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i Advances in DNA Detection Yajing Song KTH Royal Institute of Technology School of Biotechnology Stockholm 2014
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Advances in DNA Detection
Yajing Song
KTH Royal Institute of Technology School of Biotechnology
Stockholm 2014
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ISBN 978-91-7595-053-2 ISSN 1654-2312 TRITA-BIO Report 2014:4 © Yajing Song, 2014 Universitetsservice US AB, Stockholm
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To my family. Forever.
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Yajing Song, 2014: Advances in DNA detection Division of Gene Technology, School of Biotechnology, Royal Institute of Technology (KTH), Stockholm, Sweden. ISBN 978-91-7595-053-2 Abstract DNA detection technologies have an increasing importance in our everyday lives, with applications ranging from microbial diagnostics to forensic analysis, food safety evaluation, and environmental monitoring. Currently, as the associated costs decrease, DNA diagnostic techniques are routinely used in research laboratories, in clinical and forensic practice. The first aim of this thesis is to unravel the potential of DNA detection on cellulose filter paper and further investigate the filter paper as a viable candidate for DNA array support. In Paper I, we studied the method of functionalizing the surface of filter paper and the possibility to detect DNA on the active paper using fluorescence. In Paper II, we addressed visual detection with magnetic beads and increased the detection throughput on the active filter paper, which required no instrumentation. Second, in pursuit of a rapid, sensitive and specific pathogen diagnosis in bloodstream infection (BSI), we explored the possibility of rare DNA detection in the presence of a high amount of background DNA by an enzymatic reaction, which can remove background DNA while enriching the rare DNA fraction. In order to overcome the challenge of the second objective, we developed a chemical fragmentation method to increase the efficiency of enzymatic digestion and hybridization. In addition, DNA library preparation for massively parallel sequencing may benefit from the chemical fragmentation. Paper III and Paper IV introduce this work. The findings in Paper I showed that XG-NH2 and PDITC can functionalize the cellulose filter paper and that the activated filter papers can covalently bind oligonucleotides modified with amino groups, while preserving the base pairing ability of the oligonucleotides. In Paper II, visual detection of DNA on active paper was achieved without instrumentation, based on the natural colour of magnetic beads. Furthermore, the possibility to increase the throughput of DNA detection on active paper was demonstrated by successful multiplex detection. In Paper III, the developed chemical fragmentation was verified to be suitable for DNA library preparation in massively parallel sequencing. The fragmentation technique is simple to perform, cost-effective and amenable to automation. In Paper IV, a limited amount of E.coli DNA was detected amid a much larger amount of human background DNA in a BSI model,
which comprises of human and E.coli amplicons with an abundance ratio of 108. Human β-actin amplicons were suppressed 105-fold, whereas the E.coli amplicons remained unaffected. The model system was applied to and improved with clinical plasma and blood samples from septic patients. Keywords: DNA detection, active filter paper, visual detection, throughput, fluorescence, superparamagnetic beads, rare DNA, Q-PCR, chemical fragmentation, DNA library preparatioin, massively parallel sequencing. © Yajing Song, 2014
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Contents Pages List of Publications........................................................................................................vii
Abbreviations...................................................................................................................viii
1 Introduction.....................................................................................................................1
2 DNA Science...................................................................................................................3
2.1 DNA History................................................................................................................3
2.2 DNA Structure.............................................................................................................4
2.2.1 Primary Structure of DNA.......................................................................................4
2.2.2 Secondary Structures of DNA.................................................................................5
2.2.3 Stability of Double-Helical DNA............................................................................7
2.2.4 Denaturation and Renaturation................................................................................7
2.2.5 Buffer............................................................................................................................8
2.3 DNA Function.............................................................................................................8
2.3.1 DNA Replication........................................................................................................9
2.3.1.1 DNA Repair............................................................................................................10
2.3.2 Transcription..............................................................................................................11
2.3.3 Translation.................................................................................................................12
2.4 DNA Detection..........................................................................................................12
2.4.1 DNA Hybridization.................................................................................................12
2.4.2 DNA Clone and Library..........................................................................................13
2.4.3 Polymerase Chain Reaction (PCR).........................................................................13
2.4.3.1 Basic Principles of PCR.......................................................................................13
2.4.3.2 PCR Variants..........................................................................................................14
2.4.3.3 Quantitative (Real-time) PCR..............................................................................14
2.4.4 Isothermal Amplification........................................................................................14
2.4.5 DNA Microarrays.....................................................................................................15
2.4.5.1 Background............................................................................................................15
2.4.5.2 Classification Based on Spatial Distribution of Support Surfaces................15
2.4.5.3 Microfluidic DNA Microarrays...........................................................................20
2.4.6 DNA Sequencing......................................................................................................21
2.4.6.1 Classical Sequencing Technology........................................................................21
2.4.6.2 The First Generation Sequencing Technology.................................................22
2.4.6.3 Next-Generation Sequencing Technology.........................................................23
2.4.6.3.1 DNA Library Preparation.................................................................................23
2.4.6.3.1.1 DNA Fragmentation......................................................................................23
2.4.6.3.1.2 Finalizing Library Preparation......................................................................25
2.4.6.3.2 NGS Strategies....................................................................................................25
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3 Specific Aims of the Studies.....................................................................................28
3.1 Paper I...........................................................................................................................28
3.2 Paper II..........................................................................................................................28
3.3 Paper III........................................................................................................................28
3.4 Paper IV.........................................................................................................................29
4 Results and Discussion..............................................................................................30
4.1 Paper I..........................................................................................................................30
4.1.1 Activation of Cellulose Filter Papers.....................................................................30
4.1.2 DNA Detection on Functionalized Papers..........................................................30
4.1.2.1 Synthetic Oligonucleotides...................................................................................30
4.1.2.2 Human and Canine Samples................................................................................30
4.2 Paper II........................................................................................................................32
4.2.1 DNA Visualization...................................................................................................32
4.2.2 Inspiration from Porous Structure of Cellulose Paper Chips...........................32
4.2.3 Multiplex Detection..................................................................................................32
4.2.4 Parameter Optimization..........................................................................................33
4.2.5 Future Application and Development...................................................................33
4.3 Paper III......................................................................................................................34
4.3.1 Principles of Chemical Fragmentation.................................................................34
4.3.2 Importance of DNA Repair...................................................................................34
4.3.3 Investigation of Fragmentation Bias.....................................................................34
4.3.4 Evaluation of Sequencing Artifacts and Errors..................................................34
4.3.5 Assessment of Sequence Capture Efficiency.......................................................34
4.3.6 Summary.....................................................................................................................35
4.4 Paper IV.......................................................................................................................36
4.4.1 Evaluation of PCR Efficiency................................................................................36
4.4.2 Q-PCR Condition Optimization............................................................................36
4.4.3 A Model Based on Amplicons................................................................................36
4.4.4 Application of the Model on Clinical Samples....................................................36
4.4.5. Future Directions....................................................................................................37
5 Conclusions and Future Perspectives....................................................................38
Sammanfattning på Svenska........................................................................................40
References.........................................................................................................................42
Acknowledgements.........................................................................................................55
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List of Publications
I Araújo, A.C. *, Song, Y. *, Lundeberg, J., Ståhl, P.L., Brumer, H, 3rd. (2012) Ac-
tivated Paper Surfaces for the Rapid Hybridization of DNA through Capillary
Transport. Anal Chem. 84(7): 3311-3317. (* Araújo, A.C. & Song, Y. contributed
equally to this work.)
II Song, Y., Gyarmati, P., Araújo, A.C., Lundeberg, J., Brumer, H, 3rd., Ståhl, P.L.
(2014) Visual Detection of DNA on Paper Chips. Anal Chem. 86(3): 1575-1582.
III Gyarmati, P., Song, Y., Hällman, J., Käller, M. (2013) Chemical Fragmentation
for Massively Parallel Sequencing Library Preparation. J Biotechnol. 168(1): 95-100.
IV Song, Y., Giske, C.G., Gille-Johnson, P., Emanuelsson, O., Lundeberg, J.,
Gyarmati, P. (2014) Nuclease-Assisted Suppression of Human DNA Background
in Sepsis. Manuscript.
Paper I is reprinted with permission of Analytical Chemistry. Copyright 2014
American Chemical Society.
Paper II is reprinted with permission of Analytical Chemistry. Copyright 2014
American Chemical Society.
Paper III is reprinted with permission from Elsevier. Copyright 2014.
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Abbreviations
A adenine
AFA Adaptive focused acoustic shearing
APTES aminopropyltriethoxysilane
BER base excision repair
BSI bloodstream infection
C cytosine
CAE capillary array electrophoresis
cDNA complementary DNA
CRTS cyclic reversible termination sequencing
ddNTP 2’,3’-dideoxynucleoside triphosphate
DNA deoxyribonucleic acid
dNTP 2’-deoxynucleoside triphosphate
dsDNA double-stranded DNA
E. coli Escherichia coli
emPCR emulsion PCR
ENCODE the Encyclopedia of DNA Elements
FDA food and drug administration
FP filter paper
G guanine
HDA helicase-dependent amplification
HGP the Human Genome Project
MMR mismatch repair
mRNA messenger RNA
NER neucleotide excision repair
NGS Next generation sequencing
-OH hydroxyl group
8-oxoG 8-oxo-7,8 dihydroguanine
PCR polymerase chain reaction
PDITC phenylenediisothiocyanate
PE phycoerythrin
PPi pyrophosphate
Q-PCR quantitative (real-time) polymerase chain reaction
RCA rolling cycle amplification
RNA ribonucleic acid
rRNA ribosomal RNA
SBL sequencing by ligation
SBS sequencing by synthesis
SFC sheath-flow cuvette
SMRT single molecule real time sequencing
SMS single molecule sequencing
SOLiD sequencing by oligonucleotide ligation and detection
SSB single-stranded DNA binding protien
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ssDNA single-stranded DNA
T thymine
tRNA transfer RNA
UV ultraviolet
XG xyloglucan
ZMW zero-mode waveguide
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1 Introduction
What is life (Schrödinger, 1944; Ernberg et al., 2010)? Where do we come from and
where are we going to? Man’s curiosity in the exploration of life has led to the
discovery of DNA and the development of DNA technologies. At the end of the
19th century, DNA was found in white blood cells (Dahm, 2005; Dahm, 2008). In
the mid 20th century, it was realized that DNA molecules possess a unique duplex
structure and carry genetic information (Hershey & Chase, 1952; Watson & Crick,
1953). At the start of the 21st century, the first human genome draft was announced
and genome drafts of other species were accomplished. At present, DNA science is
in undergoing considerable advancement due to improvements and developments
in related fields, such as biology, chemistry, biotechnology and physics.
Microarray technologies were established in the 1990s based on dot blot and reverse
dot blot techniques. They are important tools for analyzing biomolecules because
of their high-throughput capacity. Thousands to millions of features can be
analyzed simultaneously on a single microarray. This property meets the needs of a
variety of large-scale data-gathering studies in biology (omics), e.g., genomics,
proteomics and microbiomics, as this technology is mature and flexible. Researchers
and clinicians can either use commercial microarray products directly, or design and
fabricate arrays based on their specific needs. For example, clinicians sometimes only
need low-throughput arrays to detect a couple of pathogens. To open up new
possibilities, there is a great interest in improving and further developing microarray
systems. Part of the work described in this thesis aimed to develop a novel DNA
microarray method that is user friendly, cost-effective, timesaving and allows real-
time visualization.
One significant challenge in DNA detection is how to perform rapid and robust
detection of rare DNA amongst a huge amount of background DNA. For instance,
pathogen DNA detection in BSI. Normally, blood is sterile and when bacteria are
occasionally present, the host immune system can detect and remove them.
However, if the inflammatory immune response becomes out of control, the BSI
may go into a life-threatening phase: sepsis, which typically has a low survival rate
that strongly depends on the early initiation of effective therapy after the onset of
sepsis. Therefore, rapid and accurate diagnosis and early effective treatment are
crucial to increase the survival rate of this disease. In an attempt to achieve this, we
developed a new diagnostic method based on enzymatic reactions (e.g. DSN & BAL
31) and a molecular biological technique, i.e. quantitative (real-time) polymerase
chain reaction (Q-PCR).
One problem we addressed was the bottleneck of DNA library preparation in
massively parallel sequencing: random fragmentation of a flexible volume or/and
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amount of DNA. For this scenario, we developed another method - chemical
fragmentation, based on Fenton chemistry: Fe2+- EDTA4- + H2O2 → Fe3+- EDTA4-
+ ·OH. As shown in this study in comparison with other common fragmentation
methods (e.g. mechanical fragmentation and Nextera technology), this method
displayed the capacity to simplify DNA library preparation, and may be specifically
suitable for de novo sequencing and metagenomics studies. Furthermore, chemical
fragmentation helped solve the problem of removing high background DNA for
rare DNA detection.
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2 DNA Science
2.1 DNA History
What is life? In pursuit of an answer, DNA was discovered from the nuclei of white
blood cells by Friedrich Miescher, a German doctor in 1869, which was termed
'nuclein' (Dahm, 2005; Dahm, 2008). In 1952, it was proved that DNA is the
molecule of heredity by the Hershey-Chase Blender experiment. This study
comprised two parts and the work was carried out side-by-side by Alfred Hershey
and his assistant Martha Chase. In this experiment, the protein capsule and the DNA
core of a phage were labelled with radioactive sulphur (35S) and phosphorus (32P),
respectively. After the phage had infected the bacteria and the bacterial cells had
been cultured, centrifugation was used to separate the phage, which was left in the
supernatant, from the infected bacterial cell fraction. 35S was detected mainly in the
supernatant, whereas 32P from the phage was found predominantly in the infected
bacterial cells, from which the new generation of phage was generated. This study
demonstrated that DNA but not protein was the molecule carrying hereditary
information (Hershey & Chase, 1952).
What is the structure of this molecule of heredity? This chemical puzzle, which
remained elusive for more than 80 years, was finally solved by James Watson and
Francis Crick in 1953 (Watson & Crick, 1953), who announced the "double helix"
structure of DNA (Figure 1). It was the most important discovery in biology in the
20th century. The physical basis of DNA was revealed via X-ray crystallography.
Maurice Wilkins and Rosalind Franklin obtained X-ray diffraction photographs of
DNA in 1951 that strongly suggested the helical nature of DNA structure and
prompted Watson and Crick to publish their Nature paper about the first duplex
DNA model ( helix). Related to this discovery, another major breakthrough in this
century in biology was the development of sequencing technologies (Maxam &
Gilbert, 1977; Sanger et al., 1977). The first entire bacterial genome-Haemophilus
influenza-was published in 1995. In 1996, the first eukaryote genome sequence-
Saccharomyces cerevisiae-was released. In 1990, the Human Genome Project (HGP) was
initiated with the goal of understanding the human genome and genetic background
of human diseases. A further objective was to serve human health and human
evolution by making the human genome publicly available for researchers. In 2001,
the first draft of the DNA code of the human genome was published, representing
the start of the DNA era (Micklos et al., 2003), and with the announcement of the
complete genome in April 2003, the HGP came to an end. However, the accurate
number of genes in the human genome is still being debated (IHGSC, 2004). The
understanding of the function of the non-protein coding genes of human genome
sequence is also not fully uncovered, despite the concerted effort towards this goal.
One of the most comprehensive projects aiming for the characterization of
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functional elements in the human genome is the Encyclopedia of DNA Elements
(ENCODE) project (ENCODE Project Consortium et al., 2007; 2012).
Figure 1 DNA double helix structure. Two strands are antiparallel and complementary
to each other.
2.2 DNA Structure
2.2.1 Primary Structure of DNA
By the 1900s, the basic chemistry of the DNA polymer had been worked out. The
basic building block of DNA is the nucleotide, which is composed of three distinct
subunits: 2’-deoxyribose, a nitrogenous base and an acidic phosphate group. 2’-
deoxyribose is a pentose with a hydrogen group on the 2’-carbon of ribose. The 1’-
carbon of the sugar is the position that the base attaches to. There are four
nitrogenous bases, two single-ring pyrimidines (cytosine, C, and thymine, T) and two
double-ring purines (guanine, G, and adenine, A). Following base-pairing, the DNA
double helical structure forms with two base-pairing combinations bonded via
hydrogen bonds inside the DNA molecule: C base-paired with G by three hydrogen
bonds and T base-paired with A by two hydrogen bonds. Thus, in each sample of
double helical DNA, the amount of G equals the amount of C, the amount of A
equals the amount of T and the ratio of purines and pyrimidines is 1:1, giving rise
to the famous " Chargaff ’s rules" (Chargaff et al., 1952; Elson & Chargaff, 1952).
