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Microchip Methods in Diagnostics
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For other titles published in this series, go to
www.springer.com/series/7651

ME T H O D S I N MO L E C U L A R BI O L O G Y ™
Microchip Methods in Diagnostics
Edited by
Dr. Ursula Bilitewski
HelmholtzCentreforInfectionResearch,Braunschweig,Germany

Editor
Dr. Ursula Bilitewski
Helmholtz Centre for Infection Research
Braunschweig
Germany
ISBN: 978-1-58829-955-0 e-ISBN: 978-1-59745-372-1
ISSN: 1064-3745 e-ISSN: 1940-6029
DOI: 10.1007/978-1-59745-372-1
Library of Congress Control Number: 2008939907
© Humana Press, a part of Springer Science+Business Media, LLC 2009
All rights reserved. This work may not be translated or copied in whole or in part without the written permission of
the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013
USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of
information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology
now known or hereafter developed is forbidden.
The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified
as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights.
While the advice and information in this book are believed to be true and accurate at the date of going to press,
neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that
may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein.
Printed on acid-free paper
springer.com

Preface
The continuously increasing degree of miniaturization of electronic circuits and the devel-
opment of corresponding fabrication technologies stimulated progress also in other fields,
such as analytical chemistry. The ideas of “labs-on-chips,” in which all manual procedures
required to obtain an analytical result are automatically performed on a chip, were pre-
sented almost 20 years ago, and fabrication technologies for DNA chips, which allowed
to obtain genetic information in a highly parallel manner, were suggested already in the
beginning of the nineties. These early dreams of miniaturized highly integrated analyti-
cal devices were based on the combination of developments in very different fields. The
development and industrial fabrication of integrated electronic circuits had shown that
by photolithography silica and glass could be precisely structured in all three dimensions
on the micrometer or even nanometer scale. In biology, amplification methods such as
the polymerase chain reactions (PCR), gene sequencing technologies, and biotechnologi-
cal production methods for proteins were established. In organic chemistry, methods of
combinatorial solid phase synthesis were developed, which made peptides and oligonu-
cleotides easily accessible, and analytical separation methods were developed in which
columns or planar surfaces were replaced by capillaries, such as in capillary electrophoresis
or gas chromatography. This was accompanied by improvements in detectors, which had
to deal with lower amounts of analyte molecules, a side effect of miniaturization.
Nowadays the field of microchip methods is rather heterogeneous, and there is even
no common definition for the different approaches and devices. In this book microchip
methods are analytical methods, which are based on miniaturized systems. This covers
not only arrays for the simultaneous determination of several analytes (DNA microarrays
and protein or peptide microarrays) or the simultaneous analysis of several samples (cell
arrays), but also labs-on-chips, for which phrases such as µTAS (micro total analytical
systems) or MEMS (micro electronic and mechanical systems) are also used. Whereas
miniaturization of arrays involves the sizes and densities of spots, labs-on-chips are mini-
aturized fluidic systems and are not necessarily multidimensional.
The present book wants to illustrate the diversity of possibilities, as they are applicable
now in medical diagnostics. Thus, only those approaches are included, which have reached
a certain degree of maturation so that they are applicable in practice also by the nonexpert,
and for some approaches the corresponding systems are even commercially available.
As mentioned earlier three types of microchips were chosen: DNA microarrays, protein
microarrays, and labs-on-chips in particular related to cell analysis. There is one chapter
for each of these areas as an introduction to the fundamentals of the respective technol-
ogy, followed by chapters describing methods related to specific applications. However,
the chosen examples are by no way comprehensive and the methods are easily applicable
to other diagnostic areas. This is in particular true for the field of DNA microarrays,
which is at present the dominating microchip technology allowing the analysis of gene
sequences and of gene expression. There is only one chapter for each of these applica-
tions (Chap.3 for gene expression analysis, Chap. 4 for gene sequence analysis), though
there are numerous publications on different diagnostic problems using different types of
arrays. However, the basic procedures are identical for all these applications, and are not
v

vi Preface
dependent on the particular diagnostic application and even not on the array platform.
Moreover, the increasing experience with these arrays shows that the comparability of
results among array platforms and laboratories is improved, if experimental protocols
are harmonized. Thus, the presentation of different protocols would be counterproductive
with respect to harmonization, if not the need for the deviations is discussed. The same
idea of representative examples was followed for the choice of contributions dealing with
protein arrays and fluidic or cell-based chips. However, compared with DNA microarrays
the number of commercially available systems is much less, and thus, the fabrication of
those systems is also included (in particular Chaps. 9 and 12, but also protein and peptide
arrays in Chaps. 7 and 8).
Although the application of DNA microarrays has developed into an essential tool for
biomedical research the applicability of this new technology in practical diagnostics is still
debated. Thus, Chap. 2 was included to discuss the comparability of diagnoses based on
microarrays and on established methods. Again leukemia profiling is to be considered just
as an example.
This handbook wants to support the introduction of diagnostic methods based on
microtechnologies, as miniaturization leads to a reduction of sample volumes for a single
analysis or allows the parallel determination of several analytes without the need for more
sample or time. Moreover, novel molecular information could be made available from
patient samples, such as more comprehensive information about gene or protein expres-
sion, improving diagnostic possibilities. Whether these expected benefits for patients prove
to be true can be verified only by application and validation in practice.
Ursula Bilitewski
Braunschweig, Germany

Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1. DNA Microarrays: An Introduction to the Technology . . . . . . . . . . . . . . . . . . . 1
Ursula Bilitewski
2. Discussion of the Applicability of Microarrays: Profiling of Leukemias . . . . . . . . 15
Torsten Haferlach, Ulrike Bacher, Alexander Kohlmann,
and Claudia Haferlach
3. Expression Profiling Using Affymetrix GeneChip Microarrays . . . . . . . . . . . . . . 35
Herbert Auer, David L. Newsom, and Karl Kornacker
4. Genotyping of Mutation in the Beta-Globin Gene
Using DNA Microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Martin Dufva and Lena Poulsen
5. Antibody-Based Microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Christer Wingren and Carl A.K. Borrebaeck
6. Application of Protein ArrayTubes to Bacteria, Toxin,
and Biological Warfare Agent Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Ralf Ehricht, Karin Adelhelm, Stefan Monecke,
and Birgit Huelseweh
7. Detection of Known Allergen-Specific IgE Antibodies
by Immunological Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Barbara I. Fall and Reinhard Nießner
8. Peptide Microarrays for Serum Antibody Diagnostics. . . . . . . . . . . . . . . . . . . . . 123
Heiko Andresen and Frank F. Bier
9. Microchips for Cell-Based Assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Martin Dufva
10. Bio-Cell Chip Fabrication and Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Honggu Chun, Dong Soon Lee, and Hee Chan Kim
11. Microchip Capillary Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
Elaine T.T. Tay, Wai S. Law, Sam F.Y. Li, and Larry J. Kricka
12. Detection of Enteropathogenic Escherichia coli
by Microchip Capillary Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Wai S. Law, Sam F.Y. Li, and Larry J. Kricka
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
vii

