Mechanical Streching and Lightscattering on DNA

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DNA

Dissertation

zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften

an der Universit¨at Konstanz

Mathematisch-Naturwissenschaftliche Sektion Fachbereich Physik

Lehrstuhl Prof. Dr. G. Maret

vorgelegt von

Juha Koota

Tag der m¨undlichen Pr¨ufung 7. Juli 2006

Referent: Prof. Dr. Georg Maret Referent: Prof. Dr. Joachim R¨adler

Konstanzer Online-Publikations-System - URL: http://www.ub.uni-konstanz.de/kops/volltexte/2006/1893/

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1 General introduction 7

1.1 DNA and its conformations . . . 7

1.2 Requirements for a successful experiment . . . 8

2 Confocal Microscopy and Mechanical Stretching of DNA 11 2.1 Introduction . . . 12

2.2 Materials and methods . . . 12

2.2.1 DNA and its end-modification . . . 12

2.2.2 Preparing the surfaces for DNA end-graft . . . 16

2.2.3 Working with fluorescent dyes . . . 18

2.2.4 Confocal Microscope . . . 18

2.2.5 Observing stretched DNA with confocal microscopy . . . . 19

2.3 Results and Discussion . . . 20

2.4 Conclusions . . . 30

3 Preparation and characterization of end-modified DNA samples 31 3.1 Introduction . . . 32

3.2 Material and methods . . . 32

3.2.1 Field inversion gel electrophoresis . . . 32

3.2.2 The dot-blot technique . . . 34

3.2.3 Preparation of Cy3-modifiedλ - DNA samples for the microarray reader . . . 35

3.2.4 PCR and the preparation of monodisperse DNA samples . 35 3.3 Results and discussion . . . 40

3.3.1 Amount of concatemers in DNA samples . . . 40

3.3.2 Dot-blot reveals a filtering problem . . . 41

3.3.3 The fluorescently modified λ - DNA and the sample purity 44 3.3.4 High quality PCR DNA for force measurements . . . 46

4 Covalent end-attachment of end-modified DNA on a solid sub- strate 49 4.1 Introduction . . . 50

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4.2.1 Different ways of attaching a λ - DNA on a functionalized glass slide . . . 51

4.2.2 Silanization of glass surfaces . . . 60

5 Characterization and density enhancement of DNA carpets 67 5.1 Introduction . . . 68

5.2.1 Sample preparation for the microarray reader . . . 68

5.2.2 Combing on hydrophilic surfaces . . . 69

5.2.3 Entropy-driven grafting density enhancement . . . 70

5.2.4 Electrophoretic grafting density enhancement . . . 71

5.3 Results and discussion . . . 73

5.3.1 Surface densities of DNA carpets with confocal microscope 73 5.3.2 Surface densities of DNA carpets with microarray reader . 74 5.3.3 Entropy driven grafting density enhancement . . . 80

5.3.4 Density enhancement by electrophoretic grafting . . . 81

5.3.5 Multiple combing with spermidine . . . 83

6 Evanescent wave dynamic light scattering on DNA carpets 91 6.1 Introduction . . . 92

6.2 Theory . . . 92

6.2.1 Evanescent waves . . . 92

6.2.2 Emitted fluorescence as a measure of the penetration depth 94 6.2.3 Evanescent-wave dynamic light scattering . . . 95

6.3.1 Setup . . . 98

6.3.2 DNA samples for the EWDLS measurement . . . 101

6.4.1 Estimation of the penetration depth . . . 101

6.4.2 EWDLS with colloids . . . 102

6.4.3 EWDLS with a end-grafted DNA carpet . . . 105

6.4.4 Characterization of the sample cell surface . . . 110

Summary 115

Zusammenfassung 117

A List of chemicals and material suppliers 119

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B Functionalizing λ - DNA with oligos 123 B.1 FIGE protocol . . . 125 B.2 Functionalization of glass slides . . . 126 B.3 Thiolation of streptavidin . . . 128 C Partial heterodyning formulas in auto- and crosscorrelation

mode 129

C.1 Autocorrelation mode . . . 129 C.2 Crosscorrelation mode . . . 130 D Characterizing the single-mode fiber and the PMT 133

Acknowledgments 137

Bibliography 139

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General introduction

1.1 DNA and its conformations

DNA (deoxyribonucleic acid) (see Fig. 2.1) posses the blueprints for biological development of life, but without proteins DNA would be merely a storage for data. It is the protein-DNA interaction what regulates the development of cells and therefore to understand these interactions better is of prime biological in- terest. Proteins are the active media inside cells and as some of them read the genetic code from DNA they have to locally deform DNA. This makes the elastic properties of DNA biologically relevant. The starting point for our project was laid in the works by Smith et al. [1] and Cluzel et al. [2] who both studied elastic properties of DNA by measuring a force-extension curve for single λ - DNA molecule. For example the measurement of Cluzel et al. [2] was done so that a DNA molecule was end-grafted from both of its ends on two separate surfaces and by moving one of the surfaces the response of the applied force over the molecule was then detected from the other surface. Without going more in to detail how this measurement was done, we show simply the force-extension curve in Fig. 1.1.

First, at extensions much lower than the contour length of the molecule, work against the entropy has to be done but since entropic forces are rather low (in the order of k_BT) there is no response detected in the force-extension curve. As the molecule is stretched further out, close to the contour length, the DNA molecule first elongates as any material in its elastic regime would. As the stretching is going further a force plateau is reached where the molecule can be stretched up to 60 % – 70 % over its contour length without applying any additional force.

This plateau has been interpreted as a coexistence of B-form¹ DNA and a new S-form [2] (S stands for stretched). The force-extension curve has been measured several times since but up to now there is no experiment revealing the structure of the S-DNA. The suggestions about the structure vary from strong base in- clination with little unwinding [2] [3] to ladder like fully unwounded DNA [3]

1B-form is the natural form of DNA as it is found inside the cell

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depending on the way how the DNA molecule is stretched. Additionally also DNA melting (strand separation) has been suggested [4] as an explanation of the force-extension plateau.

force[pN]

length [L₀]

Figure 1.1: Force-extension curve for singleλ- DNA molecule. First the gaussian coil is stretched out and very little force is needed and as the extension approaches the contour length (L₀) the force diverges. At higher forces a plateau is found and at this plateau the DNA molecule can be stretched with a constant force up to (1.6−1.7)×L0. After the plateau the force increases rapidly and the extension ends up at a point when either the molecule or the surface chemistry is not withstanding the force. Figure taken from the work done by Cluzel et al. [2]

Furthermore similar overstretched DNA states have also been observed with DNA binding proteins such as recombination protein RecA [5] or TATA box binding protein [6] and still very little is known about structure of these protein- DNA complexes [7] where the DNA is severely deformed. In order to study the S-DNA and the DNA-protein interactions our goal was to build a setup where we can stretch DNA and simultaneously gain structural information by X-ray diffraction, by light scattering or by birefringence of the stretched states. At the moment we have already build two versions [8] [9] of such a setup, but up to now we have not been able to reproduce force-extension curve or gain structural data.