The DNA helix has a deoxyribose-phosphate backbone formed by phosphodiester
bonds on the outer part of the molecule. The two strands run in opposite directions
and the diameter of the helix is 2.37 nm (Figure 2). There are major and minor
grooves on the helical DNA surface. The paired bases are almost perpendicular to
the helix axis. The length of each base pair is 0.34 nm and there are 10 base pairs
per helix turn. Thus, the pitch per turn is 3.4 nm and each base pair is rotated by 36
degrees relative to the adjacent ones. The average molecular weight of a base pair is
650 g/mol (in sodium salt).
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Figure 2 Chemical structure of DNA.
Two complementary DNA strands
form a double-stranded DNA
(dsDNA). The pink line with
phosphate and deoxyribose sugar
(left) and the blue line with phosphate
and deoxyribose sugar (right) show
the deoxyribose-phosphate backbone.
G = guanine; C = cytosine;
A = adenine; T = thymine.
Three hydrogen bonds are between G
and C; two hydrogen bonds are
between A and T.
2.2.2 Secondary Structures of DNA
2.2.2.1 A-, B-, Z-DNA
X-ray diffraction studies have revealed that the conformation of DNA is variable.
The double-helical DNA that Watson and Crick reported was B-DNA with 10 base
pairs per turn, which is a right-handed double helix and the most stable
conformation under physiological conditions. A-DNA and Z-DNA are two variants
that exist under different conditions. Under low humidity and high salt
concentration (e.g. 75%, potassium), A-DNA is generated, which is also a right-
handed helix but shorter and wider than the B-form, with 11 base pairs per helical
run. Z-DNA was observed by Alexander Rich and his associates in 1979 (Wang et
al., 1979) when they tried to solve the structure of d(CG)n. Compared to the A- and
B-forms, the Z-form is a left-handed double helix that is longer and thinner with 12
base pairs in each helical run. The phosphates in the backbone take on a zigzag form.
It is still uncertain whether A-DNA exists in the cell, but there is some evidence to
suggest that Z-DNA is involved in regulating gene expression, especially for short
stretches of Z-DNA containing alternating C and G at the 5’ end of genes where
regulation of transcriptional activity occurs (Devlin, 2005). The features of different
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conformations of the DNA double helix are compared in Table 1 (Berg, 2011;
Brown, 2006; Nelson & Cox, 2013).
Table 1 Features of different DNA duplex comformations
Feature B-DNA A-DNA Z-DNA
Type of helix right-handed right-handed left-handed
Helical diameter (nm) 2.37 2.55 1.84
Rise per base pair (nm) 0.34 0.29 0.37
Distance per complete run (pitch) (nm)
3.4 3.2 4.5
Number of base pairs per complete turn
10 11 12
Topology of major groove
Wide, deep Narrow, deep Flat
Topology of minor groove
Narrow, shallow Broad, shallow Narrow, deep
Tilt of base pairs from perpendicular to helix
axis
1 degree 19 degree 9 degree
Glycosyl bond conformation
anti anti anti at pyrimidines, syn at purines
Apart from A-, B-, and Z-DNA, other variants of DNA conformation can occur
during the interaction between DNA with certain proteins, such as bent DNA,
cruciform structures, triple-stranded DNA and four-stranded DNA (Davlin, 2005).
2.2.2.2 Bent DNA
Bent DNA forms in runs of 4 or 6 adenines separated by a 10 base pair spacer or
during the interaction between DNA and certain proteins. It functions as a basic
molecule to carry out important biological processes, such as replication,
transcription and site-specific recombination. Bent DNA also acts as a signal of
DNA repair to recognize base mispairing or photochemical damage.
2.2.2.3 Cruciform DNA
Inverted repeats or palindromes may serve as molecular switches of the transition
from replication to transcription. In inverted repeats, each DNA strand is self-
complementary, which results in base-pairing within each single-stranded DNA
(ssDNA) after denaturation of dsDNA. As a result, a cruciform structure forms.
Cruciform DNA is important for genomic stability and certain basic biological
regulatory processes in the cell (Brázda, 2011). It has been found that many proteins
have specific structures for cruciform DNA binding in the cell (Brázda, 2011).
2.2.2.4 Triplex and Quadruplex Forms of DNA
Triplex helices can be formed by parallel binding of the third homopyrimidine
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strand to the homopurine strand of B-DNA through Hoogsteen hydrogen bonds,
resulting in T-A-T and C+-G-C triplets, as well as antiparallel binding of the third
homopurine strand to the homopurine strand of B-DNA via reverse Hoogsteen
hydrogen bonds to form A-A-T and G-G-C triplets. Four-stranded DNA usually
occurs in G-rich regions of DNA through Hoogsteen hydrogen bonds. Four Gs
interact with each other to form a guanine tetrad, and then several guanine tetrads
stack upon each other to form tetraplexes, or G-quadruplexes, which damages the
DNA helix. The structure is stabilized by cations, especially potassium, with
coordination by the four O-6 oxygens in the central channel of the quadruplex. The
first evidence that four-stranded DNA exists in cells was reported in 2009
(Lipps & Rhodes, 2009). In 2013, Shankar Balasubramanian and his colleagues
published strong evidence that the G-quadruplex does exist in the DNA of living
cells (Biffi et al., 2013; Lam et al., 2013), lending support to its proposed biological
functions.
2.2.3 Stability of Double-Helical DNA
The stability of the DNA molecule is crucial as it carries important genetic
information. Generally, base-stacking and hydrogen bonds are thought to be the
main forces acting to stabilize the duplex of DNA. In contrast to stacking forces,
hydrogen bonds were believed to primarily orient base pairing until 1963, when the
importance of the stacking interaction in stabilizing the double duplex was verified
through experiments with different reagents (Levine et al., 1963). Base-stacking, also
termed π-π interaction, originates from hydrophobic forces and van der Waals
interaction. The stacking energy for each adjacent stacked pair of nucleobases is
around 2-3 times stronger than that in each hydrogen bond. Electrostatic forces also
affect the conformation and stability of duplex DNA because the phosphodiester
groups are negatively charged under physiological conditions, and therefore repel
each other. Cations and proteins can help to decrease repulsive forces between
ionized phosphate groups (Davlin, 2005).
2.2.4 Denaturation and Renaturation
The hydrogen bonds and stacking interactions in the centre of DNA are disrupted
under certain conditions. This separation process between complementary DNA
strands is termed denaturation, helix-to-coil transition or melting. A minute energy
is needed to create one or more bubbles in dsDNA or DNA-RNA complexes. These
bubbles or stretches will immediately pair up at room temperature to reform a stable
duplex structure, at elevated temperature, the range of stretches increases and the
kinetic energy eventually overcomes the forces stabilizing the duplex, resulting in
single strand formation. Significant changes in pH can also disrupt the double helix
of DNA, e.g. alkali deprotonates specific positions on the DNA base, disrupting
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hydrogen bonds, whereas acid protonates hydrogen bond acceptors, blocking
hydrogen bond formation. Alkaline denaturation is usually used to prevent damage
to the nucleosides or sugars that happens at either low pH or high temperature
(Davlin, 2005). This technique was applied in Papers I and II. In the process of
denaturation, a hyperchromic effect occurs, namely, the DNA absorbance of
ultraviolet (UV) light at 260 nm increases. This is because the bases in duplex strands
have a lower ability to absorb light than that in single strands.
The denatured complementary DNA strands can reform a double helix under
suitable conditions. This is termed renaturation, coil-to-helix transition or
reannealing. The renaturation kinetics mainly depends on the strand separation level,
DNA complexity, original DNA concentration (Co) and duration (t). (Wilson &
Walker, 2010). If the separated strands are part of a dsDNA molecule, the
renaturation process is usually completed relatively quickly. However, for completely
denatured DNA strands, a much longer time is required to restore the original
structure, especially for the first base-pair formation. However, after formation of
a short double helix with a few base pairs, the remainder anneals quickly due to
neighbouring pair effects (Devlin, 2005). The DNA complexity influences the
reannealing process, such as the GC content in one DNA molecule. The higher the
GC content, the more energy is required for reannealing. Renaturation is also a
concentration-dependent process, namely, in a given environment and time, there
are more chances for higher concentration complementary sequences to find and
bind to each other than for lower concentration complementary strands per unit
volume. Therefore, the Cot value is used to evaluate the degree of reannealing
(Wilson & Walker, 2010). The Cot effect is applied in Paper IV.
2.2.5 Buffer
Significant changes in pH disrupt DNA structures as well as enzyme functions. Thus,
it is important to maintain a stable pH in certain biological processes, such as
polymerization and transcription. Buffers are solutions which alleviate pH changes.
In biological systems, there is an important buffer based on phosphoric acid
(H3PO4), which can be deprotonated to form H2PO4-, HPO4
2- and PO43- sequentially.
H2PO4- is a proton donor and HPO4
2- is a proton acceptor. At about pH 7.4, a
mixture of equal concentration of H2PO4- and HPO4
2- exists, which prevents the
formation of either acid or base at pH values between about 5.9 to 7.9 (Berg, 2011).
These mechanisms were employed in molecular biological studies of denaturation,
renaturation and hybridization (Papers I, II).
2.3 DNA Function
Studies on DNA function have established the “central dogma” of molecular
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biology, which generally states that genetic information flows from DNA to RNA
and from RNA to proteins. In this process, genetic information is not only stored
and transferred in DNA by precise replication, but also selectively expressed by
transcription and translation. To some viruses (e.g. retroviruses), whose RNA stores
their genetic information, the flow of the genetic information is from RNA to DNA
(Baltimore, 1970; Temin & Mizutani, 1970).
2.3.1 DNA Replication
The DNA structure determines its function. The discovery of the DNA double
helical structure by Watson & Crick in 1953 suggested that semi-conservative
replication of DNA was the possible mechanism of genetic information transfer
during cell division: “It has not escaped our notice that the specific pairing we have
postulated immediately suggests a possible copying mechanism for the genetic
material” (Watson & Crick, 1953). This assumption was supported by the Meselson-
Stahl experiment later in 1958. Briefly, the experiment was designed in three steps
to verify the hypothesis that DNA replication is semi-conservative and not
conservative (Bloch, 1955) or dispersive replication (Delbrück, 1954). (1) Escherichia
coli (E. coli) cells were cultured in medium with only 15N (heavy density). (2) The E.
coli generated in Step 1 were then cultured in only 14N (light density) medium. 100%
of the DNA after the first replication had a single density between that of DNA
containing exclusively 15N and 14N, which excluded the possibility of conservative
replication. (3) Further culturing of E. coli in the same medium as used in Step 2.
After the second replication, 50% of the DNA had a density between that of DNA
containing exclusively 15N and 14N, while the other 50% had the same density as
DNA containing exclusively 14N. This result excluded dispersive replication and
supported semi-conservative replication (Micklos et al., 2003). Semi-conservative
replication means that in DNA replication, each parental strand serves as a template
to produce a daughter strand with complementary bases pairing to copy the parental
genetic information, followed by the two strands winding each other. Thus, in the
newly generated DNA helix, one strand is “old” and the other one is “new”. Many
enzymes and proteins are involved in this process.
The templates of DNA replication are ssDNAs. Thus, the DNA helix must be
unwound and the two strands separated from each other to generate two templates
before replication. It is carried out by DNA helicase and topoisomerase. Next,
single-strand binding proteins (SSBs) attach to the single DNA strands to prevent
DNA renaturation. An RNA primer is necessary to initiate DNA replication via its
free 3’ hydroxyl group (-OH). This determines the direction of DNA synthesis from
5’ to 3’. The RNA primer is made by the primase before DNA replication and
replaced by DNA with the aid of DNA polymerase I after replication. Finally, DNA
ligase connects the fragments to form a strand complementary to the template
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(Figure3).
Figure 3 Schematic view of DNA semi-conservative replication (see text for details).
2.3.1.1 DNA Repair
DNA repair refers to a process whereby DNA damage is monitored and corrected.
DNA replication must be exceedingly precise to preserve the genetic information in
DNA through different phases of cell division. However, DNA damage does occur
during replication and other processes of cell division due to various endogenous
(metabolic) and exogenous (environmental) agents. Therefore, all organisms have
evolved a variety of DNA repair mechanisms to maintain DNA integrity (Ferrier,
2013; Houtgraaf et al., 2006).
2.3.1.1.1 DNA Damage
The two main sources of DNA damage are environmental agents and species
generated during intracellular metabolism. In the former case, DNA-damaging
agents include chemical mutagens, UV light, microbial toxins, alkylating agents,
among others. In the latter case, cellular metabolic products, such as endogenous
reactive oxygen species, i.e. superoxide radical anions, hydroxyl radicals, hydrogen
peroxide, can cause DNA damage. The main types of DNA lesions include altered
bases, e.g. oxidation of bases (8-oxo-7,8 dihydroguanine (8-oxoG)), alkylation of
bases (6-O-methylguanine) and deamination (C converts to U); loss of bases, e.g.
depurination, depyrimidination; mismatch bases, e.g. G-A; insertion and deletion;
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pyrimidine dimers, e.g. T-T; single-strand breaks and double-strand breaks; and
intra- or inter-strand cross links (Houtgraaf et al., 2006; Tuteja et al., 2001).
2.3.1.1.2 DNA Repair Mechanisms
DNA repair studies were initiated in the early 1960s (Hanawalt, 2013 and references
reviewed therein). The most important known DNA repair pathways are base
excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR)
and double-strand break repair (HR, NHEJ) (Christmann et al., 2003; Ferrier, 2013;
Friedberg, 2003; Houtgraaf et al., 2006). In this thesis, BER and NER are described.
2.3.1.1.2.1 Base Excision Repair
Different types of base lesions (e.g. base alteration and loss) can be repaired by the
BER pathways (Kubota et al., 1996). Generally, there are four steps in this pathway:
(1) Removal of abnormal bases and formation of AP (apyrimidinic/apurinic) sites.
N-glycosylase hydrolytically cleaves the glycosidic bond between the damaged base
and deoxyribose-backbone of the strand to form AP sites. (2) Recognition of AP
sites. A specific AP-endonuclease identifies AP sites and creates incisions at the 5’
end of AP sites. (3) Removal of deoxyribose-phosphate residues. AP lyase (a
deoxyribose phosphate lyase) catalyzes this step. (4) Nucleotide insertion and
ligation. DNA polymerase and DNA ligase are needed (Ferrier, 2013; Paper III;
Tuteja et al., 2001).
2.3.1.1.2.2 Nucleotide Excision Repair
NER is one of the pathways for repairing bulky DNA adducts. In bacteria, four
excision endonucleases (UvrA, B, C and D) recognize and unwind damaged strands,
produce dual incisions at the ends of damaged strands and then cleave the
oligonucleotides containing the damaged strands. DNA polymerase and ligase
catalyze the gap filling and strand ligation. The NER process in human cells is very
complex, involving approximately 30 proteins (Ferrier, 2013; Friedberg, 2003; Tuteja
et al., 2001).
2.3.2 Transcription
In order to transfer genetic information to synthesize a variety of proteins, the copy
process from DNA to RNA is also important. This process is termed transcription,
which is initiated by RNA polymerases to create an RNA strand antiparallel with
and complementary to a DNA template. U in the new RNA molecule replaces T in
this template. The synthesis direction is 5’→3’. Transcription generally consists of
three steps: (1) initiation, proceeds via formation of an initiation complex, which is
made up of RNA polymerase, cofactors and a core promoter sequence in the
genomic DNA; (2) elongation, the process in which the RNA polymerase complex
traverses the template molecule to synthesize an RNA strand; (3) termination, which
12
occurs when the RNA polymerase complex recognizes the terminator and finally
releases the newly synthesized RNA strand. The transcribed products include
messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA),
regulatory RNAs (e.g. microRNA) and other RNA molecules. There are also some
proofreading mechanisms that occur during transcription, but they are not as
effective as DNA replication (Berg et al., 2011).