Contributors
KARIN ADELHELM • CLONDIAG Chip Technologies, Jena, Germany
HEIKO ANDRESEN • Fraunhofer Institut für Biomedizinische Technik,
Institutsteil Potsdam, Potsdam, Germany
HERBERT AUER • Columbus Children’s Research Institute, Columbus, OH, USA
ULRIKE BACHER • Bone Marrow Transplant Unit, University Hospital
of Hamburg-Eppendorf, Hamburg, Germany
FRANK F. BIER • Fraunhofer Institut für Biomedizinische Technik,
Institutsteil Potsdam, Potsdam, Germany
URSULA BILITEWSKI • Helmholtz Centre for Infection Research,
Braunschweig, Germany
CARL A.K. BORREBAECK • Department of Immunotechnology, Lund University,
Lund, Sweden; CREATE Health, Lund University, Lund, Sweden
HONGGU CHUN • Department of Biomedical Engineering, College of Medicine,
Seoul National University, Seoul, Korea
MARTIN DUFVA • Fluidic Arrays Systems And Technology (FAST), DTU Nanotech,
Department of Micro and Nanotechnology, Technical University of Denmark,
Kongens Lyngby, Denmark
RALF EHRICHT • CLONDIAG Chip Technologies, Jena, Germany
BARBARA ISABELLA FALL • München, Germany
CLAUDIA HAFERLACH • MLL Münchner Leukämielabor GmbH, München, Germany
TORSTEN HAFERLACH • MLL Münchner Leukämielabor GmbH, München, Germany
BIRGIT HUELSEWEH • German Armed Forces Scientific Institute for Protective
Technologies NBC Protection, Münster, Germany
HEE CHAN KIM • Department of Biomedical Engineering, College of Medicine,
ALEXANDER KOHLMANN • Roche Molecular Systems, Inc., Pleasanton, CA, USA
KARL KORNACKER • Columbus Children’s Research Institute, Columbus, OH, USA
LARRY J. KRICKA • Department of Pathology & Laboratory Medicine, University of
Pennsylvania Medical Center, Philadelphia, PA, USA
WAI S. LAW • Department of Chemistry, National University of Singapore, Singapore,
Republic of Singapore
DONG SOON LEE • Deptartment of Biomedical Engineering, College of Medicine,
SAM F.Y. LI • Department of Chemistry, National University of Singapore, Singapore,
Republic of Singapore
STEFAN MONECKE • Faculty of Medicine “Carl Gustav Carus”, Institute for Medical
Microbiology and Hygiene, Technical University of Dresden, Dresden, Germany
ix

x Contributors
DAVID L. NEWSOM • Columbus Children’s Research Institute, Columbus, OH, USA
REINHARD NIEßNER • Institute of Hydrochemistry, Chair for Analytical Chemistry,
München, Germany
LENA POULSEN • Microarray Group, Department of Micro and Nanotechnology,
Technical University of Denmark, Kongens Lyngby, Denmark
ELAINE T.T. TAY • Department of Chemistry, National University of Singapore,
Singapore, Republic of Singapore
CHRISTER WINGREN • Department of Immunotechnology, Lund University, Lund,
Sweden; CREATE Health, Lund University, Lund, Sweden

Chapter 1
DNA Microarrays: An Introduction to the Technology
Ursula Bilitewski
Summary
DNA microarrays allow the comprehensive genetic analysis of an organism or a sample. They are based
on probes, which are immobilized in an ordered two-dimensional pattern on substrates, such as nylon
membranes or glass slides. Probes are either spotted cDNAs or oligonucleotides and are designed to be
specific for an organism, a gene, a genetic variant (mutation or polymorphism), or intergenic regions.
Thus, they can be used for example for genotyping, expression analysis, or studies of protein–DNA inter-
actions, and in the biomedical field they allow the detection of pathogens, antibiotic resistances, gene
mutations and polymorphisms, and pathogenic states and can guide therapy. Microarrays, which cover
the whole genome of an organism, are as well available as those which are focussed on genes related to
a certain diagnostic application.
Key words: Specific probes, Immobilization, Hybridization, 2D pattern, Gene expression analysis,
ChIP-chip
DNA arrays are two-dimensional substrates on to which differ-
ent nucleic acids are immobilized as spots in an ordered pattern
(Fig.1). Each spot contains one type of nucleic acid and each
nucleic acid is a polymer of nucleotides of defined length, which
differs from the other nucleic acids in the array in the sequence
of the bases adenine (A), cytosin (C), guanine (G), and thym-
ine (T) (1,2). Depending on the diameter, density, and number
of spots the arrays are called macro- or microarrays (3). Typical
dimenisons of macroarrays are up to 22-cm length per side of
the substrate with up to approximately 1,200 spots per array
(www.clontech.com). They are made by spotting cDNAs with
1. Introduction
Ursula Bilitewski (ed.), Microchip Methods in Diagnostics, vol. 509
DOI: 10.1007/978-1-59745-372-1_1
1

2 Bilitewski
a length of 400–1,000 base pairs (bp) on positively charged
nylon membranes. Microarrays typically have the size of a micro-
scopic slide with up to approximately 100,000 spots/cm² and
are made of glass substrates onto which oligonucleotides are
spotted or synthesized. When similarities to electronic devices
such as the high degree of miniaturization and the application
of corresponding fabrication technologies are highlighted, DNA
microarrays are also called DNA chips.
DNA arrays are considered to be one of the major analytical
tools, with which the information from the various gene sequenc-
ing programmes can be explored. Nowadays sequences that are
characteristic for specific organisms or a particular feature of an
organism, or sequence variations [mutations or single nucleotide
polymorphisms (SNPs)] associated with disease are known. Thus,
it is possible to distinguish pathogenic from non-pathogenic
microorganisms and identify features such as antibiotic resistances
not only by microbiological methods but via the detection of the
corresponding genes without the need to cultivate the respective
organism (4). DNA samples can be analysed simultaneously for
several mutations in genes, which could increase the probability
for certain diseases (see Chap. 4) or influence the metabolism of
drugs (5). Most of these data were accessible in the past using
methods such as Southern blots, gel or capillary electrophoresis
in combination with PCR and restriction fragment length anal-
ysis, etc. With the introduction of DNA microarrays, however,
it became possible to simultaneously analyse several hundreds of
genes, even all genes in an organism, i.e. the whole genome. Applica-
tions range from genotyping of organisms, gene expression analysis for
example to classify patients according to the type of disease (see Chaps.
2 and 3) or to guide therapy, and gene–protein interaction analysis,
Fig. 1. Scheme of a DNA microarray. Each spot represents a specific probe, which is
characterised by sequence and length and can be a chemically synthesised oligonucle-
otide or a cDNA resulting from PCR. The scale of arrays can be adjusted to the applica-
tion, i.e. all ORFs identified in the genome of an organism can be represented in the
array by specific probes to allow comprehensive gene expression analysis, or a limited
number of genes is queried by probes in the array for a focussed application.
Spot
1a
Spot
1b .......
.......
.......
.......
Spot
1c
Spot
1d
Spot
1e
Spot
2a
Spot
3a
Spot
3b
Spot
2b
Spot
2c
Spot
2d

DNA Microarrays: An Introduction to the Technology 3
which is relevant for the identification of binding sites of proteins
that regulate gene expression (6). Among these application areas
gene expression analysis or “transcriptomics”, as it is called, when
it is performed on a genome-wide scale, is the best established,
whereas genome-wide analysis of interactions between proteins
and DNA is a more recent extension of chip applications (7, 8).
These investigations are called ChIP-chip experiments as they
are based on the combination of chromatin immunoprecipita-
tion (ChIP) and DNA chips. The following section is focussed
on the technological fundamentals of DNA chips as background
information for the application-oriented following chapters of
this book.
DNA arrays are based on the specific base pairing of comple-
mentary nucleotides (A-T and C-G) leading to double-stranded
sequences of nucleic acids. Unlike traditional analysis of blots the
strand being specific for the gene under investigation is immo-
bilized as capture probe and the corresponding counterpart, the
target, is isolated from the organism or cell culture and present in
solution. Thus, probes have to be designed that allow unambigu-
ous identification of genes, as no separation of nucleic acids with
respect to size occurs, and the target usually has to be labelled
prior to detection. In macroarrays, probes are cDNAs with a
length of 400–1,000 bp (e.g. www.eurogentec.com; www.clontech.
com), which leads to a high specificity for the targets. In micro-
arrays, probes are oligonucleotides with lengths <200 bp (e.g.
www.eppendorf.com; www.operon.com; www.ocimumbio.com;
www.agilent.com; www.affymetrix.com), which are chemically
synthesized either separately or directly on the chip (www.affyme-
trix.com). Synthesis on the chip is possible only for rather short
oligonucleotides of 25 bases. As the specificity of gene detection
is reduced, when short oligonucleotides are used, on those chips
the recognition of a single gene is based on the combination of
several probe oligonucleotides covering an extended sequence of
the target gene (see Chapts. 2.1 and 2.2) and control oligonu-
cleotides with a mismatch base in the centre of the sequence.
When longer probes are used (at least 50 bp), for each gene a
single specific probe is designed. All probes are immobilized on
the substrate in an ordered two-dimensional pattern (Fig. 1),
and on a single slide all open reading frames (ORFs) of a genome
or only a subset of genes can be represented (2).
2. Technology

