biochemical (3.3), and oligonucleotides (3.4).

Chapter 4 describes the methods for performing the experiments from chapters 5 - 7. Section 4.1 presents the final protocol for immobilizing oligonucleotides to different surfaces. Section 4.2 shows the process for the fabrication of DNA microarrays, section

4.3 the replication of such an array. Section 4.4 explains PCR reactions in standard tubes, on DNA microarrays, and in the picowell sequencing chip PTP. Section 4.5 shows a technique for the visualization of surface-bound DNA molecules. Section 4.6 presents general post-PCR methods. Section 4.7 closed with the detection of signals derived from surface-bound DNA.

Chapter 5 presents different methods related to DNA microarrays. Section 5.1 presents a protocol for attaching PCR primers onto various materials in a microarray format and its performance in solid-phase PCR. Section 5.2 presents a protocol for the parallel transfer of DNA sequences from a master- onto a target microarray.

Chapter 6 describes solid-phase PCR reactions in different configurations in the sequencing chip PTP. Section 6.1 investigates the basic reaction setup for amplifying DNA template molecules in a PTP and immobilizing the PCR products onto a target array. Section 6.2 describes an identical reaction but using DNA beads whereto the template DNA is immobilized. Section 6.3 presents a digital solid-phase PCR reaction where single DNA molecules are amplified and immobilized to a target array and here also into the wells of a PTP.

Chapter 7 examines two different strategies to remove existing DNA and prevent DNA contaminations. Section 7.1 presents a chemical strategy for the decontamination of surfaces like work benches and laboratory equipment. Section 7.2 deals with the enzymatic decontamination of carry-over DNA in the PCR reaction mix.

Chapter 8 summarizes the achievements and presents future projects and applications, based on the results of this thesis.

2 Theoretical background

2.1 Immobilization of DNA

Nucleic acids immobilized onto surfaces are used for a variety of applications: In next generation sequencing systems, DNA molecules are immobilized to surfaces and decoded [16,17,20,21,23,30,31]. On DNA microarrays, DNA is immobilized to a substrate in an array format for the identification of complementary target sequences [4-7]. Many different variants of these microarrays exist for the detection of SNPs [9,32,33], solid-phase PCR [34-41], or the identification of aptamers [42-44].

The large variety of immobilization chemistries differs strongly due to their chemical structure, components, and number of modification steps. Common characteristics are covalent and oriented bonds while unspecific interactions between the negatively charged DNA backbone and a substrate is controlled. Surface bound oligonucleotides are desired to withstand reaction conditions like high ionic strength, extreme ph values and temperatures up to 100 °C. To be compatible to various interaction and reaction mechanisms, the chemistry must ensure high reactivity and availability of the oligonucleotide. Fundamental approaches for the immobilization of nucleic acids to surfaces are summarized in Figure 2-1.

illustration not visible in this excerpt

Figure 2-1: Schematics of four different chemical strategies for attaching nucleic acids to substrates. Usually, a substrate (a) is functionalized with an organosilane (blue semi-circles, b). (A) Unmodified nucleic acids can attach to a functionalized substrate via its negatively charged backbone. (B) In direct immobilization methods, nucleic acids bind covalently and oriented to a functionalized substrate. (C, D) In indirect immobilization methods, bifunctional linking molecules (c) connect modified DNA (d) with a functionalized substrate (a). (C) Homobifunctional molecules feature two identical chemical moieties, (D) heterobifunctional two different moieties.

2.1.1 Functionalization of substrates

This section describes methods to activate chemically inert substrates for the subsequent immobilization of nucleic acids. Glass is frequently used as substrate material because of its inertness, homogeneous chemical surface, and low autofluorescence [7,45-50]. Besides, synthetic polymers like COP [51,52], COC [53,54], PP [55], PS [56], PMMA [52,57,58], PC [52,57], or PDMS [59,60] are more and more investigated due to well adjustable surface properties, low cost, availability, and formability. Regardless of the material, each functionalization protocol starts with precleaning of the substrate to remove macromolecular compounds and interfering organic or inorganic molecules. Substrates are now ready for the modification with organic groups via organosilanes as already shown in 1969 by Wheetall [61]. The term “silanization” describes the covalent linkage of organic groups (organosilanes) to hydroxylated organic or inorganic substances via a condensation reaction. Hydroxyl groups are introduced into a surfaces by oxygen plasma [41], UV ozone treatment, piranha acid, or treatment with concentrated sodium hydroxide for activating the surface. Hydroxylated substrates can react with trifunctional organosilanes like the widely used silane 3-aminopropyltriethoxysilane (APTES) as shown in Figure 2-2 B. In general, silanes feature a central silicon atom connected to a functional group (R1) and three reactive organic groups (X). As shown in Figure 2-2 A, (X) can be a hydroxyl-, methoxy-, or ethoxygroup. (R1) can be an aldehyde-, mercapto-, amino-, or epoxy group (from top to down) attached via an alkyl chain in varying length to the silicon atom. In a coupling reaction, the organic group (X) is first hydrolized to silanol (-OH) groups. To trigger hydrolization, an addition of 5 % H2O into the silanization solution can increase the silane density bound to a surface by 400 % compared to an anhydrous solution [62]. Silanol groups are connected to the hydroxyl groups on a substrate via hydrogen bonds. A subsequent curing step at temperatures between

70 °C and 120 °C in a humid atmosphere covalently connects the silane network to the substrate via a condensation reaction. Roughly 20 % of the trifunctional silanes get directly attached to the surface due its branched nature, whereas 80 % are cross-linked with the unbound moieties of other silanes forming a 3-dimensional network as shown in Figure 2-2 C [63].

