Download Stretching DNA Fibers out of a Chromosome in Solution

中國機械工程學刊第三十卷第四期第289~295頁(民國九十八年)
Journal of the Chinese Society of Mechanical Engineers, Vol.30, No.4, pp.289~295 (2009)
Stretching DNA Fibers out of a Chromosome in
Solution Using Electro-osmotic Flow
Min-Sheng Hung*, Osamu Kurosawa**, Hiroyuki Kabata*** and
Masao Washizu****
Keywords ： electro-osmosis, DNA, chromosome,
laser-induced heating.
ABSTRACT
This research develops a bio-nanotechnology
for mechanically handling individual DNA fibers.
The current study particularly focuses on surgery of a
chromosome, which is a long strand of DNA tightly
wound on proteins. Since DNA wound on a
chromosome is several centimeter long, it is too long
to observe under a microscope. Therefore, it is
necessary to develop a method for partial unwinding.
This work uses laser-induced local heating. Focusing
a laser beam onto an aimed location on a
chromosome immersed in protease solution, induces
temperature to rise and locally activates the
enzymatic reaction. This work demonstrates
stretching DNA fibers out of a chromosome using
electro-osmotic flow. Results show a chromosome
immobilized onto a glass surface with released DNA
fibers as long as 150μm.
INTRODUCTION
Chromosomal
DNA
encodes
genome
information of all the inheritable characteristics of an
organic structure. In the stage of post genome
handling, DNA information of individual person is
suitable to be used for protection or treating diseases.
To meet this requirement, the development of
bio-nanotechnology allows one to consider
conducting bioanalytical assays at very small
volumes to increase the speed of these assays and to
reduce the amount of material and reagents needed.
Paper Received August, 2008. Revised March, 2009, Accepted
April, 2009, Author for Correspondence: Min-Sheng Hung.
* Assistant Professor, Department of Biomechatronic Engineering,
National Chiayi University, Chiayi, Taiwan 60004.
** Researcher, ASTEM RI, Kyoto, Japan.
*** Sysmex Co., Kobe, Japan.
**** Professor, Department of Bioengineering, The University of
Tokyo, Tokyo, Japan. JST, CREST, Japan.
Fluorescent in situ hybridization (FISH) is a
common technique for locating specific genes on a
chromosome (Langer-Safer et al., 1982). The
principle of FISH is the hybridization of DNA by
using a fluorescence labeled oligonucleotide probes
which having the sequences complementary to the
unknown DNA sequences. The spatial resolution of
FISH depends upon the conformation of the sample
DNA. For a closely condensed chromosome, the
spatial resolution of FISH may be a few mega base
pairs (Alberts, et al., 2002). Heng et al. (1992)
demonstrated that the free chromatins released from
interphase nuclei for gene mapping. Their results
show that the spatial resolution of FISH can approach
10kbp (kilo base pairs) when DNA is extended to a
straight fiber. Therefore, physical manipulation of
DNA is a useful technique for studying genomic
DNA regions.
In fact, manipulation of single DNA molecules
has been recognized as an important technique in
molecular biology. This technique provides a new
analytical method completely different from
conventional analytical methods in biology and
biotechnology. Washizu et al. (1990) proposed and
demonstrated a microfluidic technology to stretch and
immobilize DNA in the solution by dielectrophoresis.
Recently, several studies have focused on
manipulation of DNA or physical properties of DNA
by using different methods. Wuite et al. (2000)
developed and integrated laser trap/flow control
video microscope for mechanical manipulation of
single biopolymers. For the purpose of understanding
their traits, they studied the elasticity of DNA using
the combined laminar/optical mode or the flow
control system alone to exert the force. Maier et al.
(2000) measured the replication rate by a single
enzyme of a stretched single strand of DNA. In their
study, a strong magnetic field gradient was generated
to exert on the magnetic bead and its tethered DNA
molecule. Further, the extension of DNA was
measured by real-time video analysis of the bead’s
image. Bennink et al. (2001) measured
force-extension relationships in polymer chains. To
be effective, they used optical tweezers setup to
attach a streptavidin-covered polystyrene bead to
each end of a biotin end-labeled single DNA molecule.
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J. CSME Vol.30, No.4 (2009)
Murayama and Sano (2001) measured the elastic
force for a single DNA molecule during a transition
between an elongated coil and a collapsed globule
state by using dual-trap optical tweezers. Hirano et al.