4 Bilitewski
The design of probes and the choice of hybridization conditions
are crucial points for chip development and application and
still offer room for improvements (9). Here, only some general
aspects are mentioned:
The most important aspect is the specificity of the probe for
the gene of interest. In gene expression analysis RNAs are isolated
from the cells and labelled cDNAs are produced by reverse tran-
scription. The whole mixture is applied to the array and allowed
to hybridize (Fig. 2) (10). Thus, each probe should bind only the
transcript of the respective target gene among the presence of all
other transcripts. To acieve this degree of specificity with a single
probe per gene a minimal length of 50 nucleotides per probe was
reported (11). When shorter oligonucleotides are used, several
probes should be combined for each gene. Bioinformatic tools
and services are offered, which help to design probes of suit-
able sequences and lengths. The detection of mutations or SNPs
in genes does not allow the free choice of the probe sequence,
2.1. Probe Design
Fig. 2. Scheme of the experimental procedure.
Isolation of RNA
Labelling
Incubation with array
.......
.......
.......
.......
.......
.......
.......
.......
Control cell culture Sample cell culture
Fluorescence detection:
mRNA present only in control, mRNA present only in sample,
mRNA present in both cultures, mRNA present in none of the cultures

because this is given by the sequence neighbouring the variable
region in the target gene (6).
The hybridization reaction of nucleic acids is characterized
by the affinity constant and by the kinetic constants of the asso-
ciation and dissociation reaction. The affinity between two single
strands (i.e. probe and complementary sequence on the target)
is influenced by the sequences of the nucleic acids and by experi-
mental conditions. The guanine – cytosine (G – C) base pair con-
tains three hydrogen bonds, the adenine – thymine (A – T) base
pair only two, so that G – C-rich sequences are more stable than
A – T-rich sequences of the same length. Moreover, as each base
pair contributes to the strength of the overall binding, the affin-
ity increases with the length of the interacting sequences, i.e. the
number of matching nucleotides (12). If the sequence of matching
base pairs is interrupted by a mismatch, this does not totally pre-
vent hybridization of both strands but leads to a reduced stability
(13). The degree of destabilization is dependent on the lengths
of the remaining perfectly matching sequences and, thus, on the
position of the mismatch. Usually double strands are less stable,
when the mismatch is localized in an internal position compared
with mismatches at the end of the sequence (13, 14). Thus, signal
intensities increase with the length of the probe and decrease, if
mismatches are present in the centre of the sequence. If, how-
ever, variations in nucleotide sequences have to be detected, such
as in the analysis of SNPs, too long probes are not suitable, as it
is more difficult to find hybridization conditions, which allow
the distinction between completely matching sequences and sin-
gle mismatches. Thus, for SNP detection another assay format
was developed, called minisequencing, in which the specificity
of DNA polymerases for completely matched double strands as
substrates in the extension reaction of primers is utlized. The 3′-
ends of corresponding probes are the nucleotides of interest, so
that the primer is extended by a labelled nucleotide only in the
case of a perfect match. With different probes for each SNP to be
investigated, which query the different possibilities for the SNP,
identification of the respective sequence is possible (15). How-
ever, there is no free choice of probe sequences; only the length
of the probes is a variable, which can be used for the adjustment
to experimental conditions.
Even for given nucleic acid strands the affinity between the
two complementary strands can be influenced by additives to the
hybridization solution (16). It was found that formamide inhibits
the formation of hydrogen bonds, and thus it reduces the sta-
bility of the double-stranded helix. Moreover, nucleic acids are
negatively charged due to the phosphate groups of the nucleotide
backbone. Without compensation of this charge by counterions
even complementary single strands are electrostatically repelled
and monovalent cations (Na+
) are added to enable formation of

6 Bilitewski
double strands. Another important experimental parameter is the
hybridization temperature (14, 16), as double strands are sepa-
rated into single strands by increasing the temperature, a reaction
called “melting of DNA”. The temperature, at which 50% of the
double strands are dissociated, is called the melting temperature
Tm
of the sequence and is used for characterization of the stabil-
ity of the double strand. For oligonucleotides it can roughly be
calculated from the sequence by using the approximation
( ) ( )
= ° × − + ° × −
m 2 C A T 4 C G C ,
T (1.1)
with (A–T) being the number of A–T pairs and (G–C) the number
of G–C pairs in the sequence.
For longer hybrids the approximation
[ ] ( )
m 81.5 C 16.6log 0.41 %G C 500 /
Na
T c n
+
= ° + + − − (1.2)
is used with n being the length of the hybridising strand and cNa+
the concentration of Na+
ions.
If Tm
exceeds the hybridization temperature by 10–15°C,
efficient hybridization is observed. If the hybridization tempera-
ture is much lower than Tm
, hybridization of strands containing
mismatches or being only partly complementary will also occur.
However, the length of the probe is important not only for
the specificity of the hybridization and the stability of the double
strand, but also for the kinetics of the reaction. The hybridiza-
tion rate is mainly determined by the access of the targets to
the immobilized probes, which is influenced by the density of
the immobilized probes (17), the length and complexity of the
sequence, and by the diffusion rate, which depends on tempera-
ture, concentrations, and viscosity of the target solution. In longer
probes secondary structures may be formed and the complex-
ity of the sequence, i.e. the degree of non-repetitive sequences,
increases; both also lead to a reduction of the hybridization rate.
It was found that hybridization equilibrium is reached only after
at least 24h hybridization time, even if oligonucleotide probes
are used (14).
Probes, which are prepared by PCR or by chemical synthesis of
oligonucleotides, are applied to the substrate surfaces with spotters,
which allow the deposition of pL to nL volumes (10, 18). Contact
spotters dip pins into the probe solution and place the adher-
ing liquid to the substrate by touching the substrate surface.
Non-contact spotting relies on the generation of drops from a
capillary with piezoelectric pumps. The major disadvantage of
contact spotters is the need for precise adjustment of the height
of the pins so that contact forces and area are the same for all
spots. This is difficult to achieve in particular, when a number
of pins are combined for the simultaneous deposition of several
2.2. Immobilization

probes. Non-contact spotting requires a clean environment to
prevent blocking of capillaries by ambient dust. In any case the
low volumes rapidly evaporate, so that fast-binding reactions are
a prerequisite, though evaporation times can be prolonged by
additives, for example glycerol. Additionally, environmental con-
ditions, such as temperature and humidity, should be controlled
to increase the reproducibility of spot quality. Spots typically have
a diameter of 100–200 μm with spacings in the same order of
magnitude (200–300 μm), so that up to 244,000 features (spots)
are combined on a single slide (www.agilent.com).
Nucleic acids (oligonucleotides or PCR products) are immo-
bilized on glass slides or nylon membranes (3). However, systems
based on beads or tubes are also available (www.illumina.com;
www.clondiag.com).
As nucleotides are negatively charged, they interact by elec-
trostatic attraction with positively charged surfaces as delivered
by nylon membranes or glass slides pre-treated with poly-L-lysine
[e.g. (10); http://cmgm.Stanford.edu/pbrown/protocols] or
aminopropyltriethoxysilane (APTS or GAPS) [e.g. (19); www.
corning.com]. If PCR products are used, they are denatured
either prior (19) or after spotting. Usually, UV irradiation is re-
commended as an additional cross-linking step, but heating to 60
and 120°C is also possible (10). As each nucleotide contributes
with an additional charge, the electrostatic forces increase with
increasing length of the nucleic acids, which makes this immo-
bilization method applicable mainly for longer probes, such as
cDNAs. Zammatteo et al. (19) showed that for a 255bp capture
probe the efficiency of this electrostatic attraction exceeded the
efficiency of even covalent attachment.
The alternative to immobilization via physical interactions is
covalent binding. This requires the availability of suitable func-
tional groups on both the probe to be immobilized and the
immobilization substrate, usually glass. Amino-functionalized
nucleic acids can be coupled easily to epoxy- or aldehyde-modified
glass surfaces (10). Resulting Schiff bases can be reduced by
sodium borohydride. This method proved to be highly effective
and specific and suitable for the application of very small volumes
of liquid (19).
As solid-phase synthesis of oligonucleotides is well established,
oligonucleotide capture probes can also be synthesized directly on
the chip surface. This was described by Pease et al. already in 1994
(20), and later (1996) by Weiler and Hoheisel (21) and Blanchard
et al. (22). The basic reaction is the reaction of phosphoramidite-
activated deoxynucleosides with suitable functional groups, usu-
ally hydroxyl groups, on the glass or polypropylene surface. At
Affymetrix these hydroxyl groups are generated at selected sites
by illumination of the chip through an appropriate mask (1, 18
20). In a first step the solid support is derivatized with a covalent