Figure 2-2: Illustration of the structure of organosilanes and its reaction. (A) General chemical structural formula of an organosilane featuring a central silicon atom connected to three organic groups (X) and a functional group (R1). (B) The ethoxy groups of the widely used 3-aminopropyltriethoxysilane (APTES) are hydrolyzed to hydroxyl groups transforming the silane into an activated state. (C) Different structural modes of the reaction of a silane with a hydroxylated surface forming a 3-dimensional interconnected silane network on a surface.

2.1.2 Immobilisation of DNA to substrates

Modified or unmodified nucleic acids can be attached to functionalized substrates via absorption (bond DNA-substrate), directly (selective bond DNA-substrate), or indirectly (selective bond DNA-linker-substrate) as summarized in Figure 2-1. Yield of each immobilization chemistry is dependent on the sequence itself [64] as well as the length of the nucleic acid [65].

Absorption

Unmodified nucleic acids can absorb in an undefined conformation to surfaces if certain chemical prerequisites are fulfilled. Molecular interaction is induced by the negatively charged backbone, hydrophobic elements of the base environment, or primary amino groups of the bases adenine, guanine and cytosine. Call et al. activated glass slides using 3 M H2SO4 and subsequently 3 M HCl, spotted unmodified DNA, and post-baked at 130 °C for 30 - 60 minutes [66]. Belosludtsev et al. spotted unmodified nucleic acids onto a 3-aminopropyltrimethoxysilane (APTMS) silanized glass slide and post-baked at 50 °C. [67]. Tan et al. spotted unmodified nucleic acids to an aminated glass slide. DNA from the liquid phase is thermally cross-linked to the slide during incubation at 80 °C in a humidified chamber for 3 h [68].

Direct immobilization

UV radiation causes numerous changes in the structure of DNA [69]. Especially the bases thymine and cytosine are prone to form radicals, ions, or dimers. At high radiation densities and energies, DNA molecules are denatured wherefore it is used for the decontamination of carry-over DNA (section 2.8.1). Modified or unmodified nucleic acids are immobilized to surfaces by two different strategies employing UV irradiation. Either a chemical group at a DNA molecule or a chemical group at a surface is activated. Rühe et al. investigated the latter strategy by spotting benzophenone containing hydrogels with unmodified DNA to polymeric or silanized surfaces. By subsequent UV irradiation, benzophenone molecules are activated linking the hydrogel to the surface and nucleic acids to the hydrogel, simultaneously [70-72]. Other researcher utilized the strong increase in reactivity of thymines towards hydroxyl, amine, or carbon containing surfaces through UV irradiation [52,73]. Church et al. attached polyT tailed nucleic acids to nylon membranes by UV radiation at wavelength of 254 nm for sequencing applications [30]. Kimura et al. attached polyT tagged oligonucleotides onto the polymers PMMA, PET, and PC performing hybridization experiments [57]. Sun et al. arrayed poly(C,T) tagged oligonucleotides on polymeric substrates for the identification of multiple targets via solid-phase PCR [74].

Various other chemistries for the direct immobilization of DNA to surfaces are investigated like the EDC chemistry, where 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) mediates the linkage of aminated nucleic acids to hydroxylated substrates [75,76]. Other strategies utilize strong interactions between aminated nucleic acids and epoxy [66,67,77], aldyhde [76,78] or carboxy [79-81] modified surfaces. A selective and direct bond DNA - substrate should be guaranteed when nucleic acids feature chemical moieties which are naturally not present like biotin, Acrydite™, or thiol groups. The protein streptavidin has four highly affine binding sites for the vitamin biotin exhibiting a binding constant of about 10-15 mol-1 [82,83]. Biotinylated nucleic acids can be attached to streptavidin coated surfaces [83,84]. Acrydite™ labelled oligonucleotides are co-polymerized into a polyacrylamide gel applied to a methacryloxy silanized substrate [85-88]. Thiolated nucleic acids strongly adhere to gold coated surfaces by chemisorption [89-97]. Nevertheless, Wolf et al. observed non-specific interactions between gold surfaces and nucleic acids, which are minimized by the reagent mercaptohexanol (MCH). This small molecule can reduce unspecific interactions between DNA and a substrate and additionally saturates the gold surface [95].

Indirect immobilisation

Modified nucleic acids can be indirectly attached to substrates by bifunctional linking molecules. They have a chemically inert spacer in common, e.g. a carbon chain, and reactive groups at both ends. Homobifunctional linkers feature two identical reactive groups, heterobifunctional linkers different reactive groups each targeting a different chemical moiety.

Guo et al. first investigated the homobifunctional linking molecule 1,4-phenylene diisothiocyanate (PDITC) for the immobilization of aminated DNA onto aminated surfaces. He demonstrated selectivity of the bond, thermostability, and an adjustability surface density [98]. The general PDITC chemistry for DNA immobilization is visualized in Figure 2-3.

illustration not visible in this excerpt

Figure 2-3: Immobilization of nucleic acids to substrates using PDITC chemistry. (A) On the surface of an unmodified substrate, hydroxyl groups (B) are generated by oxygen plasma. (C) The aminosilane APTES reacts with the hydroxyl groups, leaving an amine terminated surface. (D) The homobifunctional PDITC molecule binds to the amine groups, terminating the surface with thiocyanate groups. (E) An aminated oligonucleotide binds covalently to the thiocyanate groups.

