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Page 1: Advances in DNA Detection - diva-portal.org705792/FULLTEXT01.pdf · Yajing Song, 2014: Advances in DNA detection Division of Gene Technology, School of Biotechnology, Royal Institute

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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

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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

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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).

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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

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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

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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

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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

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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 &

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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

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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

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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

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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

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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.

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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).

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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

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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,

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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).

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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,

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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.

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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

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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.

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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

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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.

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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

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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.

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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

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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.

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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.

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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.

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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

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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.

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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.

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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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