1.2 Requirements for a successful experiment

In contrast to the experiments of Smith et al. [1] and Cluzel et al. [2] we have to stretch, not a single molecule, but several thousands of molecules in order to get measurable signal for the structure determining measurement. In a successful experiment DNA molecules are stretched between two separate surfaces and simultaneously the structure of the DNA is determined for example by X-

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ray diffraction as presented in Fig. 1.2. What are then the requirements for a successful experiment?

Let us look carefully at the DNA sample preparation:

• First of all we have to introduce end-modifications on both ends of the DNA molecule so that there is a handle for end-grafting.

• The end-modifications have to be covalently linked to the rest of the molecule so that force can be applied over them.

• The end-modification process should also be efficient so that both ends of all the molecules really carry their modifications.

• The sample preparation should provide DNA molecules which are monodisperse in length so that the S state is reached simultaneously by all the molecules as the two surfaces are moved apart.

• The substances used to prepare the DNA samples can distort the experiment in later phase so an effective purification method has to be also developed.

There are also very hard criteria for successful DNA end-grafting surface chemistry:

• The surfaces have to carry functional groups which are first of all strongly enough, i.e. covalently linked to the surface.

• The functional groups have to be able to react with the DNA end- modifications creating bonds that are also stable enough so that the S state can be reached.

• The functional groups on the surface are allowed to react only with one kind of DNA end-modification at a time. When both of the end-modifications would react already with one surface, we would have loops of DNA, instead of DNA molecules spanning from one surface to the other.

• The functionality should stay reactive so long that dense DNA carpets can be formed. It is clear that even the best surface chemistry will not help when the end-grafting densities of the DNA carpets are not high enough to produce measurable signal for the structure determination.

In this work we will concentrate to fulfill the criteria explained above since we believe that the existing apparatus build by M. Clausen [9] meets from the mechanical point of view the needs of the experiment. In chapter 2 we show that the previously developed surface end-grafting methods [8] are not stable enough for the multi-molecule stretching experiment as described above. Furthermore in

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generator : KaCu = 0.154 nm

Sample to detector distance Rotating anode -

generator : KaCu = 0.154 nm

30 cm < D < 1 m Pinhole :

5 mm diameter

Confocal optics Surface Force Apparatus Detector (image plate) Flux :

1.38 x 10⁶ photons/seconds X-rays

Figure 1.2: In a successful experiment dense carpet of both ends grafted DNA molecules are being stretched and simultaneously X-ray diffraction is performed revealing the structure of the overstretched DNA or DNA-protein complex (with the courtesy of A. Zinck).

chapter 3 we show that previously used sample preparation protocol [8] is not optimal: we have instead of homogeneous both ends modified λ- DNA molecules very inhomogeneous samples. The problems of the sample inhomogeneities and the stable surface end-grafts are treated and solved in chapters 3 and 4. Fur- thermore in chapter 4 we give also additional possibilities to covalently end-graft DNA on various types of surfaces. In chapter 5 we present methods to characterize the surface end-grafted DNA carpets, and additionally we show also methods how to enhance the end-grafting density. Finally in chapter 6 we show that a scattering signal, which is inevitable for structure determination, can be recorded from an end-grafted DNA carpet.

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Confocal Microscopy and

Mechanical Stretching of DNA

Abstract

In this chapter we will present basic techniques to modify DNA molecules with oligonuleotides, silanize surfaces and finally end- graft modified DNA molecules specifically on functionalized surfaces. Furthermore we will present an experimental setup with which we studied the mechanical stability of the DNA-surface an- choring by stretching the molecules and observing the rupture lengths. We present rupture length distributions for λ - DNA molecules which were end-grafted from one end with gold-thiol linkage and from other end with biotin-streptavidin linkage to the surface. Additionally we study the effect of multi-biotin end- modification in the end of theλ - DNA molecules, instead of single end-modification, on the rupture length distribution. Our results show that the present end-grafting techniques are not fulfilling the conditions that are needed for the multi-molecule stretching experiment as presented in the general introduction 1.

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2.1 Introduction

There are only a few experimental techniques available to study the quality of the specific end-graft of a single DNA with a surface. The possibilities presented in the literature rely almost always to the fact that DNA is attached on one end to a colloidal bead or to a AFM tip and on the other end to a solid support or to a another colloidal bead. The bead/tip can then be manipulated with a optical/magnetical tweezer [10] [11], with a atomic force microscope (AFM) [12]

or with a micropipette [2] while the other end of the DNA molecule is held fixed.

The common feature for all of these experiments is the fact that they are all single molecule experiments. Single molecule experiments are done in order to see the actual behavior of molecular individuals while the classical macroscopic experiments tend to average over the molecular conformations. For example the B-S transition of stretched DNA reported first by Cluzel et al. [2] and Smith et al. [1] are beautiful examples of single molecule experiments where the force- extension behavior of single DNA is measured but no structural information of the stretched state is gained. In order to gain the structural information classical macroscopic experiments have to be done. Our goal is basically to combine these two point of views: (i) to stretch a single molecule with both ends grafted and (ii) to do it on large number of molecules in parallel. So the requirements for the DNA functionalization and the surface preparations are very demanding.

In this chapter we review the basic experimental techniques used already by Lehner [8] to modify DNA and end-graft it onto a surface. Furthermore we present a simple but effective setup with which we study the quality and the possible problems in the DNA end-grafting to the surface.

2.2 Materials and methods

2.2.1 DNA and its end-modification

The nucleotide (see Fig. 2.1), which is a monomer unit of DNA polymer, consist of three different chemical moieties: the deoxyribose sugar, the phosphate group and one of the bases (adenine (A), thymine (T), guanine (G) or cytosine(C)). The backbone of a single strand of DNA is a repetitive chain of phosphate groups and sugars. The phosphate groups are joined to the 5⁰ or 3⁰ hydroxyl groups of the sugar with phosphodiester linkage [13]. Two of such a single DNA strands are joined together over hydrogen bonds between the bases [14]. Such a pair of linked bases is called a base-pair and base-pairing can only occur between A and T or between C and G. In most common biological form (B - form) the two strands are coiled around each other forming the double-helix [13]. Such a double-helix has diameter of 2 nm and the distance between adjacent base-pairs is 0.34 nm.

In most of our experiments we are using, unless otherwise mentioned, the DNA

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Adenine

Thymine

3`

5` 5`

3`

Cytosine

Guanine

Nucleotide

Sugar Phosphate

Figure 2.1: The chemical structure of DNA: The nucleotide, which is a monomer unit of DNA polymer, consist of three different chemical moieties: the deoxyri- bose sugar, the phosphate group and one of the bases (adenine (A), thymine (T), guanine (G) or cytosine(C)). The backbone of a single strand of DNA is a repet- itive chain of phosphate groups and sugars. The phosphate groups are joined to the 5⁰ or 3⁰ hydroxyl groups of the sugar with phosphodiester linkage [13]. Fur- thermore the two chains of nucleotides are linked together over hydrogen bonds between the bases forming finally the double-helix. The original figure is taken from Wikipedia (http://en.wikipedia.org/wiki/DNA)

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of a bacterial virus called bacteriophage λ, hence the name λ - DNA¹. The viral DNA is carried in bacteriophage particles as a linear double-stranded molecule with single-stranded complementary termini 12 nucleotides in length [15]. From the viruses extracted DNA is commercially available and we use it as received.