Several groups further investigated the PDITC chemistry [47,62,99-103]. Erdogan et al. [45] and Kranaster et al. [47] showed successful solid-phase PCR on a DNA microarray realized by PDITC chemistry on glass. Another well investigated homobifunctional linker is glutaraldehyde, which links aminated DNA to aminated surfaces [40,49,104]. Liu et al. successfully demonstrated real-time PCR on a glass surface featuring TaqMan probes immobilized by glutaraldehyde [49].

Unlike homobifunctional linkers, heterobifunctional linkers feature different reactive moieties at their ends, each targeting a different reactive group. Hence, heterobifunctional linker promises to diminish unwanted reverse reactions between the linker and reactive groups on a surface. Adessi et al. investigates the linkers s-SIAB, sMBS, s-GMBS, s-MBP for the immobilization of oligonucleotides onto glass and its performance in solid-phase PCR [39]. A prominent example is the linker SSMCC which features a N-hydroxysulfosuccinimide (NHS) ester and a maleimide functionality reactive towards amines and thiols, respectively [94,105-108]. Kumar et al. introduced the concept of attaching reactive silanol groups to nucleic acids for its immobilization to hydroxylated substrates. Employed heterobifunctional crosslinkers are N-succinimidyl3-(2-pyridyldithiol)-propionate (SPDP) or succinimidyl-6-(iodoacetyl-amino)-hexaonate (SIAX) [109].

2.1.3 Detection of immobilized DNA

For the detection of immobilized oligonucleotides, a common method is to introduce labelled nucleotides into a nucleic acid sequence during chemical synthesis [110]. For quantification of DNA densities, measured signals can be aligned to a reference curve [52,60,111]. The use of radiolabelled DNA offers absolute data but suffer from hazardous waste disposal issues [65]. Another commonly used method is to detect signals from fluorescently labelled nucleic acids [39,47,87,94,101,102,112]. Fluorescent labels are widely used in biochemistry to visualize DNA, RNA, proteins and other structures of interest. Several fluorophores exist, which do not only differ in their spectral properties but also in the way they interact with other molecules. Spectral properties of fluorophors can be influenced by pH, ionic strength, temperature or the presence of other molecules. Besides these environmental factors, stoichiometric or surface derived interactions influence the activity of fluorophors. Additionally, the DNA sequence itself has a strong influence on the activity of the fluorescent molecule [113]. Therefore, an exact quantification via florescent signals is not possible and more or less semiquantitative.

To exclude these interdependencies, surface coverage of DNA can be indirectly determined by external analysis of DNA related signalling molecules. Pal et al. cleaved fluorescent molecules from immobilized nucleic acids using alkaline phosphatase for quantification off the surface [97]. Zammatteo et al. analysed colorimetric signals derived from horseradish peroxidase attached to nucleic acids [76]. Hurst et al. cleaved fluorescent nucleic acids off the surface using dithiothreitol (DTT) [114]. Demers et al. hybridized fluorescent probes to immobilized nucleic acids for subsequent dehybridization, measurement, and alignment to a reference curve [115].

Besides labelling techniques as discussed above, label-free techniques are also used for the characterization of surface-bound oligonucleotides like X-ray photoelectron spectroscopy (XPS) [91-93,116,117], surface plasmon resonance (SPR) [89,94,108,108,118], RIFS [119], ellipsometry, or electrochemistry/amperometric [59,90].

In general, detection methods for immobilized DNA can be classified by the kind of molecules to be sensed, assays to be performed, the number of molecules to be detected, the employed immobilization method, its stability and reactivity under reaction conditions, and its density to name only a few. Surface density is an often discussed and measured parameter [62,89]. It correlates with the sensitivity and the availability for biochemical reactions [120]. Therefore, surface density is measured by a large variety of methods as presented above. Density of immobilized DNA sequences can be increased by chemically inert spacer molecules [121] [122], poly(dT) tails [98,123], poly(dA), or Poly(dG) tails [117]. Beside a reduction of steric hindrance by spacer molecules, density can be increased using branched linking molecules like dendrimers [62] or three-dimensional immobilization strategies such as agarose gels [112,124,125], polyarcylamid gels [85-88,126,127], or the usage of porous substrates [85,128,129]. As a closing remark, a high density of nucleic acids does not necessarily lead to high hybridization efficiency [89,130,131] and can even lead to a unwanted signal reduction due to quenching of fluorescent signals [132,133].

2.2 Fabrication of DNA microarrays

There are two major strategies to fabricate DNA microarrays: deposition of previously synthesized probes onto a substrate using spotting technologies [134,135] and the insitu synthesis of probes on a substrate [136-142].

DNA probes can be pre-synthesized by phosphamitide chemistry [110], PCR [143], or reverse transcription [144]. Synthesized probes are subsequently spotted onto a functionalized substrate either by contact or noncontact printing technologies [6,145,146]. These technologies can have drawbacks like a finite number of probes that can be spotted, missing spots (clogging of channels), or blurred spots (wrong humidity, misalignment) and are mainly used in research environment.