(2002) developed a manipulation technique for native
DNA molecules based on laser clustering. They held
and manipulated a single DNA molecule with
surprising dexterity using a bead cluster created by
laser trap.
All of the above-mentioned studies were
designed to manipulate flexible polymer chains by
using a single DNA molecule or single chromatin
fibers as their samples. One of the prior studies of
stretching DNA as opposed to the conventional
approach was reported by Washizu et al. (2003). They
presented the application of electro-osmotic flow for
stretching out of DNA fibers from a cell. In their
study, a yeast cell wall was removed using enzymatic
reaction and the cell membrane was dissolved using
sodium dodecyl sulfate (SDS) solution wash. The cell
prepared was immobilized on a microscope slide
having a pair of platinum electrodes. Their results
indicated that a yeast cell was immobilized onto a
glass surface and the released DNA fibers of as long
as 200μm were stretched. Hung et al. (2004)
demonstrated that the laser was focused to heat a
chromosome in solution. In their study, chromosomal
DNA was unwinding by laser-induced heating when
the protein degraded enzyme were in solution.
Applying force for extending DNA must not
exceed DNA fiber mechanical strength (about
100-300pN, Bustamante et al., 2000). Prior studies
discuss several methods for extending DNA,
including glass needles, magnetic beads, and optical
traps. But the above methods cannot sufficiently
control force and the DNA extended technique is
complex. The aim of the proposed study is to stretch
the chromosome to uncurved DNA fibers. In this
study, stretching DNA fibers out of a chromosome is
demonstrated under a fluorescence microscopy by
using electro-osmotic flow. The protocol in the
present experiment starts with the locally unwinding
of chromosomes, and then stretching chromosomal
DNA fibers out of chromosomes.
Cathode
Debye
length
Figure 1 Schematic illustration of electro-osmotic
flow.
the bulk liquid are attracted to the wall and shield
these wall charges. Likewise, dissolved co-ions are
repelled from the wall. The charged high capacitance
region of ions at the liquid/solid interface is called the
electric double layer. The ions in the inner layer of
counter ions adjacent to the wall are notably
immobile. The outer diffuse part of the layer forms a
net positive region of ions that span a distance on the
order of the Debye length (λd) of the solution. The
Debye length λd is defined as
λd =
1
2
(e /(εkT))∑ n i zi2
,
(1)
i
where e is the elementary charge, ε is the permittivity
of the liquid, k is the Boltzmann constant, T is the
temperature, ni and zi are the concentration and the
valence of ith ion respectively, and the summation is
to be taken over all ionic species present in the liquid.
The Deybe length is about 10nm from the wall for
symmetric
univalent
electrolytes
at
1mM
concentration (Hunter, 1981). Applying an external
electric field parallel to the wall causes ions to move
in response to the field and drag the surrounding
liquid along the wall, called electro-osmotic flow.
The velocity profile is governed by
STRETCHING DNA BY
ELECTRO-OSMOSIS
μ
The experiment in this work adopts the
electro-osmosis method for stretching DNA fibers
from a chromosome. This study regards
electro-osmosis as a liquid streaming induced by ion
drag in the electrical double layer at a solid/liquid
interface. Figure 1, shows this charge generation is
caused by electrochemical reactions both at the
liquid/solid interface and in solid surfaces. The main
reaction is deprotonation of acidic silanol groups that
produces a negatively charged wall. Counter ions from
External field E
Anode
d 2u
dx 2
= ρEE ,
(2)
where the coordinate x is taken perpendicular to the
solid surface, E is the field strength, ρE is the charge
density, μ is the viscosity of the liquid, u is the liquid
velocity parallel to the surface, with the boundary
condition u = 0 at the surface and u = u∞ = constant at
infinity. Fig. 1 schematically shows the velocity
profile caused by electro-osmosis in a solid surface.
Because ρE exists only in nm-thickness Debye length
above the wall, it creates a very large velocity shear
with a constant hydrodynamic drag, regardless of the
vertical position of the sample. Changing the
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M.S. Hung et al.: Stretching DNA Fibers out of a Chromosome in Solution.
magnitude and polarity of E also controls the
magnitude and direction of the shear.