The λ - DNA has to be end-modified so that the specific surface attachment can be established. In our case the end-modification means that we are hybridizing to the 12 nucleotide long single stranded overhang of λ - DNA a complementary single-stranded piece of DNA. This short single-stranded piece of DNA is called an oligonucleotide or shortly just an oligo. The oligos can be chemically modified so that their ends carry a functionality such as biotin, digoxigenin, amino- or thiol- group. The end-modified oligos are commercially available and can be produced in great variety of lengths and base-pair compositions.

The preparation of end-modified DNA (see Fig. 2.2) starts by heating the stock DNA solution (i) to 75^◦C for 15 minutes so that we can be sure that most of the DNA molecules are in the linearized form (ii). This is because at such a high temperature the DNA starts to melt, in other words the strands start to separate. This happens first at the sites where the backbone is not closed. These open backbone sites are called nicks. After the heating follows a rapid cooling phase (5 minutes in ice) after which the linearized DNA is mixed with the oligos and ligase buffer providing optimal conditions for ligation (iii). This mixture is then left to react for one hour at 50^◦C, and during this time the complementary single-strands have enough time to find each other and join together. In the last step T4 ligase enzyme and adenosine triphosphate (ATP) are added (iv) to the DNA – oligo solution. The ligase is an enzyme that closes the backbone between the oligo and the DNA so that the oligo is not only over the base-pairing attached to the λ - DNA and ATP provides energy for the enzyme to operate. This last step is done at room temperature for one hour.

In the final step of the preparation we clean the unbound oligos away from the sample. This is done by gel filtration chromatography [16]. In practice the sample is pipetted into a column filled with gel beads and when the λ - DNA and oligo mixture flows through the column the oligo molecules diffuse into the pores of the gel beads whereas the λ - DNA molecules migrate quickly through the column. The DNA elution from the column can be done with purified milli- Q water with 150 mM NaCl, with PBS² or with TBE³. The collected sample is immediately aliquoted and stored frozen at −20^◦C.

1λ- DNA is 48502 bp (16.5 µm) in length.

2PBS consists of 136.9 mM NaCl, 2.7 mM KCl, 1.8 mM KH2PO4 and 10 mM Na2HPO4in milli-Q and its pH is adjusted to 8 with HCl.

3TBE consists of 130 mM TRIS, 45 mM boric acid and 2.5 mM EDTA and it has pH of 9.

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Figure 2.2: A circularλ- DNA molecule has two nicks (a missing phosphodiester bond between two nucleotides) in its backbone12nucleotides apart (i). By heat- ing the nicked DNA molecule can be opened. Rapid chilling after linearization causes λ - DNA to quench in its linear form (ii). In the following step modified (M₁ and M₂) oligos are ligated to the free single stranded overhangs (iii). Ligase protein binds the backbone of the oligo covalently to the backbone of the λ - DNA molecule (iv). (The letters 3⁰ and 5⁰ show direction of the backbone) The figure is taken from the Ph.D. thesis of R. Lehner [8]

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2.2.2 Preparing the surfaces for DNA end-graft

In order to establish specific end-graft of the DNA molecule to the surface, the surface must be treated so that it carries the matching functionality to the DNA end- modification. However before this functionalization is introduced, the borosilicate cover slips are to be cleaned thoroughly. This is done by rinsing the slides first with isopropanol (tech. garde), then with acetone (tech. grade) and directly after that the slides are soaked in milli-Q water. While the slides are in water, they are sonicated for 30 minutes and after that the slides are soaked in (1 : 1) solution of sulfuric acid (95 % – 97 % p. a.) and hydrogen peroxide (30 % p. a.) for 30 minutes. The acid cleaning is done in order to remove grease layers from the glass [17]. In the next step the slides are washed few times with ethanol (tech.

grade) and soaked in a solution which consists of 98 % ethanol (p. a.) and 2

% (3-aminopropyl)triethoxysilane (APTES). When the glass slides are exposed to water, hydroxyl groups are formed at the surface [17]. These hydroxyl groups can then react with the hydrolyzed aminosilane molecules and form a layer with amino moieties [18] (see Fig. 2.3 (i)). The silane solution is left to react with the glass slides for two hours at room temperature after which the slides are washed with ethanol (p. a.) and milli-Q water, respectively, and dried in an oven at 75^◦C.

The silanized glass surface can now be let to react with glutaraldehyde. Glu- taraldehyde is a homobifunctional crosslinker carrying aldehyde terminals in both of its ends. This means that the crosslinker can link molecules containing amino groups together [19]. Upon the reaction of glutaraldehyde with aminosilanized surface, a Schiff base is created between glutaraldehyde and amino group of the aminosilane molecule while the other end of the glutaraldehyde stays reactive as can be seen in Fig. 2.3 (ii). We added typically a drop of 8 % glutaraldehyde solution directly on a amino silanized surface and left it to react for 30 minutes in a humid box in order to avoid drying of the surface.

With glutaraldehyde derivatized amino reactive surface can then again react with amino group containing molecule (see Fig. 2.3 (iii)). Silva et al. [20] has suggested that glutaraldehyde can react only with lysine⁴ which are found at the surface of streptavidin⁵. Typical incubation time of streptavidin was roughly one hour in PBS buffer and after that the unspecific binding sites at the glass surface were blocked with bovine serum albumin (BSA). BSA is a protein which prevents unspecific adhesion of DNA to the coverslip surface but lets the streptavidin untouched. BSA was left to react for 30 minutes also in PBS. In the final step the streptavidin coated coverslip surface was left to react with biotin-modified DNA. The biotin binds with high affinity to the surface attached streptavidin and forms a stable linkage [19] (see Fig. 2.3 (iv)). The last step was done in TBE

4Lysine is one of the amino acids which are the building blocks of proteins.

5See for example Protein Data Bank for the structure of streptavidin (http://www.rcsb.org/pdb/Welcome.do).

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+

APTES

Streptavidin

OH OH OH

C H₃

Si O O O

NH₂

H₂N

NH₂

SASA H₂N

(i)

(iii)

biotin modified DNA

+

SH

Biotin - thiol-modified DNA

Biotin

N Si N

O O O

H₂N

NH₂

SASA

(iv)

Binding site for biotin

N Si N

O O O

H₂N

NH₂

SASA ^S

(v)

+

Au Au Au

+

Glutaraldehyde

NH₂ Si

O O O

O O

(ii)

N Si N

O O O

+

Figure 2.3: The clean glass surface is silanized with APTES (i). In the following step the amino moiety found at the surface is crosslinked with glutaraldehyde (ii) to the amino moiety found at the surface of streptavidin (SA) (iii). The biotin- modified λ - DNA molecules are end-grafted over biotin – streptavidin linkage (iv). The double-sided end-graft of the λ - DNA molecules is finally achieved over thiol-gold linkage (v).