Instead of spotting pre-synthesized oligonucleotides, probes can be synthesized directly on the surface of a functionalized substrate [136-142]. Here, the nucleotides are assembled by a cyclic process to generate the sequence of interest as illustrated in Figure 2-4.

illustration not visible in this excerpt

Figure 2-4: Illustration of the process chain for the in-situ generation of a DNA microarray as used by the companies Affymetrix, NimbleGen, and Febit. The whole surface of a substrate is coated with photolabile protective groups. (A) In a first cycle, protective groups are removed at selected areas either by local irradiation or by the addition of a liquid reagent. (B) The first nucleotide, containing a protective group, is applied to the surface and binds to the de-protected areas. (C) In the second cycle, protective groups are selectively removed again before adding the second nucleotide (D).

Coupling efficiency of nucleotides differs between 98.5 % and 99.5 % for a light induced synthesis by Zhou et al. [137,147]. As an example, only 37 % of all DNA strands of a 200 base pair long sequence contain no errors synthesized at a coupling efficiency of 99.5 %. Current technologies are restricted to length 60 [136,137,139] and 100 - 200 bases [138,139], respectively.

In 1994, the first in-situ synthesized DNA microarray is presented by Fodor et al. using a photolithographic technique that is the base for the still today sold GeneChip™ from Affymetrix [148]. An aminated glass surfaces is coated with a nucleotide containing a protected 3’-hydroxyl group. Such groups are locally activated by irradiating the substrate through a photomask defining a pattern of UV light. In repetitive cycles, protected nucleotides are applied to the whole substrate, reacted with the previously de-protected areas, subsequently de-protecting another pattern before the next coupling step (Figure 2-4). The first publication demonstrates the fabrication of an array of 256 different combinatory DNA probes in length of 8. Using this technique, an array comprising 200 base long DNA probes, 800 individual photomasks are required. Dill et al. from the company Combimatrix published in 2001 a CMOS chip for the synthesis of DNA probes on a semiconductor surface [138]. The chip features 1,024 individually addressable microelectrode spots below a porous reaction layer. Nucleotides are added in repetitive cycles and electrochemically coupled to the electrically activated spots. In 2001, Hughes et al. presented a method for the in-situ synthesis of DNA microarrays based on the inkjet printing technology [139]. A substrate is covered with unprotected, reactive groups as starting layer. In repetitive cycles, protected nucleotides are spotted (maximum 25,000) to distinct areas and reacted with the surface. After each printing cycle, the whole substrate is chemically deprotected for the selective attachment of the next nucleotide. The company Agilent sells DNA microarray fabricated by the so-called SurePrint technology. Nuwaysir et al. presented in 2002 another maskless photolithographic system for the light-directed synthesis of 195,000 DNA probes [142]. The technology is based on a digital light processor (DLP) which is capable of generating patterns of UV light. Compared to the technology of Affymetrix, this digital light mask eliminates the need for expensive and timeconsuming photomasks. DNA microarrays produced by this technology are today sold by the company NimbleGen, which was acquired in 2007 by Roche. Baum et al. presented in 2003 a similar system synthesizing 56,000 individual DNA probes by a light activated process within the channels of a microfluidic channel system which was distributed by the German company Febit [141].

2.3 PCR

Polymerase chain reaction (PCR) is an in-vitro method for clonal amplification of DNA relying on repetitive thermal cycles for DNA melting and enzymatic amplification [143]. The sequence to be amplified (“target”) of a template DNA is defined by two oligonucleotide sequences (primers). Double-stranded DNA is denatured first, so that primer and polymerase can anneal to the single strand. Starting at the 3’-end of the primer, the polymerase incorporates nucleotides that are complementary to the target sequence. PCR follows a cyclic temperature protocol, which is 94 - 96 °C for denaturation, 50 - 65 °C for annealing and 72 °C for extension (three-step protocol). Extension can also occur at the annealing temperature, so that an additional step is not required (two-step protocol). In the first PCR cycles, the number of copies N synthesized after n PCR cycles theoretically follows equation (2-1), whereas N0 represents the initial amount of template DNA and E stands for the amplification efficacy. The efficacy of liquid-phase PCR is typically in the range of 0.8 [36] to 1 [38].

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In real-time or quantitative PCR (qPCR), the amplification process is monitored online by measuring an increase of a fluorescent signal after each cycle [149,150]. Here, PCR is performed in the presence of either an intercalating dye or a targetspecific fluorescent probe. Intensity of intercalating dyes increases the more doublestranded PCR product is generated. Signals from fluorescent probes are released due the 5’ 3’ exonuclease activity of a polymerase, separating fluorophore and quencher increasing a fluorescent signal [151,152]. The cycle number (Cq value), at which a certain intensity threshold is exceeded, depends on the initial concentration of template DNA. The comparison of Cq values from different reactions with known standards allows for a quantification of the initial template DNA concentrations. Under ideal conditions (amplification efficiency = 1), a shift of 3.3 cycles indicates a difference of one order of magnitude in the initial template concentrations and theoretically allows for the detection of a single DNA molecule.