2.3.3 Translation
Translation is the next step of gene expression. In this process, mRNA, tRNA and
ribosomes work together to produce proteins from amino acids. Ribosomes,
consisting of a large subunit and a small subunit, are the sites where amino acids are
assembled into proteins. mRNA carries genetic information from the transcription
and attaches to the small subunit of the ribosome. tRNA identifies specific amino
acids by specific anti-codons and transfers the specific amino acids to the large
subunit of the ribosome via binding its anti-codons to the code sequences of
mRNA (condons, a triplet of bases complementary to antiocodons in tRNA). As
the ribosomes move along the mRNA, the information in mRNA is translated into
a polypeptide until they encounter a termination codon, and the polypeptide is
released.
2.4 DNA Detection
2.4.1 DNA Hybridization
In many methodologies, such as PCR, blotting or microarrays, hybridization is the
basis of DNA detection, which is a process whereby complementary polynucleotide
strands associate with each other. The polynucleotide strands can be either
homogenous or heterogeneous. Schildkraut and his colleagues introduced a
technique in 1961 where a probing oligonucleotide was used to detect the
complementary ssDNA sequence (Schildkraut et al., 1961). These oligonucleotides
are called the “probe” and “target” in array technologies (see 2.4.5), which have been
applied to nucleic acid detection and quantification, e.g. genotyping, gene expression,
phylogeny and genetic mapping. Usually one of them is immobilized on a solid
matrix, whereas the other is labelled with fluorophores or biotin (Figure 4), then
detection of the bound labels is used to quantify the target sequences. In PCR, they
are called the “primer” and “target” or “template” (see 2.4.3). Hybridization
techniques are fundamental tools in molecular biology (Papers I, II, III, IV).
Depending on the particular study goals, hybridization studies are usually influenced
by several factors involved in a complex network of cause and effect, e.g.
hybridization temperature (Papers III, IV), hybridization buffer composition (Papers
I, II, III, IV), hybridization time (Papers IV), complexity of probes/primers or
13
targets/templates (GC content, secondary structure, strand length) (Papers I, II, III,
IV), concentration of probes/primers (Papers I, II, III, IV), amount of
targets/templates (Papers II, III, IV), washing solution composition, washing
temperature and time (Papers I, II).
2.4.2 DNA Clone and Library
Originally, the term clone referred to the process where a single cell type is
reproduced to create a population of identical cells. Regarding DNA, a clone
represents a multiple identical copy of a particular DNA segment. This technology
is called recombinant DNA technology. The goal of this technology is to amplify
target DNA, which can be used for downstream studies. This technology commonly
consists of six steps: (1) Generation of target DNA with restriction endonucleases.
(2) Selection of a cloning vector capable of self-replication, which is treated with
the same restriction endonuclease. Typically, a vector is a plasmid or viral DNA. (3)
Creation of recombinant DNA by inserting target DNA into the vector using DNA
ligase. (4) Transfer of recombinant DNA into the host cell for replication. (5)
Selection of the host cells containing target DNA. (6) Analysis of the biological
properties of target DNA (Nelson & Cox, 2013).
A DNA library is a collection of DNA clones, e.g. genomic DNA and cDNA
libraries. A genomic DNA library aims to contain the whole genome of an organism,
whereas the purpose of a cDNA library is to understand gene and protein function
(Nelson & Cox, 2013).
2.4.3 Polymerase Chain Reaction (PCR)
2.4.3.1 Basic Principles of PCR
The idea to replicate a short DNA fragment with an enzyme and primers was first
described in 1971 by Kjell Kleppe (Kleppe et al., 1971). By 1983, a complete in vitro
PCR technique had been invented by Kary B Mullis (Bartlett et al., 2003; Mullis et al.,
1986). This technique mainly comprised three steps: (1) dsDNA denaturation at a
high temperature, normally > 90°C; (2) primer annealing at lower temperature (50-
75°C); and (3) extension at a certain temperature (commonly 72°C). These three
steps constitute one cycle of a PCR. After the first two to three cycles, the target
sequences are amplified exponentially. Therefore, after 30 cycles, billions of
amplicons are produced under ideal reaction conditions. This standard PCR can be
followed by agarose gel electrophoresis to evaluate the amplicon size(s) by
comparing them to a DNA ladder, a molecular weight marker of known size. In
contrast to cell culture techniques, PCR enables more rapid, sensitive and robust
detection and analysis. The PCR technique rapidly became an accepted and
important tool in the research field (Strachan et al., 2003).
14
2.4.3.2 PCR Variants
Many efforts have been made to enhance the efficiency and specificity of classical
PCR, resulting in different variations of this technique.
2.4.3.2.1 Hot-Start PCR
Hot-start PCR is a modification of the classical PCR that increases the specificity
and specific product yields. In this system, Taq DNA polymerase is inactivated by
different methods until the reaction reaches a high temperature. For example, Plat-
inum® Taq DNA Polymerase is a hot-start polymerase where the ability of the pol-
ymerase is blocked by an antibody until the initial denaturation step. In classical PCR,
Taq DNA polymerase has some activity at low temperature. Therefore, unspecific
priming may be extended to produce unspecific amplicons, which reduces the target
yields.
2.4.3.2.2 Nested PCR
In this variant, two primer pairs are used instead of one. The products amplified
from the first pair of primers serve as the templates for the second pair of primers.
Therefore, the sensitivity of nested PCR is increased compared to classical PCR.
Ideally, the specificity is increased at the same time because the unspecific products
from the first pair of primers will not be amplified by the second pair of primers.
However, it depends on the design of the primers.
2.4.3.3 Quantitative (Real-Time) PCR
Real-time PCR, a further development of classical PCR, was first reported in the
early 1990s (Heid et al., 1996; Higuchi et al., 1992; Higuchi et al., 1993; Holland et al.,
1991). Following the basic principles of classical PCR, real-time PCR combines the
use of a thermal cycler and fluorimeter to monitor the progress of the reaction in
real time with the use of a fluorophore(s) (Mackay et al., 2002). Compared to classical
PCR, real-time PCR increases the rate of the reaction and reduces the risk of
amplicon spread since both detection and analysis are carried out in a closed tube
during an amplification, which prevents carry-over contamination of PCR products
in post-PCR steps, e.g. agarose gel electrophoresis. The quantitative PCR techniques
can be divided into two types based on the fluorescent principle: non-specific
fluorophore without probe, e.g. SYBR Green I (Paper IV), and fluorophore labelled
specific probe, e.g. TaqMan chemistry (Holland et al., 1991) and Molecular Beacon
chemistry (Tyagi & Kramer, 1996).
2.4.4 Isothermal Amplification
Amplification via PCR requires a precise thermal cycler to control the temperature
changes in the different steps of every run. In contrast, isothermal amplification
15
only uses one temperature for the entire amplification reaction without thermal
cycling steps. This facilitates its operation and is beneficial for on-site detection
systems. Isothermal amplification was investigated as a diagnostic approach at the
beginning of the 1990s (Guatelli et al., 1990). There are various isothermal methods
available, such as rolling cycle amplification (RCA) (Fire & Xu, 1995; Larsson et al.,
2004; Lizardi et al., 1998) and helicase-dependent amplification (HDA). RCA is an
amplification technique that uses one primer and circular DNA. HDA uses a
helicase to unwind double-stranded DNA for two primer annealing (Chang et al.,
2012).
2.4.5 DNA Microarrays
DNA microarrays are a high-throughput DNA detection technology based on the
hybridization of DNA libraries and PCR products which can screen many
thousands, even millions of features from one or more sample(s) simultaneously
(Nelson & Cox, 2013). Therefore, this technology has dramatically improved DNA
identification in many different fields, such as gene expression, genotype
determination, SNP analysis, medical diagnosis, environmental monitoring,
forensics, and evolutionary studies.
2.4.5.1 Background
The basis of the present array technology was established during the late 1970s to
1990s through experiments on dot blots and reverse dot blots (Southern, 2001).
With the development of DNA hybridization techniques (e.g. Southern blots and
DNA cloning), the first progress toward a microarray technology was achieved by
Hoheisel et al., who printed a high density of clones on filters by robotics and used
an imaging method to measure the parallel signals (Hoheisel et al., 1994). The
principles of DNA microarrays were introduced in 2.4.1, i.e a fluorescence- or
biotin-labelled target (Papers I, II) or probe hybridizes to a complementary probe
(Papers I, II) or target immobilized on a solid surface (Figure 4), and the amount of
target sequences is evaluated from the particular signal intensity, e.g. fluorescent
signal intensity.
2.4.5.2 Classification Based on Spatial Distribution of Support Surfaces
Hybridization between the immobilized probe and target oligonucleotides takes
place on a solid support surface, which may be either two or three dimensional.
2.4.5.2.1 Two-Dimensional Support Surface
Many different two-dimensional support materials have been investigated for DNA
detection, such as nitrocellulose and nylon membranes (Conner et al., 1983;
Meinkoth & Wahl, 1984; Saiki et al., 1989), polystyrene (Rasmussen et al., 1991),
polypropylene (Matson et al., 1994), and glass (Maskos & Southern, 1992). To
16
achieve a suitable support for oligonucleotide detection, several requirements have
to be considered regarding the support properties, such as the material stability and
complexity, amenability to chemical modification, scattering and non-specific
background fluorescence, loading capacity and cost (Guo et al., 1994).
2.4.5.2.1.1 Glass Surfaces
2.4.5.2.1.1.1 Development
Compared to various other supports, glass quickly became commonly accepted as a
microarray material as it met all the above needs (Guo et al., 1994; Southern, 2001).
On microscope glass slides, tens of thousands of probes can be printed in one spot
for one target detection. In 1991, Maskos developed the first array on glass to
analyze nucleic acid sequences (Maskos, 1991). In 1995, Schena et al. reported gene
expression profiling on a glass microarray (Schena et al., 1995). The first eukaryotic
genome analysis on a microarray was published in 1997 by Lashkari et al. (Lashkari
et al., 1997).
2.4.5.2.1.1.2 Support Surface Treatment for Glass Slides
In microarray techniques, support surface treatment is crucial. The goal of support
surface treatment is to attach oligonucleotides efficiently and stably by coupling
chemistry via a suitable linker. There are several criteria for an ideal linker: the
linkage should be stable, sufficiently long to avoid steric effects from the support,
sufficiently hydrophilic and provide specific binding to the support (Guo et al., 1994).
Isothiocyanate and epoxysilane are applied to activate glass slides and provide
functional groups to bind the oligonucleotide targets with their modifications (Guo
et al., 1994; Lamture et al., 1994). Amino groups are routinely used to modify
oligonucleotides to bind the functional groups of linkers. In this way,
oligonucleotides can be immobilized onto isothiocyanate-coated or epoxysilane-
derivatized glass slides (Figure 4).
2.4.5.2.1.1.3 5’ Amino Modifiers
An amino group can be added to the 5’ terminus, 3’ terminus or an internal part of
DNA. To minimize steric and electrostatic interactions, amino groups need to be
converted to amino-modifiers, which are amino groups with a couple of carbon
spacers (e.g. amino-C6 and amino-C12) to create a distance between the
oligonucleotides, ligands and surfaces. The optimal distance between the
oligonucleotide and surface depends on the goals of the study. Sometimes, an even
longer spacer is needed than an amino-C12 modifier (Integrated DNA
Technologies). For example, in Papers I and II, a polyT spacer was integrated into
each surface probe between amino-C6 and oligonucleotides-NH2. This section
focuses on 5’ amino modifiers. A variety of amino-modifiers can be added to the 5’
terminus of oligonucleotides in the last step of oligonucleotide synthesis because
17
the synthetic direction is from 3’ to 5’.
Figure 4 Functionalization of a solid support and the principle of microarray.
1. Support surface modification. 2. Covalent binding of functional linker to surface modifiers. 3.
Immobilization of molecules by covalent binding to functional groups of linkers. 4. Detection of
fluorophore-targets or bead-targets via interaction between target molecules and immobilized
molecules, e.g. oligonucleotide hybridization.
2.4.5.2.1.1.4 Attachment of 5’ Amino-Modified Oligonucleotides
An acylating reagent is needed for the immobilization of amino-modified
oligonucleotides, such as isothiocyanate. Carbodiimide is another common acylating
agent for amino-modified oligonucleotide attachment, which finally generates an
amine carbonyl group. The kinetics of the reaction between an acylating reagent and
an amine is strongly pH dependent, with an optimal rate at around pH 8.5-9.5
(Papers I, II). 5’ amino-modified oligonucleotides can also covalently attach to
epoxysilane-derivatized glass (Lamture et al., 1994).
2.4.5.2.1.1.5 Fabrication: Spotted or In Situ Synthesized Microarrays
If the probes are printed onto the glass surface, there are two main ways to position
them onto the solid array surface using a robotic printer: either by direct contact or
with an ink-jet method (Goldmann et al., 2000). If the probes are synthesized in situ
on the array surface, conventional phosphoramidite chemistry (Beaucage &
Caruthers, 1981) and photolithography (Fodor et al., 1991) can be used. Both
methods employ protective groups during the cycled reactions. In photolithographic
synthesis, phosphoramidite chemistry is used with photolabile reagents. (Fodor et al.,
1991).
2.4.5.2.1.2 Silicon Chips
18
The demand for high-density microarrays led to the development of gene chip
technologies in the 1990s (Fodor et al., 1991), which now occupy a dominant
position in the field (Affymetrix, Santa Clara, CA). In this technique, oligonucleotide
probes are synthesized on the surface of quartz wafers directly by semiconductor-
based photochemical synthesis. Such chips represent a significant improvement over
the density of printed probe microarrays (about 10,000 to 30,000 features) as more
than 1 million synthetic probes can be synthesized on an array of around thumbnail
size (Dalma-Weiszhausz, 2006; Miller & Tang, 2009).
In the in situ array synthesis technique of Affymetrix GeneChips, three principal
steps are needed: masking, optical deprotection and coupling of suitable nucleotides
in each synthetic cycle. Linkers with light-sensitive protected groups are attached to
the surface of a quartz wafer. Photolithographic masks are carefully set according
to the probe sequences. The masks determine the activated positions on the gene
chip in each cycle. Upon exposure to UV light, the linkers become deprotected and
free hydroxyl groups are created suitable for the attachment of nucleotides labelled
with light-protecting groups. The probes are synthesized by repeating these basic
steps (Dalma-Weiszhausz et al., 2006; Fodor et al., 1991; Miller & Tang, 2009). In the
in situ array synthesis technique of Roche NimbleGen (Madison, WI), digital masks
produced by programmable micromirrors take the place of the masks in Affymetrix
GeneChips, thereby this technique is called markless array synthesizer technology
(Miller & Tang, 2009).
2.4.5.2.1.3 Paper Chips
Paper mainly comprises cellulose fibres forming a porous structure, which offers a
large surface area and high penetrating capacity to fluids without the need for
pumping. These properties of paper are of interest not only for academic research
but also for industrial applications as paper-based detection has a huge potential
commercial value. Paper chips can be fabricated with functional components during
the process of manufacturing or can be printed or coated with functional chemicals
by spotting or printing after manufacturing. Many applications of different bioassays
have been investigated, such as pathogen diagnosis, water and food quality
monitoring (Aikio et al., 2006; Hong et al., 2008; Martinez et al., 2010; Zhao et al.,
2008).
2.4.5.2.1.3.1 Paper Test Strips
The objective of paper tests is to create a low-cost, easy-to-use, accurate and rapid
test method. The development of paper test strips dates back to the 19th century.