In the flow field, when one end of a
chromosomal DNA has a length larger than λd and is
immobilized on the solid surface, the DNA fibers
extend along the flow with the degree of extension
controllable by the applied field magnitude, should a
large shear force be applied.
USING LASER-INDUCED
TEMPERATURE RISE TO
PARTIALLY UNWIND A
CHROMOSOME
The total length of DNA wound on a
chromosome ranges from several hundred
micrometer of bacterial or eucaryotic cells to several
meter of higher species. Since DNA is only 2-nm
thick, it is fragile and easily broken even by a gentle
flow of the surrounding medium during handling.
The present study attempts to extend the whole
chromosomal DNA, requiring a method for partially
unwinding a chromosome and stretching DNA fibers
out of the chromosome.
The chromosome is one of the small,
rod-shaped, deeply staining bodies that become
visible in the eucaryotic cell nucleus at mitosis. Most
interphase chromosomes are too far extended and
entangled for clearly observing their structures. In
contrast, chromosomes from nearly all eucaryotic
cells are readily visible during mitosis when they coil
up to form highly condensed structures. The
chromosome is an intricately folded nucleoprotein
complex with many domains, in which the local
chromatin structure is devoted to maintaining genes
in an active or silenced configuration to
accommodate DNA replication, etc. Chromatin is a
complex of DNA and proteins in which the genetic
material is packaged inside the cells of organisms
with nuclei. Thus, the fundamental subunit of
chromatin is the nucleosome, consisting of
approximately 165 base pairs (bp) of DNA wrapped
into two superhelical turns around an octamer of core
histones (two each of histones H2A, H2B, H3, and
H4), as shown in Fig. 2. Each nucleosome is
connected to its neighbors by a short segment of
linker DNA and this polynucleosome string is folded
into a compact fiber with a diameter of 30nm. The
30-nm fiber is stabilized by a fifth histone, H1,
binding to each nucleosome and to its adjacent linker
(Alberts, et al., 2002).
The molecular surgery of chromosomes
requires combining chemical and physical processing.
In order to destroy the DNA-protein complex,
enzymatic reactions can be used to proceed with this
operation; however, the timing and extent of the
reaction must be controlled in an appropriate manner.
The current investigation uses a laser-induced local
Histone
DNA
Figure 2 Schematic illustration of chromosome and
chromatin fiber. Each cylinder represents
one histone octamer protein core. The DNA
is wrapped around each octamer in a
left-handed, superhelical fashion.
Chromosome
IR Laser
Local relaxation
of chromosome
Pt Electrode
Extended DNA
Figure 3 Use of laser-induced heating for partially
unwinding chromosome and electro-osmotic
flow for the extending of a chromosome.
heating method for this purpose. Laser is often
applied to micromachining using the process of laser
ablation (Lai and Huang, 2007; Chou et al., 2008). In
the present work, focusing an IR laser beam onto an
aimed location on a chromosome immersed in
protease solution, induces temperature to rise and
activates the enzymatic reaction locally. Figure 3
depicts the chromosome process this study used
throughout the experiment.
Figure 4 schematically represents the setup
used for laser-induced heating, consisting of a
fluorescence microscope (OLYMPUS BX-60, Japan)
and optical heating with near-infrared light from a
CW Raman Fiber laser (1455nm, IPG Laser GmbH,
USA). The sample chamber was made of a coverslip
and a microscope slide, glued together at four corners
of the coverslip with manicure and mounted on a
stage which could be moved manually. To visualize
the chromosome, the sample was stained by DNA stai-
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J. CSME Vol.30, No.4 (2009)
SIT／CCD
RM
IP VCR
BE
DM2
Infrared
laser
EM
EX
Hg lamp
DM1
Objective
Slide / Cover glass
Figure 4 Schematic diagram of optical system of
laser-induced heating. The infrared laser
beam is expanded by beam expander (BE)
for adjusting the laser focus to an
observation plane of the objective. DM1
and DM2, dichroic mirrors; RM, reflective
mirror; IP, image processor. EX is barrier
filter to let through the excitation light only
and EM the emission light only.
ning
fluorescence
probes
YOPRO-1
and
4'-6-Diamidino-2-phenylindole (DAPI) (Molecular
Probes Inc.). The transmitted light was collected with
an objective (OLYMPUS, IR 100×, oil immersion,
NA 1.35, Japan) and imaged onto a SIT (Silicon
Intensify Target) camera (Hamamatsu C2400-08,
Japan) and CCD camera (Hamamatsu 5810, Japan).