2.4 Small-volume PCR

The first primer-directed PCR published in 1988 by Saiki et al. was performed in a reaction volume of 100 μL [143]. Over the last decades, many efforts have been made to scale down PCR reaction volumes and simultaneously increase the number of reactions [153-161]. And for good reason: small volume PCR in large-scale systems decreases costs per reaction. When e.g. loading Saikis 100 μL of reaction mix into the wells of a GS FLX Titanium sequencing chip, more than 5 million individual reactions can be performed in parallel. Additionally, low thermal masses and a large surface to volume ratio results in an increased thermal transfer leading to reduced cycling times. As an example, Neuzil et al. amplified an 82 bp DNA template in less than six minutes within 100 nL droplets [162]. Together with ongoing improvement of sensitivity and resolution of analytical equipment, small volume PCR allows for economical large scale analysis of genomic features, signaling pathways or expression profiles down to a single molecule level. Nevertheless, the enlarged surface to volume ratio is challenging when performing PCR within picoliter reactors because significant amounts of PCR components are adsorbed to surfaces. As an example, a 10 μL PCR (performed in a standard 100 μL PCR tube) has a surface-to-volume ratio of 0.002 μm-1. In contrast to that, an 18.5 pL reactor has a surface-to-volume ratio of 0.209 μm-1, which is ~ 100 times higher. This challenge can be circumvented by passivation of the respective surfaces, by adding components to the PCR mix, which compete with the biomolecules for adsorption, or by increasing the concentration of the PCR reaction components. Surfaces have been passivated by silianization, the treatment with polyethylene glycol and various other materials [163]. Bovine serum albumin (BSA) is used for both, the coating of surfaces prior to PCR and as a absorption reagent in the PCR mix [164,165]. Additional reagents are Tween 20 and also Tween 80, which are used as a surfactant that prevent the unspecific adsorption of DNA to a wall [155,166]. M. Krishnan et al. investigated the influence of magnesium and polymerase concentration on the PCR performance within microchannels with surface-to-volume ratios of 0.02 μm-1 to 0.13 μm-1 [167]. W. Wang et al. realized a high surface-to-volume ratio by adding oxidized silicon nanoparticles to a qPCR reaction mix showing a significantly reduced but not completely inhibited - PCR performance from ratios of 0.094 μm-1 to 0.236 μm-1 [168]. However, the optimum concentrations have to be empirically determined for each PCR platform.

2.5 Solid-phase PCR

Solid-phase PCR describes a PCR system featuring at least one PCR primer that is immobilized to a reaction compartment [34-38]. During PCR, the generated PCR product gets bound to a surface via the immobilized solid-phase primer. In 1994, Khosaka et al. investigated a PCR reaction in a microtiter plate featuring one immobilized PCR primer [37]. By different configurations of the liquid- and solid-phase primers, five different reaction mechanisms for solid-phase PCR are distinguished:

The system comprises a surface with one immobilized PCR primer and no liquidphase primers in the PCR reaction mix. Here, the template is linearly amplified and bound to the surface [38].

The system comprises a surface with one immobilized PCR primer and one liquidphase primer in the PCR reaction mix. The anti-sense template DNA strand is exponentially amplified in the liquid-phase, the template DNA bound to the surface showed an efficiency of about 0.09 [38].

The system comprises a surface with two immobilized PCR primers and no liquidphase primer in the reaction mix. In this so called bridge-amplification system, efficacy for the generation of surface-bound PCR products is reported to range between 0.2 and 0.3 [35,36].

The system comprises a surface with one immobilized PCR primer and two liquidphase primers in the PCR reaction mix. This asymmetric SP-PCR approach combines liquid- with solid-phase PCR as described in Figure 2-5 [169]. Khan et al. described a system combining the technique of nested PCR with asymmetric SP-PCR. Here, solid- and liquid-phase primers are designed with different melting temperatures shifting the amplification efficiency to the solidphase [34].

However, surface load with a PCR product is not only mechanistically but also sterically influenced. Efficient diffusion and annealing of template DNA and polymerase to solid-phase primers is important. Therefore, various spacer molecules like poly(dT) tails [169,170] or molecules based on glycol compounds [56,121] are investigated to increase the distance between a surface and solid-phase primers. A length of five to ten base pairs as spacer is reported to be a good trade-off between increasing the diffusion and an unwanted steric shielding of neighbouring primers [38,56,121]. Additionally, spacers can diminish repellent effects of hydrophobic surfaces.

Figure 2-5: Principle of asymmetric solid-phase PCR. (A) A reaction compartment comprises solid-phase primers as well as liquid-phase forward (fwd) and reverse (rev) primers in an asymmetric ratio. (B) In the beginning, PCR proceeds preferably in the liquid phase, until the forward (fwd) primer is depleted. (C) Now solid-phase PCR dominates binding the PCR product to the solid-phase primers. (D, E) During PCR, biotin-dUTPs are incorporated into the PCR product. (F) Streptavidin-Cy5 molecules are coupled to the biotin molecules for subsequent visualization of the surface-bound PCR product.

2.6 Single-molecule PCR

2.6.1 Digital PCR

In Digital PCR, a DNA sample is adjusted to either 0 or 1 DNA sequences per reaction volume. By PCR amplification and endpoint detection, the presence (= 1) or absence (= 0) of DNA is determined by counting the number of positive reactions [163,171-174] (Figure 2-6 A). The idea of performing assays at limiting dilutions was first described in 1982 by Fazekas et al. [175]. In 1999, Vogelstein et al. coined the term “Digital PCR” in analogy to the binary system [171]. dPCR offers the benefit that no sequences are preferably amplified over others simultaneously increasing amplification efficiency [163,176-178].