The first paper detection of sugar and albumin was reported in 1883. Spot test
chemistry based on the capillary action of filter paper was pioneered by an Austrian
professor, Fritz Feigl, in around the 1920s (Voswinckel et al., 1994), later described
as the “hour of birth of the modern test strip” (Voswinckel et al., 1994). Martin &
19
Synge reported a new form of chromatography theory in 1941 (Martin & Synge,
1941). The theory was exploited to separate mixtures of amino acids on a cellulose
filter paper strip by Gordon, Martin & Synge in 1943. This was the first report that
combined the use of cellulose and chromatography theory to separate molecules
(Gordon et al., 1943). Up until 1956, filter paper chromatography and
electrophoresis were utilized routinely (Comer, 1956). The concept of paper or
paper-like strip tests was investigated for pregnancy detection, and Margaret Crane
invented the first immunochromatography-based pregnancy test kit in 1968 (US
patent 3579306). Studies into immunomigration by capillary action followed in the
late 1970s and early 1980s by examining antigen-antibody interactions on a porous
material, e.g. filter paper (Glad & Grubb, 1977; Glad & Grubb, 1978; Glad & Grubb,
1981).
2.4.5.2.1.3.2 Bioactive Filter Paper Detection
The term “bioactive paper” emerged in the early 21st century with developments in
paper science, biochemistry, biotechnology, microbiology and pathogen diagnostics,
and was initiated by researchers in Canada with the aim of improving global public
health and developing a new paper fabrication process (Pelton, 2009).
Different ways to functionalize the surface of filter papers have been investigated,
e.g. physical adsorption and chemical covalent modification. The functionalized
filter papers have enabled the development of biosensors (DNA probes, antibodies,
enzymes, etc.) to bind the surface of these papers to create bioassays (e.g. pathogen
detection in food and environmental sciences) (Su et al., 2007). Studies into
(bio)paper-based microfluidic devices have also been carried out, e.g. flow channel
creation (Carrilho et al., 2009; Jahanshahi-Anbuhi et al., 2012; Martinez et al., 2007)
and reaction process control (Fu et al., 2010; Fu et al., 2011). One of the biggest
challenges in the development of a bioactive paper assay is how to attain an
immediate, sensitive and specific readout, which can ideally be identified by the
naked eye rather than requiring complex instrumentation. This challenge was
addressed in Paper II.
2.4.5.2.2 Three Dimensional Support Surface
Compared with glass microarrays or silicon chips, microbeads are ideal supports
because their surfaces are distributed in three dimensions, which provides an
opportunity to increase the concentration of oligonucleotides in the same space
compared to glass surfaces. Currently, there are several kinds of microbeads in use.
2.4.5.2.2.1 Microbeads (Illumina): Silica Beads
Silica beads of 3.4 m diameter have been assembled onto different substrates to
form high density bead arrays for use in the Illumina systems: the Sentrix Array
Matrix (SAM) or the Sentrix BeadChip. In the SAM system, there are 96 fibre-optic
20
bundles (1.4 mm diameter each), with 50,000 fibre-optic strands in each bundle.
Every single fibre forms a microwell via microelectromechanical technology. A
single bead can be loaded into each microwell (Fan, 2005; Fan, 2006; Miller & Tang,
2009). The BeadChip system can assay 1 to 24 samples at present
(www.illumina.com). The beads in the arrays randomly self-assemble into their final
locations. Thus, a “decoding” step is needed to map the bead positions (Gunderson
et al., 2004). About 700,000 identical capture oligonucleotides, as unique barcodes,
are attached to each bead to identify every bead address (Kuhn et al., 2004). The
decoding process provides quality control for intermicroarray data comparison
(Bibikova et al., 2006; Miller & Tang, 2009). Bead arrays have been used in different
applications, including SNP genotying (Fan, 2003; Gunderson, 2009; Parida, 2012),
gene expression profiling (Abramovitz et al., 2011; Fan et al., 2004; Kuhn et al., 2004)
and DNA methylation studies (Bibikova et al., 2006; Bibikova & Fan, 2009; Triche et
al., 2013).
2.4.5.2.2.2 Microbeads (Luminex): Polystyrene Beads
Polystyrene beads have been utilized in a suspension bead array, i.e. a liquid phase
array. The microbeads are separated according the different intensity peaks in their
emission spectra, red (658 nm emission) and infrared (712 nm emission). For DNA
detection using this technique, the Luminex company has developed a set of 100
fluorescent polystyrene microbeads with two spectrally distinct fluorophores. Each
unique group of probes is attached to one group of the beads with the same red-
to-infrared fluorescent intensity. Thus, in principle, 100 different analytes can be
assayed simultaneously in one single reaction (xTAG; Luminex Molecular
Diagnostics, Inc., Toronto, Canada). The microbeads can be detected and analyzed
individually with two lasers. A 635 nm laser excites the two fluorochromes and
shows the locations of the beads. A 532 nm laser excites and reports the presence
of the fluorochromes (i.e. phycoerythrin or PE) for analyzing and quantifying the
hybridization. This capacity for high-throughput detection makes this technology
attractive for clinical infectious disease detection. The first suspension bead array to
achieve FDA certification for infectious disease detection (Luminex, xTAG RVP)
obtained it in 2008 (Krunic et al., 2007; Merante et al., 2007; Miller & Tang, 2009).
2.4.5.3 Microfluidic DNA Microarrays
DNA microarray techniques have accelerated advances in molecular biology and are
still evolving in the life sciences due to the development of associated technologies,
such as the microfluidic technology. The combination of DNA microarrays and
microfluidic techniques provides an efficient method for analyzing minute volumes
or amounts of material in a short time. Samples with volumes as small as picolitres
can be handled in a microfluidic system (Liu et al., 2006; Wang & Li, 2011), compared
to the microlitre volumes handled in DNA microarrays. Hence, the microfluidic
techniques increase the target DNA concentration in microchannels. Moreover, they
21
reduce the diffusion distance, thereby improving the hybridization kinetics because,
in a limited time and distance, the increased target concentration enhances the
chance of hybridization between the target DNA and probes printed on the
substrate surface in the microchannels (Wang & Li, 2011). Recently, based on
microfluidic technique, scientists pursue an efficient and easy parallel array
hybridization on a compact disc (CD) or digital video disc (DVD) surface. This
technology unites the principles of microarray, microfluidic, centrifugal force and
CD/ DVD technologies and offers the prospect of developing a microfluidic DNA
microarray platform based on a portable lab-on-chip/DVD system (Lange et al.,
2005; Madou et al., 2006; Nolte, 2009; Potyrailo et al., 2006; Ramachandraiah, 2013)
2.4.6 DNA Sequencing
A DNA molecule stores, transfers and expresses the genetic information precisely
through the order of deoxynucleotides. DNA sequencing is a process for identifying
the deoxynucleotide order within a DNA molecule, namely, the order of the four
bases A, C, T, G.
2.4.6.1 Classical Sequencing Technology
In the late 1970s, two new DNA sequencing techniques were introduced: the DNA
chemical cleavage approach developed by Maxam & Gilbert (Maxam & Gilbert,
1977) and the “chain-terminating inhibitors of DNA polymerase” (Sanger et al.,
1977). Both methods applied the techniques of DNA terminal labelling, separation
and detection, and utilized a radioisotopic reporter to identify the four bases,
generated by four reactions in four separate lanes of one gel. The Sanger method is
still in use nowadays because of its ease-of-use, ability to sequence long pieces of
DNA, low toxicity and amenability to automation (Devlin, 2005; Nelson & Cox,
2013; Sanger et al., 1977).
2.4.6.1.1 Maxam and Gilbert’s Sequencing
Maxam and Gilbert’s sequencing technique requires a DNA molecule terminally
labelled with radioactive 32P. Four cleavage reactions targeting four specific base
combinations (G, A+G, C+T, C) within the same DNA molecule are employed to
generate different sized DNA fragments, which can be separated by gel
electrophoresis to identify the order of bases. The technique can be described by
four steps: (1) Methylation of purine destabilizes the corresponding glycosidic bond.
Thus, in this method, G is methylated by dimethyl sulphate, followed by heating to
break the glycosidic bond to release the bases, and then alkali treatment to cleave
the sugar from the phosphate groups. G methylation is faster than that of A. (2)
Cleavage of methylated A. A gentle treatment with diluted acid is performed to
release A but not G because the glycosidic bond of methylated G is more stable
than that of A, then alkali treatment cleaves the sugar. (3) Hydrazine hydrolyzes
22
pyrimidine (C+T). (4) Hydrazine cuts C only in the presence of sodium chloride
(NaCl), which prevents cleavage of T (Maxam & Gilbert, 1977).
2.4.6.1.2 Sanger Sequencing
Sanger sequencing is based on a chain termination technique during DNA synthesis.
In this system, 2’,3’-dideoxynucleoside triphosphate (ddNTP) is included besides
the DNA template, sequencing primer, DNA polymerase (I) and four different 2’-
deoxynucleoside triphosphates (dNTPs). The structure of ddNTP is similar to
dNTP except for the group on the 3 carbon of a pentose, where a hydroxyl group
is replaced by a hydrogen atom in ddNTP. Therefore, ddNTP is not able to form a
phosphodiester bond with another dNTP. Thus, when ddNTP is incorporated into
the growing chain instead of dNTP, the extension of DNA is terminated, resulting
in different lengths of DNA fragments where the terminating (last incorporated)
nucleotide is known (Sanger et al., 1977).
The classical procedure of Sanger sequencing is as follows: (1) Obtain a purified
single-stranded recombinant DNA target. (2) Generate a reaction mixture
composed of DNA templates, the DNA primers, all four dNTPs, and then divide
this mixture into four tubes, where each tube contains a unique radiolabelled ddNTP.
(3) Primer extension is performed using DNA polymerase, and ddNTP is
incorporated into the newly synthesized chain by chance, instead of the
corresponding dNTP. (4) After denaturation, a polyacrylamide/urea gel is used to
read the sequence by X-ray autoradiography in four individual lanes, one for each
nucleotide (Sanger et al., 1977).
2.4.6.2 The First Generation Sequencing Technology
The first semi-automated sequencer, the ABI 370a, was manufactured and sold
commercially by Applied Biosystems (AB) in 1987 based on the Sanger method,
using fluorescent dye labelling and detection techniques (Smith et al., 1986). The
sequencer can analyze different nucleotides labelled with four different fluorophores
in a single polyacrylamide gel lane. In the same year, based on a set of ddNTPs
labelled with four distinct fluorescent dyes, Prober and his colleagues at Du Pont
developed an automated sequencing system (Prober et al., 1987). These four labelled
ddNTPs can be used in a single reaction mixture of DNA amplification, and the
products can be separated in one lane of a polyacrylamide gel. The nucleotides can
be identified by the unique signature of fluorescence binding to each different
ddNTP (Prober et al., 1987). This technology was sold to AB after a short time of
commercialization by Du Pont (Dovichi & Zhang, 2000). To simplify the sequencing
technique, enhance signal resolution and achieve higher throughput, a capillary array
electrophoresis technology (CAE) was developed (Dovichi & Zhang, 2000;
Swerdlow et al., 1990; Swerdlow & Gesteland, 1990). The first application of CAE
in DNA sequencing was reported in 1990 (Zagursky & McCormick, 1990) and was
23
improved by adding a sheath-flow cuvette (SFC) (Dovichi & Zhang, 2000; Kambara
& Takahashi, 1993), which became commercially available as the model 3700 DNA
sequencer from AB in the late 1990s (Dovichi & Zhang, 2000).
2.4.6.3 Next-Generation Sequencing Technology
The automated Sanger technique significantly helped the development of genomics
and genetics. For example, the HGP was accomplished by this method. However,
how to increase sequencing throughput, reduce its cost, and shorten its time are still
pressing issues, both for research and industrial applications. The technologies
termed “next generation sequencing” (NGS) or “massively parallel sequencing”
were developed during the mid to late 1990s. They consist of DNA library
preparation, sequencing on NGS platforms, base calling (image analysis), and data
analysis (bioinformatics). In this thesis, the major technologies of DNA library
preparation, especially fragmentation technologies, and sequencing strategies based
on the current major NGS platforms are described.
2.4.6.3.1 DNA Library Preparation
As the starting point of massively parallel sequencing technologies, the quality of
chosen DNA library affects the quality of sequencing data. Thus, each step in DNA
library preparation is important. The early concept of a DNA library was utilized in
the technologies of DNA cloning (see 2.4.2) and traditional sequencing, which need
cloning vectors. However, on NGS platforms, the cloning vectors have been
eliminated. DNA library preparation is usually addressed in similar ways, involving
DNA fragmentation, end repair, phosphorylation, adenylation, adapter ligation and
sequencing template enrichment.
2.4.6.3.1.1 DNA Fragmentation
The overall goal of DNA fragmentation is to obtain a uniformly sized region of
genomic DNA fragments suitable for unbiased sequencing. Non-fragmented or too
long sections of DNA will not be processed further (Paper III; Solonenko &
Sullivan, 2013). An ideal fragmentation method usually has the following properties
(particularly important for genome or de novo sequencing): (1) It initiates random
DNA breaks, independent of sequence. (2) It can create a broad DNA fragment
range (e.g. from a couple of hundred to thousands of base pairs). However, under
each specific condition, the size distribution of the desired fragments should be
narrow. (3) The volume and quantity of each DNA sample are flexible. (4) DNA
repair is not required. (5) Its work flow is simple and can be performed at room
temperature. (6) It has high throughput. (7) It is fast (from seconds to minutes). (8)
It is reproducible. (9) It is inexpensive. (10) It can be automated (Paper III; Surzycki,
1990). At present, the most widely used fragmentation methods include sonication,
focused acoustic shearing, nebulization and enzymatic reactions. Each method has
its own advantages and disadvantages.
24
Sonication of a DNA solution employs a sound wave forcing the two ends of a
DNA molecule to move in opposite directions. Eventually, the DNA molecule
breaks. The process continues until the fragments cannot be broken any further.
Thus, it is a type of hydrodynamic shearing. The maximum shearing energy occurs
around the centre of the DNA molecule (Bankier et al., 1987). Sonication is a
random approach for shearing DNA molecules, which takes place without any
observable inherent bias. However, the unfocused acoustic energy used in this
technology generates heat energy, which causes thermal damage to molecules and
reduces the shearing efficiency (Quail, 2010). Adaptive focused acoustic shearingTM
(AFA) technology is an improvement to the conventional sonicator. In this
technology, the ultrasonic acoustic energy is focused on a small zone, e.g. sample
glass tubes, at high intensity and short wavelength to cause acoustic cavitation that
can shear DNA efficiently at isothermal temperature. Therefore, compared to
conventional sonication, AFA technology results in a narrower size distribution of
the fragmented DNA. Covaris Inc. markets AFA instruments (Quail, 2010).
However, the high cost of instrumentation and limited volume and amount of
DNA that can be sheared in one tube are disadvantages of this technique (Paper
III). It was also recently reported that acoustic shearing can introduce artifacts
(Costello et al., 2013).
Nebulization is another mechanical method for randomly shearing DNA by
compressing air or gas to atomize a DNA solution. In this process, bubbles formed
at the DNA solution surface help to position DNA molecules into two flow layers
with different velocities. Thus, DNA molecules break in the region of maximum
shearing force. The size of DNA fragments is inversely proportional to the gas
pressure and flow velocity, and proportional to the size of the bubbles (Surzycki,
1990). However, the low throughput and recovery rate of DNA necessitate
improvements to this technology and the development of new technologies. (Quail,
2010; Quail et al., 2008; Solonenko & Sullivan, 2013).
Enzymatic reaction can refer to endonuclease digestion, e.g. DNase I (Pihlak et al.,
2008) and NEB fragmentase (V. vulnificus nuclease and T7 Endonuclease)
(Linnarsson, 2010). To simplify DNA library preparation and minimize the input
DNA yield, Epicentre Biotechnologies has developed the Nextera technology
(Epicentre, Madison, WI, USA) using an engineered transposon and transposase
complex. This technology incorporates random fragmentation and sequencing
adapter insertion into one step, followed by PCR for template enrichment. The
library can be constructed from nanogram quantities of DNA. However, the
insertion bias, resulting from the preference of transposon insertion site, can result
in incomplete sequencing (Adey et al., 2010; Green et al., 2012; Marine et al., 2011,
Paper III).
25
2.4.6.3.1.2 Finalizing Library Preparation
Following fragmentation, three main steps remain: (1) end repair, which aims to
produce blunt ends of the DNA fragments by removing the 3’ overhang and
extending the strand at the 5’ end; (2) adapter ligation, in which universal adapters
are joined to both blunt ends of DNA fragments for downstream template
enrichment and sequencing (to make ligation possible, phosphorylation and
adenylation are performed to produce a phosphate group at each 5’ end of
fragments and an A overhang at each 3’ end of fragments); and (3) enrichment of
sequencing templates by PCR, using the adapters as primers to produce a sufficient
amount of DNA for sequencing.