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buffer with 0.5 M NaCl.

In order to achieve double-sided attachment the DNA molecules have to be end-grafted from both of their ends. The attachment of the other end is achieved over gold - thiol bond (see Fig. 2.3 (v)). Experimentally gold - thiol bond is rather easy because the only requirement for the surface is that it must carry freshly evaporated gold layer. On this layer the thiol-modified DNA can be directly deposited. We incubated typically DNA in TBE buffer and left it to react for one hour. No further steps are needed and the DNA carpet is ready for use after the unbound DNA is washed away. The gold surfaces have nevertheless their downside. The gold has to be freshly evaporated otherwise the DNA will not bind – 15 minutes of waiting is usually already too long.

2.2.3 Working with fluorescent dyes

The visualization of single DNA molecules is done with fluorescent dyes. Through all this work we are using cyanine dye called YOYO-1. YOYO-1 is bis-intercalator meaning that it intercalates with high affinity between subsequent base-pair pockets [21]. Furthermore it experiences 500 fold fluorescence enhancement upon binding to the DNA [22]. The loading concentration that we are using is roughly 1 dye molecule to 5 base pairs. In practice this means that we mixed 100 ng of DNA to 1.5µl of YOYO-1 in concentration of 0.02 mM and we left the YOYO-1 to react with DNA at least for 15 minutes at room temperature before we diluted the mixture.

In order to prevent the problems of photobleaching and photodamage, we mixed an antibleaching solution consisting of 0.1 mg/ml glucose oxidase, 0.04 mg/ml catalase, 5 mg/ml glucose and 0.1 M dithiothreitol in TBE buffer. The antibleaching solution without DTT was stored frozen at −20^◦C and the DTT added ready-to-use solution was stored also frozen and used within a few days because of degration of dithiothreitol. The ready-to-use solution was always added to the sample just before observation of it.

2.2.4 Confocal Microscope

The observation of all the samples during this work was done with a Zeiss Axiovert 200 microscope equipped with a Zeiss 100× immersion oil objective⁶ and with a PerkinElmer Nipkow-disk concofal scanner with cooled CCD camera (Hama- matsu Orca II).

The principle of the Nipkow-disk confocal microscopy is presented for one path of rays in Fig. 2.4. First a parallel illuminating beam is going through a lens, the beam is then focused onto a pinhole (Nipkow-disk). This pinhole is then imaged onto a plane where the sample is. This is the excitation light for the fluorescent

6α- Plan Fluar^r: 100×, NA = 1.45.

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Figure 2.4: The principle of a Nipkow-disk confocal fluorescence microscope (only one path of rays is displayed): The laser beam is directed through a microlens- disk after which it is focused on a pinhole-disk. The light emerging from the pinholes is exciting the fluorescent dye. The fluorescent dye emitted light is imaged onto a CCD over a pinhole-disk and a dicroic mirror. Now when we have, instead of single lens and single pinhole, a rotating microlens array and a rotating pinhole - disk then multiple points from the sample can be imaged onto the CCD simultaneously. Figure taken from Egner et al. [24].

dye. The dye emits light which is then again imaged over the same pinhole on a charge coupled device (CCD) over a dicroic mirror (DM). The dicroic mirror is filtering out the original laser light and reflects the emitted fluorescent light onto the CCD. Now when we have, instead of a single lens and a single pinhole, a rotating microlens array and a rotating pinhole - disk then multiple points from the sample can be imaged onto the CCD simultaneously. The advantage of the confocal microscope, in comparison to standard microscope, is that the axial resolution is enhanced while the out of focus emerging light is suppressed [23].

2.2.5 Observing stretched DNA with confocal microscopy

In order to check the quality of the previously described DNA end-modifications and surface functionalizations, we designed a simple setup where the double sided attachment of DNA can be studied. In Fig. 2.5 (a) the principle of the setup is shown: functionalized glass rod is brought in contact with a bottom surface (microscope cover slip) and the DNA molecules are attached between these two

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surfaces. The glass rod is driven with a feedback motor⁷ with velocity of 1 µm/s and the extension of both ends attached molecules is observed with the microscope simultaneously while driving the rod. The mechanical stability of the setup is achieved by the fact that the rod is brought in contact with the bottom surface so that the rod cannot vibrate but is supported by the bottom plate. If the rod and the plate were not in contact already small vibrations of the rod would cause such high flows that microscoping the sample would be very difficult and the DNA molecules would have difficulties to attach to the functionalized surface of the rod. Fig. 2.5 (c) shows a snapshot out of an experiment where double sided attachment has succeeded and DNA is being stretched. The experimental setup can be seen in Fig. 2.5 (b) at the microscope. By knowing the size of the field of view (66.56µm× 87.36µm) we can measure the extensions of the DNA molecules stretched between two surfaces.

Typical experiment was performed in the following way: The bottom surface was a glass slide on which roughly 1 nm of chromium and 14 nm of gold was evaporated. The chromium layer is needed to attach the gold layer on glass. The 14 nm of gold is enough for DNA to attach over thiol-gold bond and additionally it is still enough transparent for microscoping the sample. The rod was amino silanized, later treated with glutaraldehyde and streptavidin, providing biotin reactive surface for the other end of the DNA molecule, and finally coated with BSA. The carpet was prepared on the gold surface, washed thoroughly and finally incubated in TBE buffer with antibleaching chemicals. Once the bottom surface was ready at the microscope the rod was brought into contact with it, left shortly to incubate and after that the measurement was ready to be performed.

2.3 Results and Discussion

The results from the molecule stretching experiments are presented in Fig. 2.6 where a broad rupture length distribution having average rupture length of roughly 20 µm can be seen. The length of the molecules was measured from the experimental films, error being roughly±5 %⁸. The presented rupture length distribution was measured with slightly altered silanization protocol (see chapter 4.2.2). This, however, did not seem to have an effect to the result in comparison with silanization presented above (data not shown).

According to Lehner et al. [25] the contour length of a singleλ - DNA, when labelled with YOYO-1 in relation of 1 : 5 (dye molecules to base pairs), is 19.8 µm. This is also the average rupture length in Fig. 2.6. Additionally, as it can be seen from Fig. 2.6, only ∼60 % of the molecules that can be stretched upto the

7Motor: CMA-12CCCL, motion controller: ESP 300, both Newport.

8The error was determined by measuring the length of a single molecule, for 3 different molecules, 10 times and taking the standard deviation which was typically 5 % or less for given molecule length.

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buffer + antibleach

objective DNA

glass rod Dx

(a)

Glass rod Motor

Sample cell

(b)

(c)

Figure 2.5: a) The principle of the DNA stretching setup: Functionalized glass rod is brought to contact with glass plate on which DNA molecules are already attached. By moving the rod, the both ends attached molecules are stretched and this is simultaneously observed with the microscope. b) The setup at the microscope. c) A snapshot from experimental data where a single λ – DNA is being stretched. The molecule is roughly 22 µm long. Here it is important to notice that the curvature of the rod is so low that both ends of theλ – DNA are still in focus allowing reliable length determination.