After aliquoting a sample into the wells of a Digital PCR chip, some wells can contain more than one DNA molecule due to the stochastic distribution. Therefore, the number of positive wells does not necessarily reflect the number of initial DNA molecules. In order to extract sound data from the number of positive reactions, it is necessary to consider the statistical distribution of the DNA molecules within a dPCR chip. For a mathematic description, it is assumed that the distribution of DNA molecules follows Poisson statistics [157,171,179,180]. Equation (2-2) shows the Poisson distribution with P being the probability that a well contains k molecules after filling, and being the ratio between the number of DNA molecules and the number of wells:

illustration not visible in this excerpt

Figure 2-6: Schematic of the principle of Digital PCR (A) and Digital solid-phase PCR (B). (A) A PCR reaction mix containing few DNA molecules is introduced into the wells of a chip. After PCR reaction, an unbound PCR product is generated only in those wells containing a single DNA molecule. (B) In Digital solid-phase PCR, the single-molecule derived product is covalently immobilized to the wells of respective reactor.

2.6.2 Digital solid-phase PCR

Several Digital PCR platforms are developed and partially commercialized [157,160,174,181-183] offering new possibilities in research and diagnostics [173,180,184-187]. Nevertheless downstream applications are not possible since amplification products are discarded after reaction. By combining Digital PCR with SPPCR, single-molecule derived PCR products get coupled to a solid surface for downstream applications like sequencing (Figure 2-6 B). 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” [188], (B) bridge PCR: “colonies-on-surfaces” [189], and (C) emulsion PCR: “colonies-on-beads” (emPCR) [84].

illustration not visible in this excerpt

Figure 2-7: Three different approaches (A, B, C) for Digital solid-phase PCR. (a) Each method starts with a single DNA molecule (black). (b) This molecule hybridizes to one PCR primer on a surface. (c) After solidphase PCR, clonal DNA copies (red) are covalently bound to the surfaces.

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 (Figure 2-7 A) [188]. 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 (section 2.1.2). A film of this gel is applied to a silanized glass slide and polymerized thereto [88]. 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 [88], genotyping and haplotyping [190], and sequencing [31].

Colonies-on-surfaces. DNA colonies are immobilized to surfaces by the mechanism of bridge PCR (Figure 2-7 B). Forward and reverse primers are bound via their 5’-ends to a surface. Single-stranded DNA template molecules randomly hybridize to the forward primer, which is consecutively extended by a polymerase. During a subsequent denaturation step, the template molecule is removed from the surface. The extended primer remains attached to the surface - being a complimentary copy of the initial template molecule - and anneals to the complementary reverse primer for subsequent extension by a polymerase. By cyclic repetition of the previous steps, a surface-bound colony grows around the first extended forward primer [35,189,191,192]. The company Illumina uses this method to generate DNA colonies for sequencing applications.

Colonies-on-beads. DNA colonies are bound to the surface of microbeads by the mechanism of solid-phase PCR (Figure 2-7 C). A PCR reaction mix, beads featuring solid-phase primers, single DNA molecules, and oil are mixed forming a water-in-oil emulsion. After PCR thermocycling, emulsions are broken, and beads are recovered [84,193]. For the removal of empty beads, different enrichment strategies exist, which are presented in the supplements of [17] and [20]. The so called emulsion PCR (emPCR) is used in four different sequencing systems (see section 2.7) for the preparation of DNA libraries [17,20,21,23].

In the colonies-in-gel and colonies-on-surfaces approaches, DNA colonies are mechanistically constrained but can overlap resulting in colonies with mixed sequences. In the colonies-on-beads approach, droplet can merge at higher temperatures resulting in chimeric DNA sequences on the surface of the beads [193]. When comparing the three different methods with respect to its application for sequencing, it emerges that the colonies-in-gel and colonies-on-surfaces approaches require less technical effort and hands-on steps compared to the colonies-on-beads approach. Additionally, DNA colonies are arrayed directly on a chip surface. This is an advantage as long as the amplification is efficient and faultless. If not, the operator has to decide whether to discard or sequence the chip, which already consumed “a major part of overall sequencing cost” [14]. When the amplification efficiency in the colonieson-beads approach is inefficient, this reaction can be repeated before fixing the DNA colonies on a chip for sequencing.

2.7 Sequencing technologies

The first commercial next-generation system distributed by Roche/454 relies on sequencing of DNA molecules immobilized onto beads (see section 2.6: colonies-onbeads) within the wells of a picotiter plate (PTP) [17,194,195]. Wells are fabricated by anisotropic etching of optical fibre bundles [196-198]. A DNA library for sequencing is prepared by emPCR (Figure 2-8 A, B). Sequencing is done by adding one kind of deoxyribonucleoside triphosphate (dNTP) at a time (Figure 2-8 C). The incorporation process is monitored with a CCD camera, since pyrophosphate is released and enzymatically transformed into a light signal, which was termed pyrosequencing [199,200]. The intensity of the signals scales with the number of incorporated nucleotides. Initially developed by 454 Life Sciences, this technology is now owned and further developed by Roche. In the current PTPs of the GS FLX series, the walls of the wells are coated with titanium, minimizing optical cross-talk between adjacent wells, and offer an increased density of wells (3.4 million wells à 29 μm diameter) and thus a higher degree of parallelization. For medium-scale sequencing reactions, the distribution of the GS Junior system is launched in the beginning of 2011, offering 450,000 wells per chip. End of 2011, Roche introduced the GS FLX+ system, for loading DNA sequences in length of 1,000 bp onto DNA beads.