2.4.6.3.2 NGS Strategies
Different sequencing strategies have been developed based on NGS platforms.
Pyrosequencing (Roche/454), cyclic reversible termination sequencing
(Illumina/Solexa), sequencing by ligation (Life Technologies/SOLiD), single
molecule sequencing (Helico Biosciences and Pacific Biosciences) are mainly
discussed here.
2.4.6.3.2.1 Pyrosequencing (Roche/454)
Pyrosequencing is a method of sequencing by synthesis (SBS). The principle is to
evaluate the order of nucleotides by monitoring flashes of light that originate from
pyrophosphate (PPi) release, ATP generation and photons emission during the
process of DNA synthesis. This method was introduced in 1998 by Nyrén, his PhD
student Ronaghi and their colleagues (Ronaghi et al., 1998).
In 2005, Rothberg and his colleagues integrated the technologies of pyrosequencing,
bead-based emulsion PCR (emPCR), and picolitre-volume wells to develop the 454
sequencing platform. 454 sequencing significantly improved the sequencing field, as
its throughput was 100-fold higher than the automated Sanger sequencing of that
time (Margulies et al., 2005). It is suitable for de novo sequencing and metagenomics
studies because of its long read length capacity (up to 800 nt). The major challenge
of this technique originates from the pyrosequencing technology. In
homopolymeric stretches, it is difficult to determine the accurate number of
identical nucleotides due to nonlinearity between the signal intensity and length of
homopolymer for numbers greater than five (Ronaghi, 2001; Ronaghi et al., 1998).
Deletions and insertions are the major error types on this sequencing platform
(Metzker, 2010; Xuan, et al., 2013).
2.4.6.3.2.2 Cyclic Reversible Termination Sequencing (Illumina/Solexa)
Modified dNTPs can terminate enzymatic DNA synthesis. As exemplified above by
Sanger’s chain termination sequencing using ddNTP terminators. Cyclic reversible
26
termination sequencing (CRTS) is an SBS technique that uses reversible terminators
labelled with a fluorophore. The procedure includes repetitive cycles of nucleotide
incorporation, imaging and modified group cleavage. There are two types of
reversible terminators: 3’-blocked terminators and 3’-unblocked terminators. The
former has blocking groups attaching to the 3 positions of pentoses, which are
cleaved to restore a free 3’-OH for reaction with the nucleotides added after
fluorescence imaging. The 3’-OH groups of the latter are intact. However, different
chemicals can be attached to the bases, e.g. blocking groups, dNTP analogues or
large dye groups, to hinder the new CRT cycle until they are removed (Metzker,
2010 and references reviewed therein).
Work on CRT in sequencing started in the 1990s. For example, in 1994, two
independent studies were published, showing that 3’ protected dNTPs could play a
reversible terminator role in enzymatic DNA synthesis (Canard & Sarfati, 1994;
Metzker et al., 1994). Balasubramanisn & Bentley, Solexa company founders, applied
the CRT method based on 3’-blocked terminators to propose their sequencing
device in 2001 (WO 01/57248 A2). In 2006, Solexa Inc. launched the first Solexa
sequencer, the Genome Analyzer (GA), with 1 Gb output per run. Since 2007,
Illumina has acquired Solexa and gradually started to dominate the sequencing
market.
In the Illumina/Solexa SBS system, bridge PCR (Bing et al., 1996) is utilized to
prepare templates on a solid surface, e.g. glass slide, (instead of emPCR on the 454
platform). Bridge PCR is a method to amplify targets by immobilizing primer pairs
on a derivatized solid support. The process involved in the Illumina/Solexa SBS
consists of DNA library preparation, formation of template clusters by bridge PCR
and CRTS by DNA synthesis with four different fluorescently-labelled dNTPs. The
throughput of Illumina SBS has been dramatically increased compared to
conventional technologies. The limitations of this technology were the relatively
short read lengths (up to 2 × 150 bp) and amplification bias, especially in AT-rich
and GC-rich regions. The most common error type is substitutions (Metzker, 2010;
Xun et al., 2013). Recent product developments have increased the read length (up
to 2 × 300 bp for the MiSeq facility) and maximum output is 600 Gb/run (for HiSeq
2500 system) (www.illumina.com).
2.4.6.3.2.3 Sequencing by Ligation (Life Technologies/SOLiD)
Sequencing by ligation (SBL) is a sequencing technology based on cyclic dye-labelled
oligonucleotide ligation in the presence of a nucleotide template. DNA ligase is the
key enzyme, as opposed to DNA polymerase in SBS (Landegren, 1988; Merzker,
2010). In 2005, Shendure et al. described sequencing results for E. coli MG 1655 by
SBL technology. Their improvements to this technology were later incorporated
into the Polonator instrument (Merzker, 2010; Shendure et al., 2005). In 2008,
27
oligonucleotide ligation and detection (SOLiD) sequencing technology based on
probes encoded by two bases was reported by researchers at Stanford University
and AB (Life Technologies), which was later commercialized by Life Technologies.
The main advantage of this technology is the accuracy, whereas the main drawback
is short read lengths (less than 100 bp). The common error type in this technology
is substitutions due to dark nucleotide incorporation or delayed removal of the
fluorescent label (Merzker, 2010; Valouev, 2008; Xuan et al., 2013).
2.4.6.3.2.4 Single Molecule Sequencing (Helicos BioSciences and Pacific
Biosciences)
In pursuit of longer read length with retained data accuracy and without added
experimental complexity, single molecule sequencing (SMS) technology was
developed. This technology employs DNA polymerase, fluorescence-labelled
dNTPs, primers and templates, and therefore is also SBS. However, it is an
amplification-free process. Thus, the method of sample preparation is simplified,
the cost is reduced and there is no amplification bias (Milos et al., 2009). The first
SMS platform was made available by the Helicos BioSciences Corporation in 2008.
In this system, 3’-unblocked reversible terminators and flow cells are used. The
oligonucleotides immobilized on the flow cells are either primers or single-molecule
templates. However the current system needs to be improved in terms of the read
length (about 32 nucleotides) and high error rate (more than 5%) (Schadt et al., 2010;
Xuan et al., 2013). In 2009, the first report about single molecule real time
sequencing (SMRT) was published by Eid et al., and the technique was later
commercialized by Pacific Biosciences in 2011. The SMRT platform incorporates a
range of technologies - immobilization of phi29 DNA polymerase binding a single
template molecule, a zero-mode waveguide (ZMW) detector and four different
phospholinked nucleotides - to achieve real-time monitoring of the process from
nucleotide incorporation to imaging (Eid et al., 2009). SMRT has great potential
because of its unique technical features, in particular its capacity for a long read
length (around or more than 1 kb) (Bashir et al., 2012; Metzker, 2010; Rasko et al.,
2011; Travers et al., 2010) and its real-time monitoring. In summary, this technology
is suitable for de novo genome sequencing (Bashir et al., 2012) and DNA methylation
detection (Fang et al., 2012). However, the high error rate (more than 5%) on the
current platform is still a challenge (Bashir et al., 2012; Merzker, 2010; Xuan et al.,
2013).
28
3. Specific Aims of the Studies
3.1 Paper I Activated Paper Surfaces for the Rapid Hybridization of DNA
through Capillary Transport.
Ana Catarina Araújo, Yajing Song, Joakim Lundeberg, Patrik L. Ståhl, and Harry Brumer,
III. (2012). Anal Chem. 84(7): 3311-3317.
In pursuit of a cost-effective, rapid and user-friendly candidate for a microarray sup-
port and to establish a forensic analysis array model to detect human and canine
samples, a cellulose filter paper treatment was developed comprising five steps: (1)
generation of an aminated surface on cellulose filter paper; (2) functionalization of
the surface of cellulose filter papers with phenylenediisothiocyanate (PDITC); (3)
immobilization and robotic printing of synthetic NH2-oligonucleotides by covalent
bonds between amino groups and thiocyanate groups; (4) detection of synthetic ol-
igonucleotides/PCR products labelled with Cy3 that are completely or partly com-
plementary to the surface polynucleotides; (5) analysis of double strand DNA for-
mation on the functionalized surface of the filter papers using a scanner.
3.2 Paper II Visual Detection of DNA on Paper Chips.
Yajing Song, Peter Gyarmati, Ana Catarina Araújo, Joakim Lundeberg, Harry Brumer, III,
Patrik L. Ståhl. (2014) Anal Chem. 86(3): 1575-1582.
To further simplify DNA detection as well as to lower costs, we aimed to develop
the concept of visual DNA on activated filter paper chips. Superparamagnetic beads
were used to label ssDNA complementary to printed probes on the surface of paper
chips. The natural brown colour of the beads enabled DNA to be detected directly
without the need for any complex equipment. Moreover, the other main objective
of this work was to increase the multiplexing capacity of detection on the paper
chips to facilitate a potential commercial development.
3.3 Paper III Chemical Fragmentation for Massively Parallel Sequencing Li-
brary Preparation.
Peter Gyarmati, Yajing Song, Jimmie Hällman, Max Käller. (2013) J Biotechnol. 168(1): 95-
100.
In this study, we pursued an easy-to-use, automatable and cost-effective method of
DNA fragmentation for massively parallel sequencing, which would produce
continuously unbiased data. The proposed method fragments DNA via hydroxyl
radicals generated by the iron-EDTA method, and can be carried out at room
temperature without any instrumentation or limitations of DNA volume or/and
quantity. This technology could be suitable not only for NGS platforms but also for
metagenomics and clinical studies, such as on infectious diseases and cancer. Thus,
29
we also planned to use this method in Paper IV.
3.4 Paper IV Nuclease-Assisted Suppression of Human DNA Background
in Sepsis.
Yajing Song, Christian G. Giske, Patrik Gille-Johnson, Olof Emanuelsson, Joakim Lundeberg,
Peter Gyarmati. Manuscript.
To address the current challenges of BSI diagnostics - the time required for
diagnosis, the limited detectable range of pathogens, and the high false negative rate
- we aimed to develop an early, sensitive and accurate diagnostic method of BSI by
means of a molecular biological technology (Q-PCR). The method has potential to
guide BSI clinical therapy, save patients’ lives and avoid antibiotic misuse. We
intended to use a duplex-specific endonuclease (DSN) and duplex-specific
exonuclease (BAL 31) to digest the abundant human DNA background and enrich
low copies of the pathogens in a BSI model and in samples from septic patients.
The chemical fragmentation method in Paper III was going to be utilized to enhance
the efficiency of the enzymatic digestion and the hybridization.
30
4 Results and Discussion
4.1 Paper I
4.1.1 Activation of Cellulose Filter Papers
Cellulose is an easily available, cheap and abundant material. In pursuit of paper-
based DNA detection, one important challenge is how to solve the low affinity
between nucleotides and cellulose. For silicate glass slides, a general strategy is to
generate amino groups on the surface of supports by treatment with an
aminoalkyltrialkoxysilane, such as aminopropyltriethoxysilane (APTES), followed by
coupling amine-reactive functional linkers, e.g. PDITC, which provide functional
groups for the covalent immobilization of amino-modified captured targets, e.g.
NH2-ssDNA (Guo et al., 1994). This strategy was employed in this study.
Xyloglucan-NH2 (XG-NH2) was used to aminate the surface of filter papers, PDITC
with two thiocyanate groups was then added to functionalize the surface of the
cellulose filter papers by reaction with the amino groups. Finally, synthetic NH2-
oligonucleotides (ID-tag) were immobilized onto the surface of filter papers
covalently by reaction between the surface thiocyanate groups and the synthetic
oligonucletide amino groups.
This work showed that treatment with PDITC was an effective way to functionalize
the surface of cellulose. The efficiency of functionalization for PDITC in DMSO
was better than that in DMF. Treatment with XG-NH2 helped decrease the PDITC
concentration required to give the same intensity of fluorescence as with filter paper
(FP) in DMSO. There was no observable intensity difference between the XG-NH2-
PDITC-FP system and XG-PDITC-FP system at low PDITC concentration in
DMSO.
4.1.2 DNA Detection on Functionalized Papers
The porous structure of cellulose filter paper is responsible for its capillary wicking
ability. It can be used to drive targets to hybridize probes on the surface of cellulose
filter papers when the paper is in contact with the target solution.
4.1.2.1 Synthetic Oligonucleotides
Fluorophore-labelled synthetic oligonucleotides complementary to immobilized
probes were used for detection. Quantitative analysis was performed based on
measuring the fluorescence intensity. The limit of detection in this study was shown
to be approximately 0.2 pmol.
4.1.2.2 Human and Canine Samples
For robotic printing, there were six features in each layout. Four of them identified
four distinct PCR products, the other two were positive and negative controls. Cy3
31
modified the 5 termini of forward primers and ID-tag sequences were integrated
into the 5 termini of reverse primers modified with biotin. After amplification and
denaturation, a Cy3-ssDNA-anti-ID tag was generated and used to detect an
immobilized ID tag sequence on the activated paper matrix. The method succeeded
in detecting the four different targets with high specificity.
32
4.2 Paper II
4.2.1 DNA Visualization
The method described in Paper I was developed in parallel with this work.
Superparamagnetic bead labelled ssDNA was produced instead of Cy3-ssDNA to
base pair with the probes on the surface of cellulose filter paper chips. The iron
oxide core of the beads gives them a brown colour, and therefore DNA can be
detected directly without the need for complex instrumentation, e.g. a scanner. Thus,
DNA visualization on paper chips can be achieved rapidly, making use of the
capillary action of filter papers. Apart from direct visualization of the results with
the naked eye, this assay also enables quantitative measurements, as was
demonstrated in this study.
4.2.2 Inspiration from the Porous Structure of Cellulose Paper Chips
The structure of cellulose filter paper is different from that of glass and plastic. Its
porous structure is three dimensional. Thus, filter paper has a larger surface area
(Hong et al., 2007; Pelton, 2009) and the density of immobilized probes can be
increased compared to glass and plastic. A high density of printed probes makes it
easier to capture target sequences when a solution containing the targets is rapidly
flowed over the printed region of the paper chips. Thus, multiplex detection is
possible with this assay format. In this study, the average density of the printed
probes was increased to 3 pmol/mm2 compared to previous work.
4.2.3 Multiplex Detection
This work aimed to increase the multiplexing capacity of detection on paper chips.
The number of features was increased from six to ten in one array. The printing area
of each feature was increased (from 1 mm2 to 1.7 mm2) as well as the printed probe
density. The paper chips were used to detect one, two, three and four samples
separately.
Two different ways were compared for loading the solution containing the
amplicons: the first was vertical capillary transport, and the second was immersion.
Capillary transport produced strong signals that were identifiable within 90 seconds,
whereas the immersion method only produced weak signals on the same timeframe,
possibly because the paper pores were blocked by flow of solution in multiple
directions.
The detection was operated at room temperature. To ensure the accuracy of
detection, two features were included in the layout to pair with the distinct moiety
of each bead-ssDNA-(anti-internal-probe)-anti-ID-tag target. One feature
comprised synthetic oligonucleotides (ID-tag), while the other one was a section of
amplicon (internal probe). Moreover, these two probes were positioned in different
33
rows and columns to reduce spatial bias.
4.2.4 Parameter Optimization
The optimization process considered the amount of magnetic beads and amplicons.
The ideal printing area on filter paper was also investigated, as well as paper size and
density of printed probes.
4.2.5 Future Applications and Development
At present, DNA visualization on the active paper chips is performed with magnetic
beads. We showed that ten features could be detected simultaneously, but the beads
also generated a background signal. Further work is needed to increase the flow rate
and signal-to-noise ratio, e.g. by increasing the length of the filter paper chips, using
magnetic nanobeads or golden particles and/or creating microchannels on paper
chips. Based on the simple means of visualization, and its accuracy and multiplexity,
the methodology could be developed to detect pathogens and normal bacterial
communities in the human body. The paper chips could also be useful as a simple
test strip or lab-on-chip device in many applications, e.g. low throughput pathogen
diagnostics in hospital, crime scene detection in forensic investigations, screening
populations with risk genes in epidemiology, food monitoring in food safety and
water monitoring in environmental science.