8 Bilitewski
linker molecule terminated with a special, photolabile protect-
ing group. Illumination leads to deprotection and the formation
of hydroxyl groups. In the next step the nucleoside derivative
to be coupled is added, being the corresponding 3′-phosphor-
amidite and 5′-photoprotected. Illumination with another mask
generates a different pattern of hydroxyl groups allowing each
desirable sequence to be synthesized. As the number of probes
in one array is limited by the physical size of the array and the
achievable photolithographic resolution, approximately 750,000
oligonucleotides were synthesized on 1.28 × 1.28 cm² chips with
a spot diameter of only 5 μm (www.affymetrix.com). A sequence
of 200–300 bases of the gene of interest is chosen and a number
of non-overlapping 25-mer probes are designed and synthesized
on the chip together with mismatch control probes containing
a single base difference in the central position. This redundancy
should improve accuracy and improve the signal-to-noise ratio.
Hybridization of a target nucleic acid to the immobilized probe
is an affinity reaction between two complementary reaction part-
ners. Hence, this reaction was followed in real time by affinity
sensor systems, such as surface plasmon resonance (SPR) devices
[e.g. (13, 17, 18)], resonant mirrors (12), or grating coupler
systems (23). Real-time monitoring of the hybridization allowed
the analysis of the influence of probe and target length, probe and
target concentration, and the position of mismatches not only on
the resulting steady state signal but also on the association and
dissociation rates. It was shown that the affinity constants deter-
mined by SPR correlated well with melting temperatures and that
decreasing affinities due to decreasing lengths of the target influ-
enced mainly the dissociation rate (13).
Usually the detection of mRNAs utilises specific features of
nucleic acids, i.e. the possibility to synthesise a copy DNA strand
(cDNA) by a reverse transcriptase reaction, which allows the
integration of labelled nucleotides as components of the reac-
tion mixture. Suitable labels are radioactive isotopes, such as
³³P or ³²P (www.clontech.com), fluorescent dyes, biotin (www.
clondiag.com), amine groups, or micro- and nanoparticles (3, 10,
24). Labelling with biotin or with amine groups requires addi-
tional staining, e.g. with streptavidin conjugates or conjugates of
an anti-biotin antibody with horseradish peroxidase or with gold
(www.eppendorf.de) or with amino-reactive fluorescent dyes. The
advantage of gold labels is the light pink colour, which appears on
the arrays and which can be amplified by silver deposition from
the reduction of silver ions with hydroquinone, so that success-
ful hybridization is visible and can even be quantified by a simple
flatbed scanner (24). With fluorescence detection, however, it is
possible to use different dyes for the control and the sample, and
combine both labelled mixtures during hybridization so that a
2.3. Detection
Principles

direct comparison of signals on the same array is possible (Fig. 2).
As fluorescence intensities and labelling efficiencies usually are
different for different labels, a dye-switch has be performed, i.e.
the dye previously used to label the control has to be used to label
the sample and vice versa.
Consideration of the aforementioned labels usually integrated
in cDNAs (Cy3, Cy5, fluorescein, Alexa 647, phycoerythrin,
biotin) shows that the most often used detectors are fluorescence
detectors allowing the analysis of chip surfaces. Light sources are
preferably lasers with the appropriate wavelengths. Nowadays
systems are available with more than one light source, which
allow the excitation of different dyes. The emitted fluorescence is
captured by CCD cameras or by photomultipliers with the latter
showing the higher sensitivity.
A two-dimensional pattern of spots of different intensities results
from scanning the array (Fig. 2). Independent of the detection
principle signals are converted to electrical signals and, thus,
appear in a grey scaling. Typically signals from the control and
the sample are shown with different colours, which, however, are
not the true colours of the labels, but are artificially chosen.
Because of the highly regular arrangement of spots, grids
specifying the spot locations can be easily overlaid on the images.
To quantify the signal intensity of each spot the background has
to be quantified, which is usually done by measuring signals in a
circle surrounding the spot. These background values are used
for correction and filtering and only data that are significantly
above the background are considered for further evaluation. Sig-
nificant intensities are two standard deviations above the back-
ground level (10). These data are the basis for the comparison of
different samples, which requires further data treatment, such as
data transformation to a logarithmic scale, calculation of ratios,
and normalization to account for different amounts of nucleic
acids in each sample or for different labelling efficiencies. The
final outcome of data analysis should be a matrix, in which for
each gene (row in the matrix) a quantitative measure is given
for each sample (column in the matrix) (Fig. 3). There are sev-
eral challenges, which make a direct access to these data difficult,
some of which were already mentioned (different signal intensi-
ties resulting from different labels and varying amounts of nucleic
acids). In addition, a single gene may be represented by several
probes; each array may be present several times on the same slide,
and the experiments should be repeated to give biological replicates.
3. Data Analysis

10 Bilitewski
Fig. 3. Simplified scheme of data analysis. Signals are indicated just as qualitative data as “present” or “absent”,
whereas in practice quantitative information can be obtained from data acquisition. From each sample several values
exist for each gene, as experiments have to be repeated (replicates), a dye-switch may be necessary, or each gene is
represented by more than one probe on the chip. This information has to be extracted, so that finally in each sample or
experimental condition a single value can be assigned to each gene, which is represented on the chip. This results in a
matrix, in which the samples are the columns and the genes are the rows. Cluster algorithms are used to cluster genes
according to a similar behaviour in the different samples.
Samples
Sample 1
Genes
Sample 2 Sample 3
Replicates and dye-switch

All these data have to be combined to a single value for each
gene, and the procedures to achieve this goal are not standard-
ized. Thus, according to the guidelines of Minimum Informa-
tion About a Micoarray Experiment (MIAME) (www.mged.org/
Workgroups/MIAME/miame.html), which are proposed by the
Micoarray Gene Expression Database group (MGED), not only
the final data, from which conclusions were drawn, are to be
delivered, but also information about data processing routines
and the raw data (25).
Microarrays are used for genotyping, as they allow the detection
of specific genes or gene variants. Pathogenic bacteria (26) and
fungi [(27), www.clondiag.com] can be detected via species-
specific sequences within the ribosomal DNA, and for the analy-
sis of resistances against antibiotics sequences that are specific for
ß-lactamase genes can be used (4). For example 27 and 28 oligo-
nucleotides, respectively, with a length of up to 24 bp proved to
be sufficiently specific and were immobilized as probes on glass
slides to distinguish 12 Candida and Aspergillus species or sev-
eral bacterial groups (gram-negative and gram-positive) and spe-
cies (e.g. Mycobacterium tuberculosis, Listeria monocytogenes, and
Staphylococcus aureus). For pre-enrichment of organisms samples
were incubated overnight (26) or for up to 72 h (filamentous
fungi). After DNA isolation, PCR was performed to amplify the
target region of the DNA and introduce fluorescent labels. Hun-
dred and twelve out of 115 strains of bacteria isolated from food
(26), and in 16 out of 21 clinical isolates the fungal strains (27)
were correctly identified requiring just a single relatively short
cultivation step.
The detection of sequence variants, such as mutations or SNPs
that are causes of diseases, was described as early as 1989 (15, 28).
A frequently applied format for SNP analysis is minisequencing or
allele-specific primer extension, in which the 3′-end of the probe on
the microarray is the variable nucleotide. The primer can only be
extended with a fluorescently labelled nucleotide by a DNA polymer-
ase, if there is a perfect match. Thus, fluorescent spots indicate the
matching sequences. Alternatively, with high stringency conditions
hybridization to only allele-specific probes can be achieved, so that no
additional extension reaction is required. For reliable analysis sense
and anti-sense probes should be used, with different probes for all
four possible nucleotides at the target position within the sequence.
The length of the probe, the position of the target nucleotide
4. Examples for
Applications
4.1. DNA Analysis