illustration not visible in this excerpt

Figure 2-8: Description of the sequencing process of the GS systems distribute by Roche. (A) Preparation of a DNA library. Genomic DNA is fragmented in length of 400 - 1000 bp by nebulization. Blunt ends are polished and two generic primer sequences are attached. DNA fragments are bound to streptavidin coated beads (blue) via its biotin labelled primer. After melting off the non-biotinylated strand from the DNA, a single-stranded DNA sequencing library bound to beads is obtained. (B) Preparation of the DNA beads. A PCR reaction mix, the prepared DNA library, and beads are mixed. By vortexing, a water-in-oil emulsion spontaneously forms, which is PCR thermocycled. Generated PCR products are bound to beads by the mechanism of solid-phase PCR. Beads are enriched, the complementary strand is melted off, and the sequencing primer is annealed. (C) Sequencing reaction. After filling the DNA beads into a picotiter plate, enzyme- and packaging beads are added. Light signals derived from a pyrosequencing reaction are monitored for each well by a CCD camera. From overlapping regions of the single DNA fragments, the complete sequence is calculated. Pictures are taken from the GS manuals (Roche/454).

Long read-lengths are favourable in de-novo sequencing, since less computational effort is needed to aligne individual DNA fragments to a complete genome. On the other hand, especially for homopolymeric regions within a DNA sequence, insertion and deletion errors are reported for this system [12-14]. The genome of the James Watson - co-discoverer of the structure of DNA in 1953 - was decoded in 2008 using Roches/454 sequencing technology [194]. In 2011, researcher from Hitachi presented a similar but miniaturized pyrosequencing platform with 4 μm wells [23].

At the sequencing platform distributed by Illumina, DNA colonies are generated by bridge amplification on a planar chip (see section 2.6: colonies-on-surfaces) [22,201]. Sequencing is done by adding four nucleotides at a time, each featuring a univocal fluorescent label and a reversible terminating group. After incorporation of one nucleotide, the polymerase stops due to the terminating group [13]. Unbound labels are washed away and a four-color fluorescent image is acquired. Before the next cycle, terminating groups and labels are cleaved off and removed. The platform is able to sequence up to 600 giga bases at a single run for about 0.04 $ per mega base [14]. Read lengths are between 100 bp and 150 bp and can be doubled by paired-end sequencing, which means that sense- and antisense strand are deciphered [14]. The first genomes of an African [22] and of an Asian [202] are published in 2008 using Illumina sequencing technology. In 2011, Illumina systems have dominated the market, since they offer the highest throughput for a competitive prize. The short read length compared to Roche’s system is compensated by an increased computational effort [13]. Since Roche’s platform exhibits advantages for de-novo sequencing due to the higher read length, it is proposed to use Illuminas’s system together with Roche’s for more accurate de novo sequencing [203,204].

Sequencing by ligation approaches utilizes fluorescent probes [13] attached to one or two bases for the interrogation of DNA molecules. Probes hybridize to the complementary strand of a DNA bead (see section 2.6: colonies-on-beads) and are ligated by a DNA ligase. Non-ligated probes are removed and a fluorescent image is taken. Two vendors provide sequencing-by-ligation platforms. The SOLiD platform (Life Technologies/ABI) covalently binds DNA beads to a planar surface of a glass slide prior to sequencing. The system features read length of up to 100 bp (paired-end read) at a high accuracy, which is achieved by two reads per base [13,14]. The open-source platform Polonator (Dover Systems) engineered by George Church physically traps DNA beads within a polyacrylamide gel for sequencing [20]. The Polonator is currently the least-expensive platform, but lacks the ability to perform reads of more than 26 bp [13].

In 2011, a new approach that turns away from optical detection methods towards electrical sensing is introduced by Jonathan Rothberg, who already invented the 454 sequencing system [21]. The system is technological the same as from 454, but detecting the incorporation of a nucleotide chemically. DNA beads are introduced into the 4 - 10 μm wells of a CMOS chip. The bottom of each well is designed as an ionsensitive field-effect transistor (ISFET), which is able to detect hydrogen ions that are released during incorporation of dNTPs. The system is developed by Ion Torrent, meanwhile acquired by Life Technologies. Read lengths up to 200 bp are achieved and a total of 1 giga bases can be sequenced in one run. In January of 2012, Ion Torrent announced the Proton II chip to be sold in mid-May 2013 featuring 660 million wells for deciphering a human genome in two hours at 1000 $ material costs [205].