34
4.3 Paper III
4.3.1 Principles of Chemical Fragmentation
The iron-EDTA method used in this study was based on Fenton chemistry: Fe2+-
EDTA4- + H2O2 → Fe3+- EDTA4- + ·OH, namely, redox-active iron reacts with
hydrogen peroxide and dithiothreitol to create hydroxyl radicals, which can cleave
DNA strands efficiently. Cleavage mainly occurs at the hydrogen atoms in pentose.
The bases are also damaged in this process, for instance, by formation of 8-oxoG
or AP sites on the DNA strands.
4.3.2 Importance of DNA Repair
In massively parallel sequencing technology, DNA library preparation usually
includes DNA fragmentation, end repair, phosphorylation, adenylation, adapter
ligation and enrichment of target DNA. In our study, we compared results before
and after DNA repair, and after different repair times. It was shown that DNA repair
was essential between the DNA chemical fragmentation and end repair steps. The
reason for this is that DNA repair can not only restore the sugar-phosphate
backbone but also repair oxidatively damaged bases for downstream adapter ligation
and target DNA enrichment.
4.3.3 Investigation of Fragmentation Bias
Because the lambda genome is small with a length of only 48,502 bp, in this study,
the whole lambda genome library was processed and three different fragmentation
methods were compared: chemical fragmentation, mechanical shearing and
enzymatic fragmentation. The results showed that the coverage regions obtained by
chemical and mechanical fragmentation were continuous and quite similar. However,
the Nextera kit produced a region with read coverage as low as 2. Regarding the
extent of coverage, only the chemically fragmented sample showed ≥100× coverage
for 100% of the target region. It was also shown that chemical fragmentation
sheared DNA randomly. Based on the obtained data, it was concluded that chemical
fragmentation was comparable to established fragmentation technologies and is
therefore suitable for use in NGS library preparation.
4.3.4 Evaluation of Sequencing Artifacts and Errors
Unexpected mutations were investigated by means of ultradeep sequencing on
human -actin amplicons (121 bp) after chemical fragmentation and mechanical
shearing. No mutations were found in both cases and the indel rates showed no
significant differences.
4.3.5 Assessment of Sequence Capture Efficiency
To assess the capture efficiency for chemical fragmentation, exome sequencing of a
human breast cancer cell line was performed and the results with both chemical and
35
mechanical fragmentation were compared. Validation of the SNP identifications in
the two cases did not show significant differences.
4.3.6 Summary
Chemical fragmentation is a simple, rapid, cost-efficient and non-toxic method of
library preparation for DNA sequencing. It contributes to the production of more
complete and less biased data, and its throughput is flexible.
36
4.4 Paper IV
4.4.1 Evaluation of PCR Efficiency
Specific primers of human DNA and E. coli DNA were designed to target highly
conserved genes (1-deoxyxylulose-5-phosphate synthase in E. coli and β-actin in
human). The efficiencies of the two pairs of primers were shown to be high and
similar (R2 > 0.99). The human and E. coli amplicon lengths were also similar to
avoid kinetic differences.
4.4.2 Q-PCR Condition Optimization
Two target DNAs were mixed in a model system simultaneously in two parallel Q-
PCR reactions, to allow the Q-PCR reaction systems to be optimized independently
for each pair of primers. The concentration of human primers in the Q-PCR system
was 400 nm, whereas the concentration of E. coli primers was 200 nm.
4.4.3 A Model Based on Amplicons
To set up a simplified model system for sepsis conditions, a mixture of human and
E.coli amplicons was serially diluted. Based on the ratio of molecules of human and
E. coli DNA in real BSI samples, the different sizes of the human and E. coli.
genomes and limit of Q-PCR detection, the highest ratio of human and E.coli
amplicons was 108. Duplex-specific nuclease (DSN, Evrogen) was used to remove
the abundant human double-stranded amplicons on the basis of Cot effect, leaving
the rare single-stranded E. coli amplicons for downstream PCR reaction. In the
model system, around 105 human amplicons were digested; however, the
concentration of E. coli amplicons was not affected.
4.4.4 Application of the Model on Clinical Samples
4.4.4.1 Experimental Setup
The size of the human genome is approximately 1000-fold longer than that of E.
coli. To increase the digestion and hybridization efficiency, the two genomic DNAs
have to be sheared into a similar size. However, due to the limited volume and
amount of DNA per single reaction in the mechanical fragmentation method, we
needed to develop a new fragmentation method that was much more flexible in the
volume and amount of sheared DNA.
4.4.4.2 Evaluation and Development
Clinical plasma and blood samples were used, all positive for E.coli. The efficiency
of abundant human DNA amplification was reduced by 100 fold after treatment
with double-stranded specific endonuclease-DSN and exonuclease-BAL 31. This is
because the human genome size is 1000-fold larger than that of the E.coli genome.
For a mixture of one human genome and one E.coli genome, theoretically, after
shearing into uniformly sized fragments, the ratio of human and E.coli fragments is
37
around 1000:1. Therefore, the enzymatic digestion efficiency of the genome was
different from that of amplicons in this study.
4.4.5. Future Directions
The presented technique increases the chances of detecting rare DNA molecules
rapidly amid a huge amount of background DNA by means of PCR (e.g. detection
of pathogens in blood). Microarray and sequencing technologies would also benefit
from this nuclease-assisted suppression method.
38
5 Conclusions and Future Perspectives
In this thesis, we developed two methods for visual DNA detection on filter paper
chips and rare DNA detection amid a substantial amount of background DNA. We
also developed a chemical fragmentation method that provides random DNA
cleavage for massively parallel sequencing and BSI pathogen diagnosis.
First, the usability of filter paper as an array surface material was investigated. A
cellulose filter paper was efficiently functionalized with XG-NH2 and PDITC in
DMSO and the activated filter papers were applied to detect DNA via fluorescence
or directly by the naked eye with modified magnetic beads. These visualization
methods showed that filter paper is a good material for DNA array detection.
The presented technique of visual DNA detection on active filter paper provides a
simplified method for DNA detection whilst reducing the associated costs.
Moreover, it offers the possibility of increasing the throughput of DNA array assays
on active filter paper chips.
To exploit the various potential of the presented technique, both integration with
other technologies (e.g. microfluidic devices) and optimization of the parameters
considered in this study (e.g. paper size, printing region, probes density, and
detection approach) are essential. Thus, the results presented in this thesis may help
towards the goal of developing a portable, on-site device that meets the needs of
an automated detection system (Lemieux et al., 2012).
Second, we tried to solve another bottleneck in DNA detection: how to detect rare
pathogen DNA in the presence of abundant human DNA in BSI. The unbiased
chemical fragmentation and double-stranded specific nuclease (DSN and BAL 31)
treatment were applied to a BSI model system. The proposed method achieved 105-
fold suppression of background amplicons in the model and 100-fold suppression
of background DNA in samples from septic patients. This method could help to
diagnose BSI quickly and provide accurate information to guide clinical treatment
so as to reduce BSI mortality (sepsis).
A chemical fragmentation method was developed to address the limitations of
current fragmentation technologies, such as automation, instrumentation, and
sequence bias. The proposed method has many advantages over existing
technologies. For example, it can be performed rapidly at room temperature, it is
inexpensive, easy to use, unbiased and amenable to automation, and importantly, it
is flexible in terms of volume and/or amount of sheared DNA. The chemical
fragmentation could be incorporated into automated NGS library preparation
protocols to further increase throughput, simplify the method and reduce the costs.
39
This method is also suitable for de novo sequencing and metagenomics studies.
DNA detection is a broad field and the work introduced in this thesis is only the tip
of the iceberg. The present work could help researchers and clinicians make better
diagnoses and further developments of the methodology. Furthermore, these
studies open up new possibilities for academic studies and industrial applications.
For example, based on filter paper, it may be possible to develop a simple and cheap
test strip for population studies, especially for people living in poor conditions. Paper
chips could also be integrated into a microfluidic array device for DNA detection.
An automated DNA library preparation system could benefit from the chemical
fragmentation technique. The technique of rare DNA detection could be improved
to detect much rarer target DNA amid a much higher amount of background DNA.
Further technology developments will help to improve our knowledge of DNA,
challenge current assumptions, and unravel more DNA puzzles.
40
Sammanfattning på Svenska
Detektion av DNA har fått en allt ökande betydelse i våra liv, inom allt från
diagnostik av bakterieinfektioner till kriminalanalys, livsmedelsäkerhet, och
övervakning av miljön. I takt med att kostnaderna för DNA-analys har sjunkit,
används nu diagnostiska DNA-tekniker rutinmässigt inte bara i
forskningslaboratorier, utan också i klinisk och kriminalteknisk verksamhet.
Det första målet för den föreliggande avhandlingen syftar till att tydliggöra huruvida
cellulosafilterpapper har potential att vara en kandidat för DNA-matrissupport. I
artikel I (Paper I) studerade vi en metod för att funktionalisera ytan på filterpapper
samt möjligheten att på aktiverat papper detektera DNA medelst fluorescens. I
artikel II (Paper II) undersökte vi både kapaciteten för och möjligheterna till
visualisering av DNA-detektion medelst magnetiska kulor på aktiverat filterpapper,
vilket är en metod som inte kräver ett instrument (skanner) för detektionen. Det
andra målet med föreliggande avhandling är att utforska möljigheterna till en snabb,
känslig och specifik detektion av patogener (sjukdomsalstrande bakterier) vid
misstänkt blodförgiftning. Vi undersökte därför möjligheterna till detektion av
sällsynta DNA-molekyler mot en bakgrund av annat DNA, t ex förekomst av
bakterie-DNA i ett prov med stora mängder humant DNA, genom att enzymatiskt
behandla proverna. Denna behandling syftade till att avlägsna bakgrunds-DNAt (det
humana) och därmed anrika proverna för det sällsynta DNAt (det bakteriella). Som
ett led i att uppnå detta mål utvecklade vi en kemisk fragmenteringsmetod för att
höja effektiviteten hos den enzymatiska nedbrytningen av DNA och den följande
hybridiseringen. Denna fragmenteringsmetod kan också komma till nytta vid
konstruktionen av sekvensningsbibliotek för så kallad massivt parallel DNA-
sekvensering. Artiklarna III och IV (Paper III och Paper IV) behandlar detta arbete.
Resultaten i artikel I visar att XG-NH2 och PDITC kan funktionalisera
cellulosafilterpapper och att de aktiverade filterpapprena kan bilda kovalenta
bindningar med aminogruppsmodifierade oligonukleotider för DNA-detektion,
med bibehållen basparningsförmåga hos dessa oligonukleotider. I artikel II (Paper
II) påvisas att visualisering av DNA-detektion kan erhållas på aktivt papper utan
instrument, genom att använda sig av den naturliga färgen hos magnetkulorna.
Därutöver påvisas framgångsrik detektion av s.k. multiplexade prover, vilket
ytterligare styrker metodens potential att öka kapaciteten av DNA-detektion på
aktivt papper. Resultaten i artikel III (Paper III) verifierar lämpligheten av kemisk
fragmentering för konstruktion av sekvensningsbibliotek för massivt parallel DNA-
sekvensering: fragmenteringstekniken är enkel att utföra, kostnadseffektiv, och
möjlig att automatisera. I artikel IV (Paper IV) demonstreras att små mängder av
E.coli-DNA kan detekteras i ett prov med mångfalt större koncentration av humant
DNA. Detta resultat är från ett modellsystem som ska likna en
41
blodförgiftningssituation, och som består av humana och E.coli-amplikon i
förhållandet 1:108. Humana β-actin-amplikon undertrycktes med en faktor 105
medan motsvarande E.coli-amplikon inte påverkades. Modellsystemet applicerades
också på kliniska prover från patienter med blodförgiftning.
42
References
Abramovitz, M., Barwick, B.G., Willis, S., Young, B., Catzavelos, C., Li, Z., Kodani,
M., Tang, W., Bouzyk, M., Moreno, C.S. & Leyland-Jones, B. (2011) Molecular
characterisation of formalin-fixed paraffin-embedded (FFPE) breast tumour speci-
mens using a custom 512-gene breast cancer bead array-based platform. Br J Cancer.
105, 1574-1581.
Adey, A., Morrison, H.G., Asan., Xun, X., Kitzman, J.O., Turner, E.H., Stackhouse,
B., MacKenzie, A.P., Caruccio, N.C., Zhang, X. & Shendure, J. (2010) Rapid, low-
input, low-bias construction of shotgun fragment libraries by high-density in vitro
transposition. Genome Biol. 11, R119. doi: 10.1186/gb-2010-11-12-r119.
Aikio, S., Grönqvist, S., Hakola, L., Hurme, E., Jussila, S., Kaukoniemi, O., Kopola,
H., Känsäkoski, M., Leinonen, M., Lippo, S., et al. (2006) Bioactive paper and fibre
products patent and literary survey, VTT Working Papers 51, VTT Technical
Research Centre of Finland, Oulu, Finland. ISBN 951-38-6603-3.
Baltimore, D. (1970) RNA-dependent DNA polymerase in virions of RNA tumour
viruses. Nature. 226, 1209-1211.
Bankier, A.T., Weston, K.M. & Barrell, B.G. (1987) Random cloning and sequencing
by the M13/dideoxynucleotide chain termination method. Methods Enzymol. 155, 51-
93.
Bartlett, J.M. & Stirling, D. (2003) A short history of the polymerase chain reaction.
Methods Mol Biol. 226, 3-6.
Bashir, A., Klammer, A.A., Robins, W.P., Chin, C.S., Webster, D., Paxinos, E., Hsu,
D., Ashby, M., Wang, S., Peluso, P., et al. (2012) A hybrid approach for the automated
finishing of bacterial genomes. Nat Biotechnol. 30, 701-707.
Beaucage, S.L. & Caruthers, M.H. (1981) Deoxynucleoside phosphoramidites: A
new class of key intermediates for deoxypolynucleotide synthesis. Tetr Lett. 22, 1859-
1862.
Berg, J.M., Tymoczko, J.L. & Stryer, L. (2011) International Biochemistry. Seventh
edition. ISBN-13: 978-1-4292-7635-1. ISBN-10: 1-4292-7635-5.
Bibikova, M. & Fan, J.B. (2009) GoldenGate assay for DNA methylation profiling.
Methods Mol Biol. 507, 149-163.
43
Bibikova, M., Lin, Z., Zhou, L., Chudin, E., Garcia, E.W., Wu, B., Doucet, D.,
Thomas, N.J., Wang, Y., Vollmer, E., Goldmann, T., Serfart, C., Jiang, W., Barker,
D.L., Chee, M.S., Floros, J. & Fan, J.B. (2006) High-throughput DNA methylation
profiling using universal bead arrays. Genome Res. 16, 383-393.
Biffi, G., Tannahill, D., McCafferty, J. & Balasubramanian, S. (2013) Quantitative
visualization of DNA G-quadruplex structures in human cells. Nat Chem. 5, 182-
186.
Bing, D.H., Boles, C., Rehman, F.N., Audeh, M., Belmarsh, M., Kelley B & Adams
C.P. (1996) Bridge amplification: a solid phase PCR system for the amplification and
detection of allelic differences in single copy genes. Paper presented at: Genetic Iden-
tity Conference Proceedings, Seventh International Symposium on Human Identification (Phoenix,
AZ, Promega Corporation).
Bloch, D.P. (1955) A possible mechanism for the replication of the helical structure
of deoxyribonucleic acid. Proc Natl Acad Sci U S A. 41, 1058–1064.
Brázda, V., Laister, R.C., Jagelská, E.B. & Arrowsmith, C. (2011) Cruciform
Structures are a common DNA feature important for regulating biological
processed. BMC Mol Biol. 12, 33.
Brown, T.A. (2006) GENOMES 3. Third edition. ISBN-10: 0815341385. ISBN-
13: 978-0815341383.
Canard, B & Sarfati, R. (1994) DNA polymerase fluorescent substrates with
reversible 3’-tags. Gene. 148, 1-6.