12 Bilitewski
(close to the centre of the probe), and hybridization conditions
have to be carefully optimized, as for high-quality SNP results
only perfectly matching sequences should give a hybridization
signal (www.febit.eu).
Transcriptional profiling, i.e. the detection of mRNAs that are
present in the cell or tissue at the time of sampling, is one of the
major application areas of DNA microarrays (3). As these data
indicate, which genes are required in a given state of the cell or
organism, the profiles are used to distinguish pathogenic from
healthy states (e.g. cancer diagnosis) and guide therapeutic strat-
egies, to examine the functions of genes or elucidate the mode
of action of drugs (29) or toxic compounds (30, 31). Suitable
microarrays can comprise oligonucleotides but also cDNAs, and
may cover all ORFs identified within the genome or just a subset
of ORFs.
Repetition of experiments in different laboratories, with dif-
ferent array platforms and even in the same laboratory usually
shows more or less significant variations. These are partly due to
different experimental protocols starting from sample prepara-
tion to data analysis and can be minimized by standardization
of as many steps as possible (31). Other fluctuations, however,
are due to biological noise, which means that the expression of
some genes is inherently sensitive to already minor differences in
cell treatment. These genes can only be identified by repetitive
analysis and should be eliminated from the analysis of transcrip-
tional profiles. Thus, the reliability of an expression signature is
not necessarily improved when the number of genes in an array
is increased.
DNA-binding proteins perform a variety of important func-
tions in cells, such as the regulation of gene expression. Thus,
the analysis of interactions of transcription factor proteins with
their respective DNA-binding sites in response to environmental
stresses or associated with the progression of diseases has become
an important issue, and several new techniques were developed,
which allow this type of analysis on a genomic scale. Among
these, ChIP-chip analysis, also called ChIP-on-chip analysis, and
the DamID technologies are based on microarrays (7, 8).
ChIP-chip is the combination of chromatin immunoprecipi-
tation with microarray detection. The cellular sample is treated
with formaldehyde so that DNA-binding proteins are cova-
lently attached to their target DNA. Shearing of the chromatin,
immunoprecipitation of the protein–DNA adduct from nuclear
extracts with specific antibodies, and reversal of the formaldehyde
cross-links leads to an enrichment of the target DNA sequences.
They are amplified, labelled with a fluorescent dye, and finally
hybridized to the microarray. Comparison to a reference sample
4.2. mRNA Analysis
4.3. Analysis of
Protein–DNA
Interaction

without enrichment shows the binding regions of the protein of
interest. This analysis was first applied to transcription factors of
the yeast S. cerevisiae and showed that binding of regulatory pro-
teins occured even several kilobases away from the transcription
start of genes. The major drawbacks of this principle are the lim-
ited availabilities of suitable antibodies and microarrays and the
low expression of some transcription factors. The first ChIP-chip
experiments were performed with microarrays spotted with PCR
amplicons covering essentially all intergenic regions. With the
utilization of microarrays with oligonucleotides designed to tile a
portion of the genome, binding sites of proteins can be defined
with higher resolution. However, this type of microarrays is avail-
able only to a limited degree. Low expression of transcription
factors or antibodies with poor affinities results in only a poor
enrichment of the target sequences. This problem is overcome to
a certain extent by the DIP-chip assays and by the DamID tech-
nology. DIP-chip assays are based on purified proteins, which are
in vitro incubated with genomic DNA fragments so that binding
occurs. The concentration of the protein is defined and known,
and as it is expressed with a suitable tag, it can easily be separated
from the sample and the bound DNA sequences are analysed
as described earlier. However, as this is an in vitro assay, results
do not reflect a physiological state (8). DamID technologies are
based on overexpression of the protein of interest as a fusion to
Dam (DNA adenine methyltransferase). Dam methylates ade-
nine in the vicinity of the protein binding site. Digestion with a
methyl-specific restriction enzyme, amplification, labelling, and
hybridization then highlight the target regions. However, defini-
tion of binding sites with high resolution is not possible, because
methylation can extend over a few kilobases from the binding
site (7).
These analyses are recent extensions of the applications of
DNA microarrays, and are yet mainly used in research.
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across platforms, Nat. Methods 2, 1–6

Chapter 2
Discussion of the Applicability of Microarrays: Profiling
of Leukemias
Torsten Haferlach, Ulrike Bacher, Alexander Kohlmann,
and Claudia Haferlach
Summary
Leukemias are classified according to clinical, morphologic, and immunologic phenotypes, caused by
specific genetic aberrations in association to distinct prognostic profiles. Usually the subtypes are defined
using complementary laboratory methods, such as multiparameter flow cytometry, cytogenetics in com-
bination with fluorescence in situ hybridization, and molecular methods such as the polymerase chain
reaction. The genetic variations of the different subtypes lead to distinct changes also in gene expression,
which is comprehensively analysed by DNA microarrays. Thus, first gene expression profiling studies
showed that analysis with whole-genome DNA microarrays leads to a prediction accuracy of 95.6%
with respect to the classical methods, and even allowed a further distinction of subtypes. It is expected
that diagnostic strategies can be optimized with this new technology and that the understanding of the
molecular pathogenesis of leukemias will be significantly improved. This could also lead to the identifica-
tion of new targets for future drugs.
Key words: Gene expression profiling, ALL, AML, CLL, Classification of subtypes
Acute myeloid leukemia (AML), acute lymphoblastic leukemia
(ALL), and chronic lymphatic leukemia (CLL) are very hetero-
geneous disorders composed by a variety of different subtypes.
These subtypes are defined by different clinical, morphologic, and
immunologic phenotypes caused by specific genetic aberrations
in association to distinct prognostic profiles. Chronic myeloid
leukemia (CML) is defined by the Philadelphia or BCR-ABL
1. Introduction
Ursula Bilitewski (ed.), Microchip Methods in Diagnostics, vol. 509
DOI: 10.1007/978-1-59745-372-1_2
15