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Figure 2.6: The rupture length distribution of stretchedλ- DNA molecules. The measurement was done with the setup presented in Fig. 2.5. The DNA molecules were end-grafted from one end with gold-thiol linkage and from other end with biotin-streptavidin linkage. The average rupture length of the distribution is roughly at 20µm. Data presented with permission from A. Andr´e.

contour length of the YOYO-1 loaded DNA or less. This result means that we have a problem if we want to parallelize the stretching of theλ - DNA molecules.

The rupture of the λ - DNA molecules occurred from both ends, but we did not see any molecules to break along their backbone. Therefore the problem of the surface end-graft should lie either in the silanization, in the crosslinking process of streptavidin to the silanized surfaces, in the biotin-streptavidin linkage, in the gold-thiol linkage or in the ligation of the oligo to the λ - DNA molecule.

The quality of the silanization is very difficult to characterize⁹ but the protocols that we are using are similar or the same as can be found in literature (see for example [26], [18] or [27]). Furthermore, as mentioned above, we did stretching experiments otherwise keeping all the same but changing only the silanization protocols and we did not observe differences between rupture length distributions.

However, this detail will be further verified in the coming experiments.

The biotin-streptavidin bond is often termed as one of the strongest non- covalent linkages in biology and it has been reported to withstand forces of ∼200 pN [28]. This, however, is not the complete picture. Merkel et al. [29] has measured the biotin-streptavidin bond strength for different loading rates between 1−10000 pN/s. From the measured rupture force histograms for various load-

9We performed some XPS measurements in order to characterize the silanization of the surfaces but these measurements were always unsuccessful due to the carbon dioxide exposure from the air which distorted the results.

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ing rates, they determined by gaussian fits to the histograms the most frequent rupture force for a specific loading rate (see Fig. 2.7 (a) and (b)). In order to understand the loading rate dependent strength of the bond we look at this detail closer. For the biotin-streptavidin bond the kinetic on and off rate constantsk_on and k_{of f} has been reported to have values of 5.13×10⁶ M⁻¹s⁻¹ and 2.8×10⁻⁶ s⁻¹, respectively [30]. With these rate constants we can calculate the equilibrium dissociation constantK_D =k_{of f}/k_on = 5.5×10¹³M. With the equilibrium dissociation constant we can estimate the binding energyE_b of the biotin-streptavidin bond as KD = exp(Eb/kBT) where kB is the Boltzmann constant and T is the absolute temperature [31]. This gives us a binding energy of roughly 30k_BT and in order to break this bond, a force (f) of roughly f =E_b/x∼200 pN is needed as we mentioned above. Here we have assumed a length of the binding pocketx to be roughly 0.6 nm. However, as the bond is non-covalent, the lifetime of the bond decreases rapidly as force is applied on it due thermal activation. Postu- lated by Bell [32] the dissociation rate of the ligand-receptor complex in solution isτ(0) =τ_oscexp(E_b/k_BT) where τ_osc is the inverse natural oscillation frequency.

Now when a constant force f acts on the biotin-streptavidin bond the energy landscape is tilted and the activation energy barrier is lowered. The lifetime of the complex is then given by τ(f) = τ_oscexp((E_b −f x_β)/k_BT) where x_β is a distance of the energy barrier from the energy minimum along the direction of applied force (see Fig. 2.7 (c)).

In the Fig. 2.8 is the force-extension behavior of YOYO-1 loaded λ - DNA (measured by Sischka et al. [10]). From this we can estimate our loading rater_f since r_f is defined as a change of applied force (df) over differential length (dl) multiplied with stretching velocity (v): (r_f = (df /dl)v). Now by taking a deriva- tive of the force-extension curve for YOYO-1 loadedλ - DNA and multiplying it with our stretching velocity∼1µm/s. We get an estimate that our loading rates were roughly 5−30 pN/s. Furthermore, asλ- DNA without YOYO-1 shows the transition from B- to S-DNA, it has over very long extension almost unchange- able force, meaning that the loading rate is very low and there is a constant force of 65 pN over the biotin-streptavidin bond. As we now look at the Fig. 2.7 (b), it is obvious that a single biotin-streptavidin bond cannot withstand the forces needed to maintain DNA is the S form.

In order to circumvent the problem of the bond stability we tried also oligos modified with four biotin molecules¹⁰. In Fig. 2.9 we show the results of both of the stretching experiments: the red histogram is measured with λ - DNA carrying four biotin molecules whereas the gray histogram is measured with λ - DNA carrying only one biotin molecule (this is the same data as already presented in Fig. 2.6). As is clearly seen from the data in Fig. 2.9 the use of the four biotin in the end of λ - DNA has shifted the average rupture length from 20 µm to 24

10The oligo with four modifications is a custom fabrication by IBA GmbH. The sequence:

(phosphate)-AGG XCG CCG CCC XCX C-biotin. Each X is an T base modified with biotin.

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(a) (b)

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Figure 2.7: a) Histograms of rupture forces for biotin-streptavidin linkage as a function of loading rate. Gaussian fits were used to determine the most frequent rupture force from the rupture force histograms. b) The most frequent rupture forces as a function of loading rate for biotin-streptavidin and for biotin-avidin linkages. The star marked data point is measured by Wong et al. [28]. c) The external force adds a mechanical potential that tilts the energy landscape. x_β (see text) is the projection of energy barrier along the direction of the applied force (x_β =hcosθx_tSi). All the figures taken from Merkel et al. [29]

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Figure 2.8: Force-extension curves for different DNA binding ligands. Our inter- est lies in the curve for YOYO-1 because with this we can estimate the loading rate which we applied to the λ - DNA molecules. Furthermore we can use the force-extension behavior also to give a rough estimate about the forces we applied to the molecules. Figure is taken from Sischka et al. [10].

µm. By comparing this with the force-extension curve for YOYO-1 loaded λ - DNA in Fig. 2.8 we notice that the rupture force has increased from∼10 pN to

∼ 50 pN. Now, if we naively take from the Fig. 2.7 (b) the biotin-streptavidin linkage strength for the lowest loading rate and multiply it by number of biotin molecules, we get roughly 80 pN. However, we estimated the rupture force to be

∼50 pN. How this difference can be explained?

We stretched our molecules always with constant velocity of∼1µm/s whereas Siscka et al. [10] measured their force-extension curve with velocity of 0.1µm/s.

This fact alone makes a crucial difference in force-extension behavior and shows us that we cannot deduce the forces we apply to the molecule out of the Fig.