The laser-beam path consisted of a 3.3× beam
expander and a dichroic mirror. The laser beam
focused on the sample using the objective, and the
diameter of the laser beam focus was approximately
2μm. The laser light was sent through the beam
expander and the objective, and a laser power meter
measured the transmitted light intensity. Samples
were made consisting of a chromosome protease
solution in 20mM MgCl2 solution. The protease
solution, used to degrade histones of chromatin, was
eukaryal prolyl endopeptidase (PEPase, Pfu
Proteinase S, TAKARA Co., LTD.). The optimum
temperature for PEPase activity was between 85oC
and 90oC with less than 5% of maximal activity
observed at room temperature and less than 30%
observed at 105oC (Harwood et al., 1997).
The process begins with enzymatic local
removal of histones from the chromosome with
laser-induced heating. The experiment demonstrates
partial unwinding of the chromosome by
laser-induced heating in PEPase solution. The
chromosome used is from human histiocytic
lymphoma cells (U-937). The cells are cultured in a
4ml medium (90% RPMI 1640 medium with 10%
fetal bovine serum) at 36.2oC. The cell culture is
treated and incubated 6 hours with 0.2μg/ml
colcemid solution to arrest sufficient cells in
metaphase, and harvested for the experiments. The
cells are subjected to hypotonic swelling, treated with
a detergent, and mechanically disrupted by passage
through a fine needle, and the chromosomes are
prepared in 20mM MgCl2 solution for the
experiments.
Figure 5 shows partial unwinding of the
chromosome before and after laser-induced heating.
The target chromosome is stained by DAPI. The
chromosome image is observed using the
fluorescence microscope at excitation wavelength
358nm and the fluorescence emission spectra is
approximately 461nm. When laser power is 22mW,
this study observes local unwinding of the
chromosome after laser-induced heating for 3
minutes, as shown in Fig. 5(b). Then laser-induced
heating is further increased to 5 minutes, and the
chromosomal DNA fibers are cut by UV excitation
light, as shown in Fig. 5(c). The excitation
wavelength for DAPI is approximately 358nm. The
short-wavelength light has stronger energy intensity
than the longer-wavelength light. The Fig. 5 result
shows that it is easy to destroy the structure for
unwinding
chromosomal
DNA
using
the
short-wavelength excitation for DAPI. Therefore, stai(a)
10µm
(b)
(c)
DNA
Figure 5 Unwinding of a chromosome (stained by the
fluorescence probe DAPI). The position of
the laser spot is indicated by close
arrowhead. The photo is taken by CCD
camera.
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M.S. Hung et al.: Stretching DNA Fibers out of a Chromosome in Solution.
E
(a)
stretched DNA
(a)
(b)
shrinking DNA
unwinding
10μm
10 μm
(b)
Photo
Figure 6 Partially unwinding of a chromosome
(stained by the fluorescence probe
YOPRO-1). The position of the laser spot
is indicated by close arrowhead. The
photo is taken by SIT camera.
ning DNA using DAPI is not suitable for stretching
DNA fibers from the partially unwinding
chromosome.
Figure 6 also shows the partial unwinding
chromosome before and after laser-induced heating.
The target chromosome is stained by YOPRO-1
(excitation 491nm / emission 509nm). The position of
the laser beam focus is the point of the arrow
indicated in Fig. 6(a). When laser power is 22mW,
this study more clearly observes local unwinding of
the chromosome after a laser-induced heating for 5
minutes, as shown in Fig. 6(b). The laser beam
focused on the chromosome in solution may attribute
to temperature increase due to optimum temperature
for PEPase activity by light absorption during focus
and subsequent heat dissipation to the bulk solution.
Fig. 5 and Fig. 6 results prove that laser-induced
heating achieves partial unwinding of the
chromosome to activate the enzymatic reaction
locally.
Figure 7 Initial stretching of chromosomal DNA.
of the electro-osmotic flow is from anode to cathode.
Figure 7 is the photo of the initial stretching
process on the left, and its schematic on the right. The
photo was taken under a fluorescence microscope.