16 Haferlach et al.
translocation, but the progress of stages is accompanied by
acquisition of additional chromosomal abnormalities (“clonal
evolution”).
In AML clonal karyotype abnormalities are detected in
~55% of all cases; in ALL in ~80%. These abnormalities cover
a broad spectrum of numerical changes and balanced and
unbalanced translocations. Karyotype is the strongest prog-
nostic parameter in AML and in ALL: In AML survival ranges
from ~75% in the patients with the favorable reciprocal rear-
rangements t(8;21), t(15;17), and inv(16)/t(16;16) to <10%
in complex aberrant karyotype, which is defined by the simul-
taneous occurrence of 3 clonal chromosomal changes. In
childhood ALL the reciprocal t(12;21) rearrangement shows
an excellent prognosis with >90% long-term survival following
standard chemotherapy, whereas Philadelphia positive ALL
with the t(9;22) shows survival of ~40% even when allogeneic
stem cell transplantation is performed.
Cases with normal karyotype can in the majority of cases be
further characterized and categorized by molecular investiga-
tions. The molecular mutations are likewise heterogeneous and
affect genes coding for transcription factors, tyrosine kinases,
protooncogenes, or tumor suppressor genes, which show specific
interactions.
In the majority of CLL cases it was as well possible to deter-
mine prognostically relevant genetic aberrations, such as dele-
tions of the 17p53 tumor suppressor gene, which are associated
to an inferior outcome. In CML clonal evolution is found in ~5%
of all patients at diagnosis only, but the acquisition of additional
karyotype abnormalities during follow-up predicts progress and
serves as a marker of progression.
This diversity of leukemia subtypes can only be mastered
by a combination of complementary laboratory methods: cyto-
morphology/chemistry, immunophenotyping by multiparam-
eter flow cytometry (MFC), cytogenetics in combination with
diverse fluorescence in situ hybridization (FISH) techniques,
and molecular methods such as the polymerase chain reaction
(PCR). This interactive approach only allows an optimized risk
stratification as basis for individualized therapeutic decisions.
Beyond that, the interplay of these methods paved the way
to targeted therapy in some leukemia subtypes, e.g., in acute
promyelocytic leukemia (APL) with t(15;17)/PML-RARA.
This AML subtype is characterized by a differentiation stop
in granulopoiesis and by aberrant promyelocytes with multiple
Auer rods (“faggot cells”). Therapy with all-trans retinoic acid
(ATRA) eliminates the differentiation stop in granulopoiesis
and can prevent the life-threatening complications that often
result from coagulopathy, which is pathognomonic for this
leukemia subtype.

Discovering Diverse Content Through
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XXIV.
IN THE DOME OF THE SKY.
There are three ways of reaching the summit of Pike’s Peak—
walking, riding a burro, or seated comfortably in one of the coaches
of the Cog Road.
It was three o’clock in the afternoon when the car was pulled out of
the yards at the foot of the Peak. The strongly-built little engine
puffed like a living thing, obedient to the task of drawing its heavy
load. The wheels moved rapidly, and we ascended the steepest
mountain railroad in the world. It wound about the mountain sides
in little curves, climbing, always climbing higher and higher, until we
shuddered at the dizzy heights as we looked down into the great
yawning chasms thousands of feet below.
The air grew cooler in the deep mountain defiles densely wooded
with fir, pine, cedar and quaking asp. A great fire once swept up
these gorges and burned away the fir and pine in patches; in their
place came the quaking asp, growing here and there in thickets.
Along the slopes and in the dells, wild flowers grew with the
luxuriant profusion of a semi-tropical clime. There were columbines
and tiger lilies growing at an altitude of ten thousand feet.
Nature has done some queer things in the mighty rocks which stand
sentinel guard along the route.
One great boulder is named the Hooded Monk, because of its
resemblance to the human head in a monk’s cowl. There is a Gog
and Magog. The Sphynx, the Lone Fisherman, and many other
images of man, bird and beast, wrought by nature’s hand in stone.
We glided by one of the loveliest glens in all the mountains; it was
called Shady Springs. Here the oriole, the raven and the big blue jay
of the mountains have builded their nests and take their morning

baths in waters clear as crystal from a spring that gushed from fern
and moss covered banks.
Farther on to the right a stream plunges in wild, mad swirl of clear
waters and dashing from rock to rock in foamy white, forms Echo
Falls. An elephant’s head in bass relief was here to be seen wrought
in stone.
We rounded Cameron’s Cone and Sheep Mountain and soon began
the ascent of the “Big Hill,” which has a rise of 1,300 feet to the
mile.
Nearing timber line, the road ahead appears to be almost at an
angle of 45 degrees.
Higher and higher; the great chasm below grew almost a mile
deeper. On one side there were masses of square rock which looked
like they were broken by human hands. Here, far above timber line,
a variety of wild flowers blossomed, while among the rocks lived
some of the strangest little animals, the whistling marmot, a fur
animal about the size of an overgrown cat, and the peka, which has
the legs of a rabbit and the head of a mountain rat; there were also
minks, weasels, porcupines and mountain rats.
At the summit was where the magnificence of the great panorama
burst upon our view. Northward, away down on the bluish haze of
the horizon, rose the Arapahoe peaks—Long and Grey’s Peak, with
their white summits glistening in the setting sun. Northwest, Mt.
Massive and Mt. Sheridan were outlined against the clear blue sky,
while the green sward of the famous South Park, a hundred miles
distant, lay between. College Range, Mt. Yale, Mt. Princeton, Mt.
Ouray and Cavenaugh reared their rugged heads far to the west,
while green mountain ranges of lesser note lay half way between
them.
Far to the southwest, far as the eye could reach, faintly outlined
against the sky, rose the snowy peaks of the Sangre de Christo and
Sierra Blanco Mountains on the other side of the grand San Luis
Valley.

Looking to the south, were the Spanish Peaks and range of
Greenhorn Mountains, and a little to the southeast rose the snow-
capped Gloriettas on the borders of New Mexico.
To the east, lay the mighty plains, stretching away to where the blue
of the sky blended in coppery tones with the billowy green.
There were dark spots here and there that were dense forests of
pine. The cloud banners hung above, in all the gorgeous colors of
sunset in crimson, purple and gold.
A dark shadow crept out upon the plain toward the east, like the
finger of a mighty giant. It moved rapidly along, covering the yellow
sand lines that mark the course of old river beds, and finally, this
shadow of Pike’s Peak was covered by the shadows of other
mountains lower down, until the plain was shrouded in the sable
garb of eventide.
But westward, the gold and crimson of the sky lingered long above
the distant peak of Mt. Ouray. The purple haze grew denser, and the
silence of the hour was made more solemn by the mountains
standing out in dark silhouette as the shadows of the night grew
deeper and denser.
At such a time as this, one feels as though he stood upon the
boundary of another world, while all about the wide white waste and
hush of space, eternity and the infinite were calling to other glories,
too great for the understanding of the human mind.
Here, in the very dome of the skies, in this clear air, the bright
worlds seem to hover over, while the vault is strewn with stars, like
isles of light in the misty sea above our heads. The purity of the
heavenly prospect awakens that eternal predisposition to
melancholy, which dwells in the depth of the soul, and soon the
spectacle absorbs us in a vague and indefinable reverie. It is then
that thousands of questions spring up in our mind, and a thousand
points of interrogation rise to our sight—the great enigma of
creation.

The harvest moon shed her yellow light over the distant plain, and
gilded with a phosphorescent light the rocks and crags of the almost
bottomless chasm below. The rocks took on fantastic shapes, while
distant mountains rose in spectral form.
I sat throughout the night, watching the ever changing panorama,
the most wondrous ever spread out to the gaze of man.
The moon and stars were bright above, while far down below storm
clouds had formed where within their inky blackness the forked
lightning played like so many fiery serpents.
There were thunderous crashes in the wild rocky pit below, where
huge rocks were shivered by lightning bolts, while echo, echoing
back the thunders of heaven’s artillery, would seem as though a
legion of imprisoned Joshuas were reaching upward again for that
sun which would stand still no more over the plains of Agalon.
The shades of night grew deeper and then the blackness was driven
back from the east by a flush of grey, gradually changing to a deep
scarlet tinged with yellow and the sun burst above a dashing sea of
clouds. There were purple and crimson waves below rising and
falling in mighty billows. A shipless and shoreless ocean whose
raging bosom claims no living thing.
An hour more and this purple sea of clouds has drifted on forever
from the sight of human eyes.
The summer sun beamed once more upon the vast panorama. Far
down upon the green mesa lay Lake Moraine, glistening in the
morning light like a molten mass of silver.
Smoke was seen to rise from Denver and Pueblo, both fully sixty
miles away. Some smelters in Cripple Creek and Victor could be seen
with the naked eye, while the streets of Colorado Springs were but
sandy marks like a checkerboard upon the plain.
I descended the peak on foot amid the beauteous scenes of green
mountain defiles, where dashing waters sing eternal symphonies

amid ferns and flowers, and the song of birds gladden the heart in
their sweet echoes from rock to rock.