2.8 (see Siscka et al. [10] Fig. 3B). Further possible error arise from the fact that the equilibration of the YOYO-1 with DNA is reported to last days at room temperature or by incubating at 50^◦C for 2 hours [33]. Furthermore, YOYO-1 has two binding modes so that the first binding (bis-intercalation) mode goes up to mixing ratio of 0.125 (dye:DNA base) and at larger mixing ratios the external binding starts to contribute [21]. This means that when incubation times are short the YOYO-1 is mainly attached to the surface of the DNA molecule and the contour length of the YOYO-1 loaded λ - DNA is not well defined as the intercalation between the base-pairs is still going on. This fact was also observed by Bennink et al. [34] who reported elongations of factor 1.1−1.2 when YOYO- 1 was incubated only 15 minutes. We incubated always at room temperature

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Figure 2.9: The rupture length distributions of stretchedλ- DNA molecules. The measurement was done with the setup presented in Fig. 2.5. The DNA molecules were end-grafted from one end with gold-thiol linkage and from other end with biotin-streptavidin linkage. The red histogram shows the rupture length distri- bution as the λ - DNA molecules were end-modified with four biotin molecules whereas in the gray histogram λ - DNA molecules had only one biotin in their ends. The gray histogram was already presented in Fig. 2.6. The average rupture length for the multi end-modification was roughly24µm whereas with single end- modification it was only 20µm. Data presented with permission from A. Andr´e.

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typically from 15 minutes to several hours, meaning that our YOYO-1 λ - DNA complex was not always fully equilibrated. Sischka et al. [10] do not report their incubation conditions or times making the comparison difficult.

As we analyze the end-grafting chemistry further we found that the crosslinker (glutaraldehyde) which we use to bind streptavidin to the surface is homobifunctional meaning that it has the same functionality in both of its ends. So upon incubation of glutaraldehyde on the amino silanized surface it can react directly with both of its ends with silane molecules and so the reactivity of the surface against the streptavidin is reduced. This leads to the fact that part of the streptavidin molecules are crosslinked over the lysine groups, one or multiple times, to the amino-silane surface and part of the streptavidin molecules are only ph- ysisorbed at the silanized surface. Further problem with glutaraldehyde is that its reaction with amino group containing compound proceeds over several possible routes (see Hermanson [19] page 119). In one of these reaction possibilities the aldehyde end of glutaraldehyde forms Schiff base linkage with amino group and the Schiff base has to be reduced before the bond is chemically stable (covalent).

Here arises the problem that the reduction has to be done after the streptavidin is crosslinked to the surface and the chemicals used for the reduction (for example sodium cyanoborohydride (NaCNBH₃)) are also reactive against streptavidin and may harm it [35]. Even if the chemicals were not reactive against streptavidin they would have difficulties finding their way to the bond to be reduced while the streptavidin is sitting on top of this bond [35].

As we already mentioned we have seen also rupture events from the gold-thiol end of the λ - DNA molecules. The gold-thiol bond itself is covalent [19] and should not break upon stretching. Where is the problem then? One potential explanation is that oligos, as they are hybridized to the λ - DNA molecules, are not covalently joined to the rest of the backbone meaning that the T4 ligase has not closed the backbone.

As we look at the data presented in Fig. 2.9 we notice a further interesting detail: we have found few molecules (∼3 %) which we can stretch over 35µm. As was already mentioned the contour length of YOYO-1 labelled λ - DNA is 19.8 µm. This would mean that we have stretched few molecules almost or more than 2 times their contour length. One possible explanation for these long molecules is that upon preparation of the end-modified DNA not every DNA - end is labelled with oligo but some of the molecules join together forming a double λ - DNA.

The remaining questions are: do we really have such double molecules and how big part of the DNA sample contains these double or more λ - DNA molecules also known as concatemers? This question will be discussed further in chapter 3.

As a further interesting detail from the stretching experiments we present Fig. 2.10 where λ - DNA molecule is being stretched between two surfaces¹¹.

11The end-grafting chemistry: Glass rod: Silanized with APTES, treated with glutaraldehyde, streptavidin and BSA. Bottom plate: silanized with GOPS and reacts directly with

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We have highlighted with a red box a part of the stretched molecule where the fluorescent dye is missing. As is clearly seen in the snapshots the dyeless area is within one molecule. However, we do not know whether we are stretching a single λ - DNA or two λ - DNA molecules joined together. This means that we cannot say if we are really stretching the molecule over its contour length. Liu et al. [36] reported that they have seen in their molecular combing¹² experiments similar missing fluorescent areas as we present in Fig. 2.10. They explained the missing fluorescence as DNA melting where nicks (backbone breaks) in one of the strands along double-stranded DNA provide a location for a melted single strand to fray back. According their explanation YOYO-1 does not bind on single-stranded DNA and therefore such a melting and back fraying would cause the fluorescent signal to vanish. This, however, is not completely true since for example Auzanneau et al. [37] reported that YOYO-1 binds also on single- stranded DNA. The melting scenario is nevertheless a valid explanation and can be accepted since when the intensity from the intercalated and from the fraying strand is missing from the total intensity, it is reasonable to expect that we cannot detect anymore the single strand of the DNA.

As such an event, where the fluorescence was missing within a stretched molecule, was very rare (less than 1 % of all the stretched molecules), we conclude that we have very little nicks in our end-modified λ - DNA. This is apparently because upon preparation end-modifiedλ- DNA, we add T4 ligase protein to the solution. The ligase protein is used to close covalently the backbone between the DNA molecule and the oligo molecule but it also repairs nicks within the whole DNA molecule [13].

Further analysis of the structure of stretched DNA–YOYO-1 complex is not possible since, as already mentioned above, YOYO-1 binds also to single-stranded DNA and as the strands are staying close together upon stretching we do not see if the DNA is melted without a nick in the backbone. Interesting question here is also why has not anybody reported such an behavior before? The answer is apparently that with tweezers one has limited stretching force of roughly 150 pN [10] whereas we do not have any limit but as already discussed above the exact analysis of the stretching force is not possible due to the missing calibration curve.

Additionally there is a thicker location ”bead” within the molecule and the gap actually arises inside of this ”bead”. We speculate that this ”bead” is apparently a coil of DNA which does not open upon stretching. It could even be that due to the coil of DNA, there is high amount of YOYO-1 concentrated at one location leading to photocleavage of a single DNA strand [38] and thereby provide an explanation for appearance of a nick in the backbone.

amino-modified DNA (see chapter 4.2.2).

12For molecular combing see chapter 5.2.2.

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(a) (b) (c) (d) (e)

Figure 2.10: A snapshot series of a single molecule as it is being stretched be- tween two surfaces. The end-grafting chemistry for the glass rod was so that it was first silanized with APTES, incubated with glutaraldehyde and finally with streptavidin and BSA. The bottom plate was silanized with GOPS and it reacts directly with amino-modified DNA (see chapter 4.2.2). Ina)andb)the molecule first elongates as it is being stretched. Inc) the molecule has reached length of

∼22µm and a gap of missing fluorescence is present in the molecule (highlighted with red box). The maximum elongation of the molecule is reached atd) where the molecule is ∼ 28 µm long. Finally after the rupture of streptavidin-biotin bond the molecule relaxes back to its end-grafting site as seen in e). We have added20µm long scale bar in (a) and the size of every snapshot is 6.5µm× 34 µm

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2.4 Conclusions

In this chapter we have presented simple but effective setup to study the quality of the end-grafts of single λ - DNA molecules on various surfaces. In order to relate the measured rupture length distributions to rupture forces we would need a force-extension calibration curve for YOYO-1 loadedλ- DNA which would have been measured in the same conditions as our measurements are done. But already now on the basis of the rupture length distributions we can say that single biotin- streptavidin linkage is not stable enough to reach the force-extension plateau with ensemble of molecules when the loading rates are below 10³ pN/s. Furthermore we found that the crosslinker glutaraldehyde does not always form covalent bonds with the amino groups and since it is a homobifunctional crosslinker it can react already alone at amino silanized surface leading to physisorption of streptavidin.