Currently, research focuses on single-molecule sequencing methods bypassing DNA colony generation to circumvent its disadvantages. Firstly, complexity of the frontend sequencing process is drastically reduced. Secondly, errors in sequencing data introduced by PCR amplification bias are overcome [206]. The first single-molecule platform [207] was offered by Helicos in 2008, but is not sold anymore [14]. Pacific Bioscience came up with another system, where single DNA molecules are captured by an immobilized polymerase and sequenced in real-time [208]. However, the system is error-prone and requires multiple reads to generate sound data [14]. Both systems still rely on the detection of fluorescent signals. Approaches to sequence single molecules without luminescent detection are under development and aim to determine the order of bases while these pass solid-state or biological nanopores [209,210]. In the mid of 2012, the company Oxford Nanopore Technology announced the MinION, a system for sequencing DNA in the format of a USB-stick.

2.8 Strategies against DNA contamination

Especially in Digital PCR systems, which are capable of amplifying single DNA molecules, carry-over DNA contaminations from previous PCR can lead to falsepositives. In sequencing, contaminations can be responsible for false results because carry-over contamination is introduced into a sequencing run. This is because all DNA molecules to be sequenced are initially flanked by two generic primer sequences, which can be therefore processed in other sequencing runs. The biggest source of contamination is the opening of a reaction carrier after PCR. A standard PCR reaction saturates at about 10[11] clonal DNA copies due to a combination of the effects: 1) rapid re-annealing of product strands; 2) depletion of PCR primers; 3) activity loss of DNA polymerase [178]. It is of utmost importance that the operator is aware of the contamination potential of each PCR step and that he strictly adheres to the following fundamental rules:

- Decontamination of surfaces before and after each PCR
- Usage of separated working areas for DNA-free preparation of reaction mixes to high-copy areas for post-PCR steps
- Centrifugation of tubes before opening
- Immersing the filter tips into chemical decontaminants after DNA handling

Fighting existent contamination and detecting the source is more difficult than taking the described preventive measures. An ideal decontamination method effectively destroys contaminating DNA without affecting the template DNA, and does not inhibit subsequent PCR reactions. A single measure cannot fulfil all these requirements shifting the focus to multi-strategies that combine different decontamination methods [211]. The following two sections present possible decontamination methods together with their strengths and weaknesses.

2.8.1 Chemical decontamination

DNA molecules can be inactivated either by high energy radiation or aggressive liquid chemicals. UV radiation at wavelengths of 245, 300 or 365 nm is commonly used to decontaminate PCR reagents [212], but affects enzymes [213] and is less effective on DNA that has absorbed and dried at a surface [214]. UV radiation randomly cracks intermolecular bonds of the DNA chain and forms pyrimidine dimers that cause the polymerase to stop. The decontaminating effect of UV radiation can be enhanced by adding psoralen or isopsoralen [215,216]. However, these reagents are carcinogenic and hamper or even inhibit PCR [217]. Alternative decontamination strategies are liquid reagents like hydrochloric acid (HCl), sodium hypochlorite (NaOCl), CoPA solution, and hydroxylamine hydrochloride (NH2OH • HCl). Beside these chemicals for self-made decontamination solutions, there are several commercial ready-to-use decontamination mixes available containing different decontaminants and detergents. These claim to increase decontamination efficiency while decreasing negative side-effects. Table 2-1 gives an overview about chemical decontamination methods, indicating their field of application, advantages, and shortcomings.

Table 2-1: Overview about chemical decontamination strategies. The description consists of a brief explanation of the application and the effect on DNA as well as advantages (+) and disadvantages (-) of the methods. Detailed descriptions are found in the indicated references.

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2.8.2 Enzymatic decontamination

Most previously mentioned decontamination methods do not differentiate between contaminating and template DNA and need to be neutralized by washing steps. Thus, these reagents cannot be used for the decontamination of PCR reagents. In contrast, enzymes like dsDNase or Uracil-N-glycosylase (UNG) can be used for the selective digestion of contaminating DNA. dsDNase digests double stranded PCR products leaving single-stranded template DNA intact [211]. UNG digests PCR products containing dUTPs leaving template DNA containing dTTPs intact [224]. In natural organisms, UNG repairs defective DNA sequences that irregularly contain uracil bases, which can be formed by the deamination of cytosines [225]. Carry-over contamination from previous PCR reactions can be eliminated utilizing the similarity between nucleotides containing either uracil or thymidine in combination with a UNG digestion step [224,226]. By replacing dTTPs with dUTPs in a PCR reaction mix, dUTPs are incorporated into the PCR product. During a UNG digestion step prior to a further PCR reaction, uracil bases are cleaved off from dUTP containing DNA sequences that are now prone to denaturation at elevated temperatures. In a hot-start PCR reaction, the temperature is initially set to 95 °C for activation of the DNA polymerase. In the same step, the UNG enzyme and the UNG treated DNA sequences are inactivated thus preventing carry-over DNA from amplification [227].

[...]

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Title
Fabrication of DNA Microarrays by Digital Solid-Phase PCR in a Next Generation Sequencing Chip
College
University of Freiburg  (Institut für Mikrosystemtechnik)
Course
Mikrosystemtechnik
Grade
1,0
Author
Year
2013
Pages
124
Catalog Number
V300928
ISBN (eBook)
9783656978275
ISBN (Book)
9783656978282
File size
4569 KB
Language
English
Keywords
fabrication
Quote paper
Dipl.- Ing. Jochen Hoffmann (Author), 2013, Fabrication of DNA Microarrays by Digital Solid-Phase PCR in a Next Generation Sequencing Chip, Munich, GRIN Verlag, https://www.grin.com/document/300928

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