XXV.
WHERE NATURE IS AT HER BEST.
If one would view the wondrous surroundings of Manitou, in all their
grandeur, let him some bright morning stroll up the long yellow road
that winds its serpentine course through Williams Canon. A little
brook with waters cold and clear as crystal, dashes along its pebbly
bed beside the road, murmuring as it were, a song of regret at
leaving its enchanted home on its journey to the sea. The road is
known as Temple Drive, named so because many towering rocks
look, at first glance, like ruined temples of India or of Egypt along
the Nile.
At times the road narrows to barely carriage room between great
high cliffs, and again abruptly brings the majestic panorama of the
canon into view. High above, among the mountain crags is the
Cathedral of St. Peter, like a massive ruin whose cornice, column and
frescoed walls had fallen with decay ages past. A little farther and
the Amphitheatre rises against the cliffs in hues of brown and yellow,
with brighter streaks of golden ochre here and there, which fairly
gleam and glisten in the morning sun. High above and in the
background on either side are hills of emerald green, studded with
cedar and pine, and dotted with flowers of gorgeous color and of
form, found elsewhere only in Alpine lands. There are towering rocks
that rise a thousand feet above the road, which resemble the ruins
of a Moorish citadel. There are towers, mosques and temples, with
turrets and battlements, needing only the white-robed figure of the
Arab in turban to make one fancy himself suddenly transported to
that enchanting and mysterious land of Sultan and slave. No sky of
Tangiers was ever deeper, clearer or bluer, and no air of Geneva was
ever purer or sweeter.
The road makes a sharp turn and traverses backward nearly half a
mile, then turns again and runs in its original direction, climbing the

mountain side like a great yellow serpent resting its head a thousand
feet among the crags, where eagles build their nests; the white and
red painted building that marks the entrance to the Cave of the
Winds, does duty as the serpent’s head. From this dizzy point of
sight, the great mountain gorge with its grey and brown rocks, and
the sloping foothills of green that stretch away to where fair Manitou
lies cradled in the valley, form a wondrous panorama.
Eastward, down on the horizon, far as the eye can reach, stretch the
mighty plains, westward the higher range of the eternal Rockies, and
above all rises the snow-capped summit of Pike’s Peak, about whose
whitened crest float the fleecy clouds of the soft, still summer
morning.
At the entrance of the Cave of the Winds one follows the guide into
the dark pathway that leads into the subterranean chambers, where
at some remote period a wild mountain cataract has whirled and
plunged its maddening waters, in swirl and maelstrom into the black
abyss of the earth. One is so suddenly transported from the
gladsome and awe-inspiring scenes without, that the lamp and
figure of the guide become spectral, his voice sounds in hollow tones
and is echoed back from cavernous depths as though titanic
monsters were repeating his words.
Knowing the cause, one bursts into a laugh, then the monsters
laugh, too, long and loud, and still others take up the laugh, way
down the black corridors, and high above in domes, as though all
the imps of darkness were there to laugh at one in revenge for
intrusion.
The guide flashes a magnesium light and the pilgrim beholds the
wonders of Curtain Hall, which nature has ornamented with
strangely colored stalactites glistening here and there on the cavern
walls, and again where they form a curtain of an intricate work and
beauty as though wrought by maiden hands, amid scenes of love
and apple blossoms. Mutely you follow the flaring lamp of the guide
into the blackness of winding passages and across bridges that span
bottomless pits opening into the very breast of the mountain, and

when the magnesium light is again flashed, one sees the arching
dome of the great canopy hall, its stalactite nymphs, Bed of
Cauliflowers, Frescoed Ceiling, Lake Basin, Grandmother’s Skillet,
Bat’s Wing, Prairie Dog Village and Fairy Scene; all presenting a
picture weird and ghost-like in the moment of stillness, and
heightened by the demoniacal, fiendish voices that repeat your every
word.
On through other crooked subterranean passages where other
demons mock the sound of your footsteps, through what the guide
calls Boston Avenue, one enters Diamond Hall. The lofty ceiling is
decorated its entire length by graceful festoons and wreaths of coral
and flowering alabaster. The walls sparkle and scintillate with the
rainbow shades, thrown back from the myriad brilliants that stud
these walls like diamonds set by hand in some antique mosaic work.
In these regions of darkness you are led by the guide until the Hall
of Beauty is lit up to your astonished gaze. Crystal flowers of the
most delicate design and exquisite workmanship hang in festoons
from every nook and corner. Sparkling incrustations that rival the
beauty of Arctic frosts and glitter in the bright light are sparkling on
every side. Most wondrous of all there are a million stalactite figures
in miniature that appear to be in a pandemonium of outlandish
contortions. Maybe, who knows, but what the goblin spirits once
lived here and worked out curious things in translucent stone,
further down the black passages of earth and caught a glimpse of
our ancestors in some of the great halls of torture way down below,
and so reproduced the scene as Jack Frost has been wont to paint
the leaves of summer on our frosted window panes.
The Magi of this dark abode, the guide in wide sombrero, black eyed
and wearing a mustache fierce as a bandit of the Corsican isle,
though harmless as a Kansas Populist, beckons on and leads the
way. Here the Bridal Chamber, and there writhing reptiles, dancing
devils, monkeys, beasts, birds in every form and riotous posture.
Then as the weird wilderness is shut out in semi-darkness, one is

inclined to ask of him with lamp and sombrero, “Mister, have I got
’em, or have you?”
The light flashes on Crystal Palace, where gems and jewels bedeck
the walls, where huge chrysanthemums or chestnut burrs stand out
in bold relief in fadeless crystal flowers moulded from tinted rock,
and all seem to mutely plead for recognition as we pass. These silent
beauties hidden away under the mountain slopes, where the rays of
sun can never reach, speak with the beauty of their creation, to the
soul with as great a love and power as the violet in the sequestered
glens.
It is mysterious. It is strange. It is one of those unaccountable things
in nature which no man can explain that here in the very bowels of
the earth, human scenes have been reproduced and human passions
portrayed.
Here perhaps centuries before man’s eyes gazed upon the scene, we
find in moulded stone, the head of a buffalo, the skeleton of a
mastodon, the drapery of a palace, the bride at the altar, the face of
sorrow, the Nymphs of Love, War and Poetry are depicted upon
these stones.
Once more the light of day, the great chasm beneath, the turquoise
skies above, and mighty plains beyond, brings one to the realm of
the outer world.
The spectral figure of an hour ago is a pleasant faced young man,
who bids you follow the winding path that leads around the
mountain side some three hundred yards and which ends at the
entrance to the Grand Caverns.
Desiring to see all, you meekly follow another guide through a dark
labyrinth and find yourself in the mighty Rotunda of the Caverns.
Here loyal hands have raised monuments to Lee, Grant and
McKinley. They are built of fragments of stone cast by visitors to the
memory of these heroes. The Imp, the guide, motions on; you are
next within a mighty auditorium and as there comes upon you the
awful silence and stillness of the hour, you hear musical notes,

swelling and cadencing louder and louder until they break in
thunderous tones within the cavernous depths, “Nearer, Nearer, My
God to Thee.” High above, mid the domes of the cavern, the light
shows the organist to be playing upon the stalactites which Nature
has attuned to the same chords as instruments made by human
hands. These stalactites are of crystal, and have the same resonant
sound as though they were of finely tempered glass. Up and down
the corridors of the cave, through winding passages and circling
galleries above, come echoes of “Nearer, My God to Thee,” in waves
and billows of sound, such as is only heard by artificial means in the
Notre Dame of Paris.
Round about somewhere, in one of the chambers, near the
entrance, the visitor is shown a human skeleton, as it was found at
the time of the discovery of the cave. It belonged perhaps to that
race of men known as the Cliff Dwellers, who once upon a time,
when the world was new, lived, loved and reared a race of men in
this fair region of the west whom Saxby, a western poet, touches
with his magic pen, and beautifies the tradition of them when he
says,
“Dismantled towers and turrets broken,
Like grim war-worn braves who keep
A silent guard with grief unspoken
Watch o’er the graves, by the canon weep,
The nameless graves of a race forgotten
Whose deeds, whose words, whose fate are one
With the mist, long ages past, begotten of the sun.”
The sun is now casting his shadows toward the east. From this point
of sight we see the Midland trains creeping from tunnels like
monster creatures of the Azotic period crawling from their lair. There
are green valleys below, and there is also a long serpentine road
leading to this side of the mountain by which visitors again reach the
pleasant shades of Manitou. Silence, and even sadness, abound in
the green-clad mountains beyond. They speak in whispers to
themselves and you can understand them if you will. They tell you in

sweet, soft voices of the song of birds, the lullaby of mountain
brooks, and by gentle winds that sing a song of peace through
cedar, fir and pine, that the love of nature, is the love of nature’s
God.