Finally we discussed also the possibility that part of the oligos are not covalently joined to the backbone of the λ - DNA molecule. This would explain low length rupture events from the gold-thiol side of the double sided end-graft.

Additionally we show that by using instead of a single biotin-streptavidin linkage but four of them, the average of the rupture length distribution shifted from 20 µm to 24µm. This is considerable enhancement but we cannot deduce, as already mentioned, the rupture forces out of our data yet since we do not have a calibration curve for YOYO-1 loaded λ - DNA.

Finally, in addition to the end-graft stability problems, we found that the DNA samples upon preparation can form concatemers and furthermore we found that the stretched DNA can locally fully loose its YOYO-1 dye. This effect was explained with the existence of nicks and DNA strand separation through melting.

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Preparation and characterization of end-modified DNA samples

Abstract

In this chapter we will show that the preparation of the end- modified λ- DNA presented in chapter 2 has problems in the form of free oligos which are still abundant after filtering in the samples.

The amount of the free oligos is estimated by using dot-blot and microarray techniques. As a result we show that through precipita- tion the oligo problem can be solved. Furthermore we estimate the amount of concatemers which were found to be present in the samples in chapter 2. This is done by field inversion gel electrophoresis (FIGE). As a result we show that the amount of concatemers is rather low on the order of few percents. Finally, in order to ob- tain high quality samples, where the molecules are to high extend both ends modified, we show that polymerase chain reaction (PCR) could also be used for sample preparation.

31

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3.1 Introduction

The preparation of end-modified DNA molecules for various single-molecule measurements has been already presented several times in the literature [2] [7] but the situation is rather different when the experiment is performed with thousands of molecules. Sample purity, homogeneity and modification efficiency are of much higher importance in such an experiment. Therefore in this chapter we will concentrate on the problems of the DNA modification with oligos.

Firstly we will look how big part of all the molecules are concatemers which we found previously in chapter 2. This is done with a field inversion gel electrophoresis (FIGE). Secondly we will study whether the filtering of our samples is good enough in terms of getting free oligos away upon preparation of end-modified DNA. This is first done by using dot-blot technique but since dot-blot is not giv- ing quantitative results, we redo the experiments with microarray reader by using fluorescent dye modified oligos. Finally we show that the use of polymerase chain reaction (PCR) could provide an alternative method for DNA sample preparation and it could circumvent most of the problems, we found earlier.

3.2 Material and methods

3.2.1 Field inversion gel electrophoresis

As was shown in chapter 2 the end-modified DNA samples, which were done over ligation of oligos, can also form molecules where for example two λ - DNA molecules are ligated together. In order to give an estimate about the amount of these concatemers, we used FIGE. The reason for the use of FIGE is that the classical electrophoresis cannot separate molecules longer than of about 50 kbp [39] and this is roughly the length of a single λ - DNA molecule. As can already be guessed from the name ”field inversion” the FIGE is actually nothing else but classical gel electrophoresis done in subsequent steps of periodically re- versing the direction of the electric field. The net movement is achieved by doing for example three pulses forward and one backward. Without dwelling in to the theory of the electrophoresis, the principle of FIGE can simply be understood so that long molecules get trapped around the agarose gel bundles while the short molecules can easily rotate around the obstacles upon the change of the field direction as presented schematically in Fig. 3.1 (a) and (b). So the inversion of the field has very little effect on the mobility of the short DNA fragments but the heavier DNA molecules are slowed down. In other words the limit mobility, that is the mobility where the molecules move with the same velocity independently of their weight, moves to higher molecular weight [39].

Experimentally FIGE is practically the same as classical gel electrophoresis, the only difference is that the classical constant current power supply has to be

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(a) (b)

Figure 3.1: The principle of FIGE:a) The mobility of the short DNA fragments is not affected by the change of flow direction as they can easily rotate around the gel bundles whereas b) the long molecules can get trapped around the gel bundles and so they are slowed down. Arrows indicate the direction of the flow induced by the electric field.

exchanged to one which has pulse controller in order to give pulses of varying length and polarity. In our FIGE setup¹ we had additionally a thermostat to control the temperature of the electrophoresis chamber. The FIGE experiments² were done in 1 % agarose gel with diluted (0.5 ×) TBE. The sample, pipetted into a single gel pocket, had a volume of 20 µl where 1/6 part of the sample was a loading dye, rest being the DNA solution. The main components of the loading dye, according to the manufacturer (MBI Fermentas), are bromophenol blue, xylene cyanol FF and glycerol. As bromophenol blue and xylene cyanol FF have blue color, it is easy to follow the progress of the electrophoresis since in 1

% agarose gels bromophenol blue comigrates with∼300 bp fragment and xylene cyanol FF with ∼ 4000 bp fragment. Furthermore the glycerol helps the DNA to sink into the gel pockets upon pipetting. The power supply was adjusted so that the difference between the electrodes was roughly 160 V and the current was roughly 70−80 mA. First the samples were driven into the gel for 10 minutes with constant current. Following 6 hours of forward/backward pulses in a ratio of 3 : 1 with increasing pulse duration from 20 to 30 seconds over the program

1Pulse controller (PC 500) and power supply (PS 500 XT), both manufactured by Hoefer Scientific Instruments, San Francisco / US.

2FIGE protocol provided by M. Leist, AG Nicotera, University of Konstanz (see Appendix B).

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run time. After that followed further two 6 hour programs where the pulse time was first going from 10 to 20 seconds and finally from 0.8 to 10 seconds. After the whole program was finished the agarose gel was incubated some 15 minutes in ethidium bromide, washed few times with water and filmed under ultraviolet (UV) light illumination.

3.2.2 The dot-blot technique

In order to study how effectively the NICK^{T M} column filters the unligated free oligos away from theλ - DNA samples, we used first a technique called dot-blot.

The dot-blot is used to detect if the samples under study contain biotin. This is done by attaching biotin containing macromolecule (here: biotin-modified DNA) to a membrane and the biotin which is abundant in the membrane after several washing steps is linked with streptavidin–horseradish peroxidase conjugate. The peroxidase is then in the final step detected over chemiluminescence.