The chromosome is on the left of the photo, and
chromosomal DNA fibers are coming out. Carried by
electro-osmotic flow, the DNA fibers move to the
right in the photo, as shown in Fig. 7(a). When the
field is removed, the flexible elastic property of DNA
fibers pulls the fibers back near the chromosome, as
shown in Fig. 7(b). Results show that electro-osmosis
successfully stretches DNA fibers and the results are
the same as Washizu et al. (2003) which stretched
DNA fibers from a cell. Figure 8 is a photo of a larger
area, the photo on the top and its schematic on the bot-
Stretched
DNA
STRETCHING CHROMOSOMAL DNA
chromosome
Stretching of chromosomal DNA by the
electro-osmotic flow is experimentally demonstrated.
For human cells, the physical length of homologous
chromosomes is approximately 4-5cm. The
chromosome prepared is immobilized on a
microscope slide having a pair of platinum electrodes.
A mild treatment with PEPase and laser-induced
heating is done to partially unwinding the
chromosome, and the field of about 1kV/m is applied.
When the voltage is applied, electro-osmotic flow is
induced, and the chromosomal DNA is pulled out of
the chromosome. It should be noted that the glass
surface in water is positively charged, so the direction
Schematic
10μm
chromosome
Figure 8 Stretching of chromosomal DNA up to
150μm. Top: photo, Bottom: schematic.
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J. CSME Vol.30, No.4 (2009)
tom. Observations show DNA fibers coming out from
the chromosome, which is stretched as long as
150μm. The extended length is much shorter than the
maximum length DNA in the chromosome. This
result may be caused by incomplete stretching due to
the chromosome not unwinding enough, or extended
DNA fibers cut by the excited fluorescence.
CONCLUSIONS
The present study investigates the extending of
whole chromosomal DNA from a singular molecular
chromosome. However, human homologous
chromosomes are complex and consist of both DNA
and proteins. Therefore, enzymatic digestion and
laser-induced heating are used to take apart the
DNA-protein to partially unwind chromosomes due
to its complexity. The stretching of DNA is made
with the use of electro-osmotic flow, which
generates a high velocity shear near the surface of
the glass where chromosomes are attached. In the
present study, the stretching of the chromosome is
experimentally demonstrated by using human
homologous chromosomes as sample. Our future
work will improve DNA fiber’s positioning and
anchoring for chromosomal DNA to extend enough
in solution to investigate FISH resolution.
ACKNOWLEDGEMENTS
This work was supported in part by BRAIN
(Seiken Kiko) Research and Development Program
for New Bio-industry Initiatives, and the National
Science Council (NSC 93-2313-B-415-003).
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NOMENCLATURE
M.S. Hung et al.: Stretching DNA Fibers out of a Chromosome in Solution.
E
e
k
n
u
T
x
electric field, [V/m]
elementary charge, [C]
Boltzmann constant, [J/K]
concentration of ion, [mol/m3]
liquid velocity parallel to the surface
temperature, [K]
Cartesian coordinate is taken perpendicular to
the solid surface
z
valence of ion
Greek Symbols
ε
permittivity of medium, [C/Vm]
electric charge density, [C/m3]
ρE
μ
viscosity of the liquid, [pas]
λd Debye length, [m]
Σ
summation of over all ionic species
Subscripts
i
with reference to ith species
以電滲流拉伸溶液中之染
色體 DNA
洪敏勝
國立嘉義大學生物機電工程學系
黑澤修
日本(財)京都高度技術研究所
加畑博幸
日本 Sysmex 股份有限公司中央研究所
鷲津正夫
日本東京大學生物工程學系
日本科學技術振興機構, CREST
摘 要
本研究為建立一套利用物理方法操作 DNA 之
生物奈米技術，主要實驗對象為真核細胞染色體。
由於真核細胞染色體是由 DNA 與組蛋白纏繞而形
成，必須將染色體上某一部位之 DNA 鬆弛，才能
進行 DNA 之操作。故本研究使用於物鏡下聚焦之
雷射間接加熱方式，在含有高溫時具較大活性之組
蛋白分解酵素之溶液中直接加熱染色體，並以電滲
流拉伸染色體 DNA。研究結果顯示染色體可被固
定於玻璃基板上，且電滲流可順利將 DNA 拉伸，
其拉伸之長度約可達到 150µm。
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