Carrilho, E., Martinez, A.W. & Whitesides, G.M. (2009) Understanding wax
printing: a simple micropatterning process for paper-based microfluidics. Anal
Chem. 81, 7091-7095.
Chang, C.C., Chen, C.C., Wei, S.C., Lu, H.H., Liang, Y.H. & Lin, C.W. (2012) Di-
agnostic devices for isothermal nucleic acid amplification. Sensors. 12, 8319-8337.
Chargaff, E., Lipshitz, R. & Green, C. (1952) Composition of the deoxypentose
nucleic acids of four genera of sea-urchin. J Biol Chem. 195, 155–160.
Christmann, M., Tomicic, M.T., Roos, W.P. & Kaina, B. (2003) Mechanisms of
human DNA repair: an update. Toxicology. 193, 3-34.
Comer, J.P. (1956) Semiquantitative specific test paper for glucose in Urine. Analytical
44
Chemistry. 28, 1748-1750.
Conner, B.J., Reyes, A.A., Morin, C., Itakura, K., Teplitz, R.L. & Wallace, R.B. (1983)
Detection of sickle cell beta S-globin allele by hybridization with synthetic
oligonucleotides. Proc Natl Acad Sci U S A. 80, 278–282.
Costello, M., Pugh, T.J., Fennell, T.J., Stewart, C., Lichtenstein, L., Meldrim, J.C.,
Fostel, J.L., Friedrich, D.C., Perrin, D., Dionne, D., Kim, S., Gabriel, S.B., Lander,
E.S., Fisher, S. & Getz, G. (2013) Discovery and characterization of artifactual
mutations in deep coverage targeted capture sequencing data due to oxidative DNA
damage during sample preparation. Nucleic Acids Res. 41, e67. doi:
10.1093/nar/gks1443.
Dahm, R. (2005) Friedrich Miescher and the discovery of DNA. Developmental
Biology. 278, 274–288.
Dahm, R. (2008) Discovering DNA: Friedrich Miescher and the early years of
nucleic acid research. Hum. Genet. 122, 565–581.
Dalma-Weiszhausz, D.D., Warrington, J., Tanimoto, E.Y. & Miyada, C.G. (2006) The
Affymetrix GeneChip platform: an overview. Methods Enzymol. 410, 3–28.
Delbrück, M. (1954) On the replication of deoxyribonucleic acid (DNA). Proc Natl
Acad Sci U S A. 40, 783–788.
Devlin, T.M. (2005) Textbook of biochemistry with clinical correlations. Sixth
edition. ISBN-13: 978-0-471-67808-3. ISBN-10: 0-471-67808-2.
Dovichi, N.J. & Zhang, J. (2000) How Capillary Electrophoresis Sequenced the
Human Genome. This Essay is based on a lecture given at the Analytica 2000
conference in Munich (Germany) on the occasion of the Heinrich-Emanuel-Merck
Prize presentation. Angew Chem Int Ed Engl. 39, 4463-4468.
Eid, J., Fehr, A., Gray, J., Luong, K., Lyle, J., Otto, G., Peluso, P., Rank, D., Baybayan,
P., Bettman, B., et al. (2009) Real-time DNA sequencing from single polymerase
molecules. Science. 323,133-138.
Elson, D. & Chargaff, E. (1952) On the deoxyribonucleic acid content of sea
urchingametes. Experientia. 8, 143–145.
ENCODE Project Consortium, Bernstein, B.E., Birney, E., Dunham, I., Green,
E.D., Gunter, C. & Snyder, M. (2012) An integrated encyclopedia of DNA elements
45
in the human genome. Nature. 489, 57-74.
ENCODE Project Consortium, Birney, E., Stamatoyannopoulos, J.A., Dutta, A.,
Guigo, R., Gingeras, T.R., Margulies, E.H., Weng, Z., Snyder, M., Dermitzakis, E.T.,
et al. (2007) Identification and analysis of functional elements in 1% of the human
genome by the ENCODE pilot project. Nature. 447, 799-816.
Ernberg, I., Blomberg, C., Jorsäter, S., Wennborg, A., Århem, P., Malmnäs, P.E.,
Aurell, E. & Cöster, J. (2010) Vad är liv i kosmos, i cellen, i människan? ISBN
9789185565337.
Fan, J.B., Gunderson, K.L., Bibikova, M., Yeakley, J.M., Chen, J., Wickham Garcia,
E., Lebruska, L.L., Laurent, M., Shen, R. & Barker, D. (2006) Illumina universal bead
arrays. Methods Enzymol. 410, 57–73.
Fan, J.B., Hu, S.X., Craumer, W.C. & Barker, D.L. (2005) BeadArray-based solutions
for enabling the promise of pharmacogenomics. Biotechniques. 39, 583–588.
Fan, J.B., Oliphant, A., Shen, R., Kermani, B.G., Garcia, F., Gunderson, K.L.,
Hansen, M., Steemers, F., Butler, S.L., Deloukas, P., et al. (2003) Highly parallel SNP
genotyping. Cold Spring Harb Symp Quant Biol. 68, 69-78.
Fan, J.B., Yeakley J.M., Bibikova, M., Chudin, E., Wickham, E., Chen, J., Doucet, D.,
Rigault, P., Zhang, B., Shen, R., et al. (2004) A versatile assay for high-throughput
gene expression profiling on universal array matrices. Genome Res. 14, 878–885.
Fang, G., Munera, D., Friedman, D.I., Mandlik, A., Chao, M.C., Banerjee, O., Feng,
Z., Losic, B., Mahajan, M.C., Jabado, O.J., et al. (2012) Genome-wide mapping of
methylated adenine residues in pathogenic Escherichia coli using single-molecule real-
time sequencing. Nat Biotechnol. 30, 1232-1239.
Ferrier, D.R. (2013) Lippincott’s illustrated review Biochemistry. Sixth edition. ISBN
978-1-4511-7562-2.
Fire, A., & Xu, S.Q. (1995) Rolling replication of short DNA circles. Proc Natl Acad
Sci U S A. 92, 4641–4645.
Fodor, S.P., Read, J.L., Pirrung, M.C., Stryer, L., Lu, A.T. & Solas, D. (1991) Light-
directed, spatially addressable parallel chemical synthesis. Science. 251, 767-773.
Friedberg, E.C. (2003) DNA damage and repair. Nature. 421, 436-440.
46
Fu, E., Lutz, B., Kauffman, P. & Yager P. (2010) Controlled reagent transport in
disposable 2D paper networks. Lab Chip. 10, 918-920.
Fu, E., Ramsey, S.A., Kauffman, P., Lutz, B. & Yager, P. (2011) Transport in two-
dimensional paper networks. Microfluid Nanofluidics. 10, 29-35.
Glad, C. & Grubb, A. O. (1977) A new method for immunochemical quantification
[proceedings]. Biochem Soc Trans. 5, 712-714.
Glad, C. & Grubb, A. O. (1978) Immunocapillarymigration--a new method for im-
munochemical quantitation. Anal Biochem. 85, 180-187.
Glad, C. & Grubb, A. O. (1981) Immunocapillarymigration with enzyme-labeled
antibodies: rapid quantification of C-reactive protein in human plasma. Anal Biochem.
116, 335-340.
Goldmann, T. & Gonzalez, J.S. (2000) DNA-printing: utilization of a standard inkjet
printer for the transfer of nucleic acids to solid supports. J Biochem Biophys Methods.
42, 105-110.
Gordon, A.H., Martin, A.J.P. & Synge, R.L.M. (1943) Partition chromatography of
free amino-acids and peptides. Biochem J. 37, xiii.
Green, B., Bouchier, C., Fairhead, C., Craig, N.L. & Cormack, B.P. (2012) Insertion
site preference of Mu, Tn5, and Tn7 transposons. Mob DNA. 3, 3. doi:
10.1186/1759-8753-3-3.
Guatelli, J.C., Whitfield, K.M., Kwoh, D.Y., Barringer, K.J., Richman, D.D. &
Gingeras, T.R. (1990) Isothermal, in vitro amplification of nucleic acids by a
multienzyme reaction modeled after retroviral replication. Proc Natl Acad Sci U S
A. 87, 1874-1878.
Gunderson, K.L. (2009) Whole-genome genotyping on bead arrays. Methods Mol Biol.
529, 197–213.
Gunderson, K.L., Kruglyak, S., Graige, M.S., Garcia F., Kermani, B.G., Zhao, C.,
Che, D., Dickinson, T., Wickham, E., Bierle, J., et al. (2004) Decoding randomly
ordered DNA arrays. Genome Res. 14, 870–877.
Guo, Z., Guilfoyle, R.A., Thiel, A.J., Wang, R. & Smith, L.M. (1994) Direct
fluorescence analysis of genetic polymorphisms by hybridization with
oligonucleotide arrays on glass supports. Nucleic Acids Res. 22, 5456-5465.
47
Hanawalt, P.C. (2013) The awakening of DNA repair at Yale. Yale J Biol Med. 86,
517-523.
Heid, C.A., Stevens, J., Livak, K.J. & Williams, P.M. (1996) Real time quantitative
PCR. Genome Res. 6, 986-994.
Hershey, A.D. & Chase, M. (1952) Independent Functions of Viral Protein and
Nucleic Acid in Growth of Bacteriophage. J Gen Physiol. 36, 39–56.
Higuchi, R., Dollinger, G., Walsh, P.S. & Griffith, R. (1992) Simultaneous
amplification and detection of specific DNA sequences. Biotechnology (N Y). 10, 413–
417.
Higuchi, R., Fockler, C., Dollinger, G., & Watson, R. (1993) Kinetic PCR analysis:
real-time monitoring of DNA amplification reactions. Biotechnology (N Y). 11, 1026-
1030.
Hoheisel, J.D., Ross, M.T., Zehetner, G. & Lehrach, H. (1994) Relational genome
analysis using reference libraries and hybridisation fingerprinting. J Biotechnol. 35,
121-134.
Holland, P.M., Abramson, R.D., Watson, R. & Gelfand, D.H. (1991) Detection of
specific polymerase chain reaction product by utilizing the 5’→3’ exonuclease
activity of thermus aquaticus DNA polymerase. Proc Natl Acad Sci U S A. 88, 7276-
7280.
Hong, J., Wang, Y., Ye, X. & Zhang, Y.H. (2008) Simple protein purification through
affinity adsorption on regenerated amorphous cellulose followed by intein self-
cleavage. J Chromatogr A. 1194, 150-154.
Hong, J., Ye, X. & Zhang, Y.H. (2007) Quantitative determination of cellulose
accessibility to cellulase based on adsorption of a nonhydrolytic fusion protein
containing CBM and GFP with its applications. Langmuir. 23, 12535-12540.
Houtgraaf, J.H., Versmissen, J. & van der Giessen, W.J. (2006) A concise review of
DNA damage checkpoints and repair in mammalian cells. Cardiovasc Revasc Med. 7,
165-172.
IHGSC (2004). Finishing the euchromatic sequence of the human genome. Nature.
431 , 931-945.
48
Jahanshahi-Anbuhi, S., Chavan, P., Sicard, C., Leung, V., Hossain, S.M., Pelton,
R., Brennan, J.D. & Filipe, C.D. (2012) Creating fast flow channels in paper fluidic
devices to control timing of sequential reactions. Lab Chip. 12, 5079-5085.
Kambara, H. & Takahashi, S. (1993) Multiple-sheathflow capillary array DNA
analyser. Nature. 361, 565–566.
Kleppe, K., Ohtsuka, E., Kleppe, R., Molineux, I. & Khorana, H.G. (1971) Studies
on polynucleotides. XCVI. Repair replications of short synthetic DNA’s as catalyzed
by DNA polymerases. J Mol Biol. 56, 341–361.
Krunic, N., Yager, T.D., Himsworth, D., Merante, F., Yaghoubian, S. & Janeczko, R.
(2007) xTAG RVP assay: analytical and clinical performance. J Clin Virol. 40 (Suppl.
1), S39–S46.
Kubota, Y., Nash, R.A., Klungland, A., Schär, P., Barnes, D.E. & Lindahl, T. (1996)
Reconstitution of DNA base excision-repair with purified human proteins:
interaction between DNA polymerase beta and the XRCC1 protein. EMBO J. 15,
6662-6670.
Kuhn, K.,. Baker, S.C., Chudin, E., Lieu, M.H., Oeser, S., Bennett, H., Rigault, P.,
Barker, D., McDaniel, T.K. & Chee, M.S. (2004) A novel, high-performance random
array platform for quantitative gene expression profiling. Genome Res. 14, 2347–2356.
Lam, E.Y., Beraldi, D., Tannahill, D., Balasubramanina, S. (2013) G-quadruplex
structures are stable and detectable in human genomic DNA. Nat Commun. 4, 1796.
Lamture, J.B., Beattie, K.L., Burke, B.E., Eggers, M.D., Ehrlich, D.J., Fowler, R.,
Hollis, M.A., Kosicki, B.B., Reich, R.K. & Smith, S.R. (1994) Direct detection of
nucleic acid hybridization on the surface of a charge coupled device. Nucleic Acids
Res. 22, 2121–2125.
Landegren, U., Kaiser, R., Sanders, J. & Hood, L. (1988) A ligase-mediated gene
detection technique. Science. 241, 1077-1080.
Lange, S.A., Roth, G., Wittemann, S., Lacoste, T., Vetter, A., Grässle, J., Kopta,
S., Kolleck, M., Breitinger, B., Wick, M., Hörber, J.K., Dübel, S. & Bernard, A.
(2005) Measuring biomolecular binding events with a compact disc player device.
Angew Chem Int Ed Engl. 45, 270-273.
49
Larsson, C., Koch, J., Nygren, A., Janssen, G., Raap, A.K., Landegren, U. & Nilsson,
M. (2004) In situ genotyping individual DNA molecules by target-primed rolling-
circle amplification of padlock probes. Nat Methods. 1, 227-232.
Lashkari, D.A., DeRisi, J.L., McCusker, J.H., Namath, A.F., Gentile, C., Hwang, S.Y.,
Brown, P.O. & Davis, R.W. (1997) Yeast microarrays for genome wide parallel
genetic and gene expression analysis. Proc Natl Acad Sci U S A. 94, 13057–13062.
Lemieux, B., Li, Y., Kong, H. & Tang, Y.W. (2012) Near instrument-free, simple
molecular device for rapid detection of herpes simplex viruses. Expert Rev Mol Diagn.
12, 437-443.
Levine, L., Gordon, J.A. & Jencks, W.P. (1963) The relationship of structure to the
effectiveness of denaturing agents for deoxyribonucleic acid. Biochemistry. 2, 168-175.
Linnarsson, S. (2010) Recent advances in DNA sequencing methods - general prin-
ciples of sample preparation. Exp Cell Res. 316, 1339-1343.
Lipps, H.J. & Rhodes, D. (2009) G-quadruplex structures: in vivo evidence and
function.Trends Cell Biol. 19, 414–422.
Liu, R.H., Dill, K., Fuji, H.S. & McShea, A. (2006) Integrated microfluidic biochips
for DNA microarray analysis. Expert Rev Mol Diagn. 6, 253-261.
Lizardi, P.M., Huang, X., Zhu, Z., Bray-Ward, P., Thomas, D.C. & Ward, D.C.
(1998) Mutation detection and single-molecule counting using isothermal rolling-
circle amplification. Nat Genet. 19, 225-232.
Mackay, I.M., Arden, K.E. & Nitsche, A. (2002) Real-time PCR in virology. Nucleic
Acids Res. 30, 1292-1305.
Madou, M., Zoval, J., Jia, G., Kido, H., Kim, J. & Kim, N. (2006) Lab on a CD. Annu
Rev Biomed Eng. 8, 601-628.
Margulies, M., Egholm, M., Altman, W.E., Attiya S, Bader, J.S., Bemben, L.A., Berka,
J., Braverman, M.S., Chen, Y.J., Chen, Z., et al. (2005) Genome sequencing in
microfabricated high-density picolitre reactors. Nature. 437, 376-380.
Marine, R., Polson, S.W., Ravel, J., Hatfull, G., Russell, D., Sullivan, M., Syed,
F., Dumas, M. & Wommack, K.E. (2011) Evaluation of a transposase protocol for
rapid generation of shotgun high-throughput sequencing libraries from nanogram
quantities of DNA. Environ Microbiol. 77, 8071-8079.