The samples were directly pipetted on the dot-blot membrane in the volume of 2 µl containing various amounts of biotin-modified DNA and biotinylated oli- gos. The membrane is made out of nylon and since it is positively charged, the negatively charged DNA binds electrostatically on to it. The membrane with the pipetted samples was left to dry on a heating block for 30 minutes at 120^◦C. After that the membrane was illuminated with UV light for 2 minutes. The UV light causes covalent cross-linking between the amino group of the nylon and thymine base of the DNA³. After the UV treatment the membrane had to be blocked, so that there was no binding sites for unspecific binding. The blocking was done with 5 % milk powder in TBS-T⁴ buffer for 30 minutes while gently shaking (the membrane was soaked in the buffer). Finally, when the blocking was ready, the membrane was incubated with streptavidin–horseradish peroxidase conjugate for 30 minutes. Due to the blocking streptavidin can bind only over biotin which was already overλ- DNA or over oligos covalently linked to the membrane. After the incubation the unbound streptavidin was washed away. This was done two times for 10 minutes with TBS-T. The streptavidin–horseradish peroxidase conjugate was then left to react with enhanced chemiluminescence (ECL) chemicals which in the presence of peroxidase give a measurable chemiluminescence signal. In practice the membrane, which was soaked with ECL chemicals, was pressed together with a photographic film and let to stay for some 10 minutes. The amount of biotin bound to the membrane was then seen as a dark spots on a developed film which was exposed to the luminescence emerging from the chemical reaction of the peroxidase and ECL chemicals.

3See Stratalinker^r UV Crosslinker (http://www.stratagene.com/manuals/7003406.pdf).

4TBS-T contains 25 mM Tris-HCl, 125 mM NaCl and 0.1 % Tween 20.

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3.2.3 Preparation of Cy3-modified λ - DNA samples for the microarray reader

We wanted to have a complementary test method to the dot-blot to estimate the free oligo content of our DNA samples. In dot-blot the biotin content is estimated over chemiluminescence. Here we estimated the oligo content of the samples by modifying the DNA molecules with fluorescent Cy3 oligos⁵ and then by measuring with microarray reader⁶ the emitted fluorescence. The end-modification of λ - DNA with oligos was already presented in chapter 2.2.1 and the same protocol was also used here but naturally when working with fluorescent dyes all kind of exposure to light have to be avoided. Furthermore it is worthwhile to notice that repeated melting of the oligos reduces their intensity. Therefore, as the oligos are delivered as a dry powder, upon dissolving the oligos in milli-Q water the oligo solution should be divided in aliquots and stored frozen. Single aliquot should be used only once. The prepared samples can then be analyzed with microarray reader which is very similar device to a fluorescence microscope where laser excites the fluorophores and the light emitted by fluorophores is collected by CCD. The difference is that in the microarray reader the sample is scanned and the sensitivity is much higher when compared with CCD since the emitted fluorescence is collected by a photo multiplier tube (PMT) (see Fig. 3.2 (a)).

Furthermore the measured intensity of a microarray reader raises linearly with the measured fluorescence so that quantitative analysis are possible.

In a typical experiment we first made a mask out of plastic tape with some 10 spots and glued it on a glass slide (see Fig. 3.2 (b)). Then the DNA samples were prepared and pipetted on the spots and left to dry since with the microarray reader only dry samples can be measured. In all the steps we tried to avoid unnecessary exposure to light which would have bleached the fluorophores.

3.2.4 PCR and the preparation of monodisperse DNA samples

With the PCR high quality both ends modified double-stranded DNA (dsDNA) can be synthesized. At the starting point of the PCR procedure the dsDNA is briefly heated so that it melts and separates in to two single-stranded DNA molecules (ssDNA). After the strands have been separated the solution is cooled down to a temperature where a short ssDNA sequence called a primer⁷, which are in large excess in the solution, hybridizes to the matching complementary base-pair combination at the long ssDNA (annealing). A protein called Taq polymerase, which is also in large excess in the solution with the four types of

5Cy3 has its absorbtion maximum at 552 nm and emission maximum at 565 nm.

6GenePix Personal 4100A, Molecular Devices.

7Primers are typically some 20−30 base-pairs long.

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Laser

CPU PMT

Objective and scanner Filter

(a) (b)

Figure 3.2: a) The principle of the microarray reader: A laser excites the fluo- rophores found at the sample and the emitted fluorescence is collected, filtered and directed to the PMT. By scanning the sample slide a full picture of the sam- ple can be made. b) Typical sample of ours: a plastic tape was glued on top of a glass slide as a mask and then on the spots Cy3-modified DNA and free oligo mixture was left to dry.

deoxyribonucleosides, starts from the location where the primer is hybridized at the ssDNA and builds the complementary DNA strand out of the four deoxyribonucleoside triphosphates. The first cycle is ready when the polymerase has finished its work and the cycle starts once again from the start and the newly synthesized fragments serve as templates for the next round. In practice in the PCR program must be enough time reserved for the Taq polymerase to finish the synthesis (extension). This parameter depends naturally strongly of the length of the desired product. In each cycle the amount of dsDNA is doubled and typically some 20−30 cycles are done [14]. The principle of the PCR is also presented in the Fig. 3.3.

The primers are commercially available with the same modifications as oligos (biotin, amino- and thiol-group) but with the advantage that when a product is successfully gained it has in both of its ends a primer with modification which needs not be the case when modifying λ- DNA with oligos. In other words when a primer does not bind then there is no starting point for the polymerase and therefore there is no product. Another question is of course if every primer really carries the modification.

The work with PCR was started by producing first short 2 kbp pieces of DNA modified only from one end⁸. As a first step a suitable template and primers⁹

8The 2 kbp DNA sample is used in chapter 6.

9End-modified primer: biotin thiol- or amino-group-5⁰-gct gcg cgt aac cac cac acc-3⁰ and unmodified primer: 5⁰-ctg cgg cca act tac ttc tga caa-3⁰.

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3`

5`

3`

5`

3` 5`

5` 3`

Heating

3` 5` 3`

3` 5`

Cooling and hybridization of primers

5` 3`

3` P 5` P

5` 3`

3`

5`

5`3`

3` 5`

DNA synthesis with polymerase

5`

DNA synthesis is ready and the cycle can start again

M

1

M

2

M

1

M

2

5`

M

₂

M

1

Figure 3.3: The principle of PCR: First the dsDNA is melted by a brief heat treatment. In the subsequent step the temperature is lowered allowing primers, which are in large excess in the solution, to hybridize to complementary sequences on the melted ssDNA strands. After that Tag polymerase (P) synthesizes the complementary strand from the four deoxyribonucleoside triphosphates (also in large excess in the solution). After the polymerase has finished its work the cycle can start again by heating up the solution and melting DNA. As in the following cycles the newly synthesized DNA strands (primer limited) will serve as a template, the DNA which is between the primers will only be copied. This leads to the fact that after several cycles essentially all the DNA have a unique length limited by primers. The end-modifications M₁ and M₂ are introduced to DNA by using end-modified primers. The both ends modified DNA is finally created when the single stranded end-modified DNA molecules are hybridized together (this step is not shown). The letters3⁰ and 5⁰ show the direction of the backbone.