50
Martin, A.J.P. & Synge, R.L.M. (1941) A new form of chromatogram employing two
liquid phases. 1. A theory of chromatography. 2. Application to the micro-
determination of the higher monoamino-acids in proteins. Biochem J. 35, 1358-1368.
Martinez, A.W., Phillips, S.T., Butte, M.J. & Whitesides, G.M. (2007) Patterned paper
as a platform for inexpensive, low-volume, portable bioassays. Angew Chem Int Ed
Engl. 46, 1318-1320.
Martinez, A.W., Phillips, S.T., Whitesides, G.M. & Carrilho, E. (2010) Diagnostics
for the developing world: Microfluidic paper-based analytical devices. Anal Chem. 82,
3–10.
Maskos, U. (1991) A novel method of nucleic acid sequence analysis, D.Phil. Thesis,
Deparment of Biochemistry, Oxford Univerity, Oxford, UK, 160.
Maskos, U. & Southern, E.M. (1992) Oligonucleotide hybridisations on glass
supports: a novel linker for oligonucleotide synthesis and hybridisation properties
of oligonucleotides synthesised in situ. Nucleic Acids Res. 20, 1679–1684.
Matson, R.S., Rampal, J.B. & Coassin, P.J. (1994) Biopolymer synthesis on
polypropylene supports. I. Oligonucleotides. Anal Biochem. 217, 306-310.
Maxam, A.M. & Gilbert, W. (1977) A new method for sequencing DNA. Proc Natl
Acad Sci U S A. 74, 560–564.
Meinkoth, J. & Wahl, G. (1984) Hybridization of nucleic acids immobilized on solid
supports. Anal Biochem. 138, 267-284.
Merante, F., Yaghoubian, S. & Janeczko, R. (2007) Principles of the xTAG
respiratory viral panel assay (RVP assay). J Clin Virol. 40 (Suppl.1), S31–S35.
Metzker, M.L. (2010) Sequencing technologies - the next generation. Nat Rev
Genet. 11, 31-46.
Metzker, M.L., Raghavachari, R., Richards, S., Jacutin, S.E., Civitello, A., Burgess, K.
& Gibbs, R.A. (1994) Termination of DNA synthesis by novel 3’-modified
deoxyribonucleoside triphosphates. Nucleic Acids Res. 22, 4259-4267.
Micklos, D.A., Freyer, G.A. & Crotty, D.A. (2003) DNA Science, a first course.
Second edition. ISBN 0-87969-636-2.
51
Miller, M.B. & Tang, Y.W. (2009) Basic concepts of microarrays and potential
applications in clinical microbiology. Clin Microbiol Rev. 22, 611–633.
Milos, P.M. (2009) Emergence of single-molecule sequencing and potential for
molecular diagnostic applications. Expert Rev Mol Diagn. 9, 659–666.
Mullis, K., Faloona, F., Scharf, S., Saiki, R., Horn, G. & Erlich, H. (1986) Specific
enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring
Harb Symp Quant Biol. 51, 263-273.
Nelson, D.L. & Cox, M.M. (2013) Lehninger principles of biochemistry. Sixth
edition. ISBN-13: 978-1-4292-3414-6. ISBN-10: 1-4292-3414-8.
Nolte, D.D. (2009) Invited Review Article: Review of centrifugal microfluidic and
bio-optical disks. Rev Sci Instrum. 80. 101101. doi: 10.1063/1.3236681.
Parida, S.K., Mukerji, M., Singh, A.K., Singh, N.K. & Mohapatra, T. (2012) SNPs in
stress-responsive rice genes: validation, genotyping, functional relevance and popu-
lation structure. BMC Genomics. 13, 426. doi: 10.1186/1471-2164-13-426.
Pelton, R. (2009) Bioactive paper provides a low-cost platform for diagnostics.
TrAC-Trends in Analytical Chemistry. 28, 925-942.
Pihlak, A., Baurén, G., Hersoug, E., Lönnerberg, P., Metsis, A. & Linnarsson S.
(2008) Rapid genome sequencing with short universal tiling probes. Nat
Biotechnol. 2008, 26, 676-684.
Potyrailo, R.A., Morris, W.G., Leach, A.M., Sivavec, T.M., Wisnudel, M.B. & Boyette,
S. (2006) Analog signal acquisition from computer optical disk drives for quantitative
chemical sensing. Anal Chem. 78, 5893-5899.
Prober, J.M., Trainor, G.L., Dam, R.J., Hobbs, F.W., Robertson, C.W., Zagursky,
R.J., Cocuzza, A.J., Jensen, M.A. & Baumeister, K. (1987) A system for rapid DNA
sequencing with fluorescent chain-terminating dideoxynucleotides. Science. 238, 336-
341.
Quail, M.A. (2010) DNA: Mechanical Breakage. In: eLS. John Wiley & Sons Ltd,
Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0005333.pub2].
Quail, M.A., Kozarewa, I., Smith, F., Scally, A., Stephens, P.J., Durbin, R., Swerdlow,
H. & Turner, D.J. (2008) A large genome center’s improvements to the Illumina
sequencing system. Nat Methods. 5, 1005-1010.
52
Ramachandraiah, H., Amasia, M., Cole, J., Sheard, P., Pickhaver, S., Walker, C., Wirta,
V., Lexow, P., Lione, R. & Russom, A.(2013) Lab-on-DVD: standard DVD drives as
a novel laser scanning microscope for image based point of care diagnostics. Lab
Chip. 13, 1578-1585.
Rasko, D.A., Webster, D.R., Sahl, J.M., Bashir, A., Boisen, N., Scheutz, F., Paxinos,
E.E., Sebra, R., Chin, C.S., Iliopoulos, D., et al. (2011) Origins of the E. coli strain
causing an outbreak of hemolytic-uremic syndrome in Germany. N Engl J Med. 365,
709-717.
Rasmussen, S.R., Larsen, M.R. & Rasmussen, S.E. (1991) Covalent immobilization
of DNA onto polystyrene microwells: the molecules are only bound at the 5’end.
Anal Biochem. 198, 138-142.
Ronaghi, M. (2001) Pyrosequencing sheds light on DNA sequencing. Genome Res. 11,
3-11.
Ronaghi, M., Uhlén, M., & Nyrén, P. (1998) A sequencing method based on real-
time pyrophosphate detection. Science. 281, 363–365.
Saiki, R.K., Walsh, P.S., Levenson, C.H. & Erlich, H.A. (1989) Genetic analysis of
amplified DNA with immobilized sequence-specific oligonucleotide probes. Proc
Natl Acad Sci U S A. 86, 6230–6234.
Sanger, F., Nicklen, S. & Coulson, A.R. (1977) DNA sequencing with chain-termi-
nating inhibitors. Proc Natl Acad Sci U.S.A. 74, 5463-5467.
Schadt, E.E., Turner, S. & Kasarskis, A. (2010) A window into third-generation
sequencing. Hum Mol Genet. 19. 227-240.
Schena, M., Shalon, D., Davis, R.W. & Brown, P.O. (1995) Quantitative monitoring
of gene expression patterns with a complementary DNA micaroarray. Science. 270,
467-470.
Schildkraut, C.L., Marmur, J. & Doty, P. (1961) The formation of hybrid DNA
molecules and their use in studies of DNA homologies. J Mol Biol. 3, 595-617.
Schrödinger, E. (1944) What Is Life? The Physical Aspect of the Living Cell. ISBN
0-521-42708-8.
53
Shendure, J., Porreca, G.J., Reppas, N.B., Lin, X., McCutcheon, J.P., Rosenbaum,
A.M., Wang, M.D., Zhang, K., Mitra, R.D. & Church, G.M. (2005) Accurate multi-
plex polony sequencing of an evolved bacterial genome. Science. 309, 1728-1732.
Smith, L.M., Sanders, J.Z., Kaiser, R.J., Hughes, P., Dodd, C., Connell, C.R.,
Heiner, C., Kent, S.B. & Hood, L.E. (1986) Fluorescence detection in automated
DNA sequence analysis. Nature. 321, 674-679.
Solonenko, S.A. & Sullivan, M.B. (2013) Preparation of metagenomic libraries from
naturally occurring marine viruses. Methods Enzymol. 531,143-165.
Southern, E.M. (2001) DNA microarrays. History and overview. Methods Mol
Biol. 170, 1-15.
Strachan, T. & Read, A.P. (2003) Human molecular genetics. Third edition. ISBN 0-
8153-4182-2.
Su, S., Nutiu, R., Filipe, C.D., Li, Y. & Pelton, R. (2007) Adsorption and covalent
coupling of ATP-binding DNA aptamers onto cellulose. Langmuir. 23, 1300-1302.
Surzycki, S.J. (1990) A fast method to prepare random fragment sequencing libraries
using a new procedure of DNA shearing by nebulization and electroporation. In
The International Conference on the Status and Future of Research on the Human Genome.
Human Genome II, p. 51. Human Genome Project, San Diego.
Swerdlow, H. & Gesteland, R. (1990) Capillary gel electrophoresis for rapid, high
resolution DNA sequencing. Nucleic Acids Res. 18, 1415-1419.
Swerdlow, H., Wu, S.L., Harke, H., & Dovichi, N.J. (1990) Capillary gel
electrophoresis for DNA sequencing. Laser-induced fluorescence detection with the
sheath flow cuvette. J Chromatogr. 516, 61-67.
Temin, H.M. & Mizutani, S. (1970) RNA-dependent DNA polymerase in virions of
Rous sarcoma virus. Nature. 226, 1211-1213.
Travers, K.J., Chin, C.S., Rank, D.R., Eid, J.S. & Turner, S.W. (2010) A flexible and
efficient template format for circular consensus sequencing and SNP detection. Nu-
cleic Acids Res. 38, e159. doi: 10.1093/nar/gkq543.
Triche, T.J., Jr., Weisenberger, D.J., Van Den Berg, D., Laird, P.W. & Siegmund, K.D.
(2013) Low-level processing of Illumina Infinium DNA Methylation BeadArrays.
Nucleic Acids Res. 41, e90. doi: 10.1093/nar/gkt090.
54
Tuteja, N., Singh, M.B., Misra, M.K., Bhalla, P.L. & Tuteja R. (2001) Molecular
mechanisms of DNA damage and repair: progress in plants. Crit Rev Biochem Mol
Biol. 36, 337-397.
Tyagi, S. & Kramer, F.R. (1996) Molecular beacons: probes that fluoresce upon
hybridization. Nat Biotechnol. 14, 303-308.
Valouev, A., Ichikawa, J., Tonthat, T., Stuart, J., Ranade, S., Peckham, H., Zeng,
K., Malek, J.A., Costa, G., McKernan, K., et al. (2008) A high-resolution, nucleo-
some position map of C. elegans reveals a lack of universal sequence-dictated posi-
tioning. Genome Res. 18, 1051-1063.
Voswinckel, P. (1994) A marvel of colors and ingredients. The story of urine test
strip. Kidney Int Suppl. 47, S3-7.
Wang, A.H.J, Quigley, G.J, Kolpak, F.J., Crawford, J.L., van Boom, J.H., Van der
Marel, G. & Rich, A. (1979) Molecular structure of a left-handed double helical
DNA fragment at atomic resolution. Nature (London). 282, 680–686.
Wang, L. & Li, P.C. (2011) Microfluidic DNA microarray analysis: a review. Anal
Chim Acta. 687, 12-27.
Watson, J.D. & Crick, F.H.C. (1953) Molecular sturcture of nucleic acids: A structure
for deoxyribose nucleic acid. Nature. 171, 737-738.
Wilson, K. & Walker, J. (2010) Principles and techniques of biochemistry and
molecular biology. Seventh edition. ISBN 9780521731676.
Xuan, J., Yu, Y., Qing, T., Guo, L. & Shi, L. (2013) Next-generation sequencing in
the clinic: promises and challenges. Cancer Lett. 340, 284-295.
Zagursky, R.J. & McCormick, R.M. (1990) DNA sequencing separations in capillary
gels on a modified commercial DNA sequencing instrument. Biotechniques. 9, 74-79.
Zhao W, Ali, M.M., Aguirre, S.D., Brook, M.A. & Li, Y. (2008) Paper-based bioassays
using gold nanoparticle colorimetric probes. Anal Chem. 80, 8431–8437.
55
Acknowledgements
I am grateful to Lanzhou University to choose me for study and work abroad, and
China Scholarship Council (CSC) to finance me for a PhD degree in Sweden, also
the support of the Swedish Research Council.
I would like to give special thanks to my main supervisor Prof. Olof Emanuelsson
for your constant encouragement. I am grateful to you to share your knowledge with
me and comment my thesis so carefully. Because of your efficient arrangement, the
work always went very well. I am also grateful to my co-supervisor-Prof. Joakim
Lundeberg. After our long talk one afternoon of July, 2010, I started my PhD work
in the Science for Life Laboratory, Stockholm. Thank you very much for your
support.
I would like to thank Dr. Peter Gyarmati, my co-supervisor, for sharing your
expertise generously. I will always remember how happy I was while we tried to
persuade each other to accept the different ideas we thought were right. My co-
authors Prof. Harry Brumer, III and Dr. Ana Catarina Araújo, thank you for your
valuable contributions to the active filter paper. Dr. Patrik Stahl, thank you for your
introduction of the cellulose project.
I particularly appreciate the helpful comments of Prof. Afshin Ahmadian to make
my doctoral thesis and licentiate thesis as accurate as possible. A big thank to Prof.
Mats Nilsson to be my licentiate examiner. Thank you very much for your good
comments and questions. To Prof. Lukas Käll, thank you so much for the advice
on my licentiate seminar.
Jan, it seems that there are never difficulties for you to deal with the imaging
techniques and also thank you very much for your good comments on my licentiate
presentation. Nick, your help always makes me feel the friendship is so nice and
Tony, it is lucky to know you and to feel your happiness. Pelin, I want to acknowledge
your help, discussions and kindness. Turkish food is really tasty. Mårten Sundberg &
Peter Nilsson, with your help, I designed the transfer files and could finish my
printing work. Emilie & Tarek, your warm help will always be in my heart. Sofie, you
are always patient and helpful. Thank you very much. Bahram, it is nice to know you.
Christian, you are always helpful and thank you for the help. Jun, wherever I am, I
will always remember your smile, our happy talks and your help. I particularly thank
Prof. Duan to take care of my health. Lanlan, thank you for your kind help. Yue,
good luck to you and enjoy your life. Yajuan & Lei, it is so nice to know you and feel
your happiness. Wish you always being happy. Mattias, hope someday I will listen to
your music.
56
It is nice to know you and have a chance to work with you all: Jingwen, Hong, Chuan,
Kristina, Lars, Fredrik, Beata, Anders, Anna, Erik, Benjamin, Sanja, David, Mahya,
Emelie, Sverker, Henrik, Simon, Nemanja, Kicki and all the people ever helped me
and encouraged me. I also would like to thank all my co-authors: Jimmie Hällman,
Max Käller, Christian G. Giske and Patrik Gille-Johnson.
Many thanks to the course organizers I ever joined, and to the book-“DNA Science,
a first course”. When trying to conceive my thesis, I occasionally read this book and
soon was attracted by the knowledge inside and the way of its writing. At that
moment, I was taking a course – “What is life? - The future of biology.” arranged
by Prof. Ingemar Ernberg. Thus, I had a blueprint about my thesis in my mind.
I started my research career in Lanzhou University and Lanzhou Veterinary Research
Institute. I would like to send my thanks to my teachers, colleagues, classmates and
friends there.
It is never enough to say thanks to my parents, 爸爸妈妈，你们赋予我生命，养
育我成长，并且鼓励我勇敢地追逐梦想，爸爸妈妈的话时刻萦绕在我的耳
边; to my sisters, 敏，你奋斗的精神一直激励我，从小到大，你是我的一个
坐标，你的重要，无人取代。波和光，你们的陪伴和支持一直都是我前行
的动力，希望你们一切都好。
The snow outside has melted, and the spring is coming. The end of one travelling
means a new start. I am grateful to this invaluable experience and all the people
helped, are helping and will help me.
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