XXVI.
WHEN THE WEST WAS NEW.
Thirty years have passed since I first crossed the plains. The buffalo
and antelope have disappeared and in their stead herds of cattle and
sheep graze in countless thousands. Farms are tilled where raging
fires swept the mighty plains in ungoverned fury; cities and towns
rear their spires where once stood Indian tepees. The westward
march of civilization has stretched across the continent and
redeemed the desert. The soil has been made to yield its harvest
and the eternal hills to give up their buried treasure. For the men
who made the trails by which these things were done, life’s shadows
are falling toward the east. They braved the vicissitudes of the
western wilderness as heroic as any soldier faced the battlefield; and
the trails over which the pioneers slowly made their way across the
desert wastes, were blazed with blood and fire. Women, too, on the
frontier, volumes might be written of her sacrifices—Indians, poverty,
years of patient toil, far from former home and friends, the luxuries
of organized society denied, all for the purpose of earning a home
and a competence for declining years.
It was my good fortune to become personally acquainted with many
early pioneers of the west and number them among my warmest
friends, and as I recall to mind some of their heroic deeds I feel that
these chapters would be incomplete without a personal mention of a
few of them.
* * * * *
Captain Jack Crawford, the poet scout, is one of those noble
characters whose memory will live so long as records exist of the
pioneers who braved the vicissitudes of the frontier and made
possible our Western civilization of today. A man of broad mind,

daring and brave and yet with all the sweet tenderness of a child of
nature, he became great by achievements alone. Others have gained
a temporary fame by dime novel writers. Captain Jack, in
comparison with others, stands out as a diamond of the first water.
He has helped to make more trails than any scout unless it was Kit
Carson. That was before the war. During that struggle he was
wounded three times in the service of his country. When the war
closed he was for many years chief of scouts under General Custer.
He laid out Leedville in the Black Hills in 1876, and was of great
service to the government in the settlement of the Indian troubles
which succeeded the Custer massacre.

Captain Jack Crawford (page 208).
Captain Jack is one of the very few thrown together with the wild,
rough element of the frontier who maintained a strictly moral
character. I knew him in the “Hills” in 1876 and have known him
ever since, and have always found him to be the same genial,
whole-souled, brave Captain Jack.
* * * * *

John McCoach, a pioneer of the sixties, was a among a party near
the headwaters of Wind River, Wyoming, in August, 1866, who
defeated a thousand warriors with the first Henri rifles used on the
plains. The story is best told in Mr. McCoach’s own language.
“Our mule trains consisting of thirty-eight wagons and forty-two
men, left Fort Leavenworth, Kansas, in April, 1866, for Virginia City,
Montana. We were all old soldiers and most of us had seen four
years of war and, inured as we were to dangers, we cared but little
for the hostile Indians of the plains.
“When we reached Fort Laramie, a big council of Indians was in
progress, Chiefs Red Cloud, Spotted Tail, American Horse and others
of lesser note were there to demand guns and ammunition from the
government, saying they needed them with which to hunt game.
Officials of high rank from Washington were there to listen to them
and among the newspaper correspondents was Henry M. Stanley,
who had been sent out by the New York Herald.
“After days of deliberation the Indians were refused the arms and
they broke camp in bad humor.
“Before allowing our party to proceed the commander of the fort had
us lined up for an inspection of our arms which were a miscellaneous
collection all the way from an old muzzle-loading rifle to a modern
musket. He told us we were too poorly armed to proceed, when the
wagon boss took him to some of the wagons and showed him 200
Henri rifles and abundant ammunition which we were freighting to
gun dealers in Virginia City. He then allowed us to go.
“I was herding the mules one afternoon near the headwaters of
Wind River, when a party of Sioux Indians, led by Little Thunder,
made a dash, intending to stampede the animals. One of them
carried a rawhide bag containing some pebbles, which made a
hideous noise. Despite their efforts, the mules broke for our camp of
circled wagons. I tried to shoot the Indian with the rattle bag but
missed. Then I dismounted and the next shot I cut the quiver of

arrows from his back when he gave a long yell and throwing himself
on the side of his pony, got away.
“When I reached camp the rifles had been distributed. We were
called from our slumbers the next morning at four o’clock and told to
keep quiet and hold our fire.
“With the first gray streak of dawn about one thousand warriors
began to encircle us, riding at full speed and like a great serpent,
drawing the coil closer about us with each revolution of the circle.
Then the order came and forty-two blazing rifles with eighteen shots
to each one dealt out death. Four years of war had taught the men
the value of a steady nerve and deliberate aim and before the
astonished Indians could retreat the plain was strewn with their
dead and wounded.
“These Indians had been at the Fort Laramie council and had seen
us drawn up in line with our old assortment of guns for inspection
and had counted on us being easy prey. They were the first Henri
rifles used on the plains and caused the Indians to speak of us in
whispers, as the white men who could load a gun once and then
shoot all day. That morning we built our fires with arrows and
cooked our breakfast. After that the Indians avoided us as though
we were devouring monsters.”
* * * * *
The experience of John McCoach’s party in surprising Little
Thunder’s braves with their Henri rifles, calls to mind a story often
told in Fort Laramie of how General W. S. Harney fooled these same
Sioux Indians under Little Thunder a few years previous to their
attack on the McCoach outfit. Jake Smith, a soldier with General
Harney in the 60’s thus relates the story:
“General Harney established his headquarters in Leavenworth,
Kansas. Little Thunder was at the head of the Sioux and sent word
that he was willing either to fight or shake hands with the white
soldier. Harney replied that if the Indian was without choice in the

matter it might as well be fight; besides, as he remembered his
orders, he was to whip some one. So Harney met Little Thunder and
about a thousand war men on the North Platte in Nebraska. He
whipped them good and some of the Indians’ friends back East tried
to make trouble for Harney because he had not had a long
preliminary confab with Little Thunder. That Sioux band was a mild-
mannered set long after Harney went back to Leavenworth.
“It was after this fight that Harney threw the Society for the
Protection of Western Savages into a particular frenzy. The wagon
trail for Oregon and California led from Fort Leavenworth to Fort
Kearney, Neb., then to Julesburg, in Colorado, from there to Fort
Laramie, through old South Pass to Badger and then to Salt Lake.
The trip by ox train took about one hundred days with good luck. I
know of a party that was on the road 300 days, delayed by Indians
and then snowbound. That wasn’t a pleasant winter for a boy of 16.
“Every now and then a band of Sioux would ride up to an ox train,
kill if they felt like it and always drive away the stock. Soldiers would
be sent out and have the pleasure of following the Indians’ trail until
the weather would make winter quarters necessary. Harney started
from Leavenworth after one band, taking about 400 cavalrymen, or
dragoons. The Indians loafed along ahead of him till they reached
the mountains, and then Harney turned back. It was the old story,
the Sioux said, and their scouts followed the soldiers until they were
well into Kansas. Then the Sioux knew the country was clear for new
operations.
“Harney stopped on the Blue River in Northern Kansas near where
Marysville now stands. A wagon train reached there from
Leavenworth and Harney had all the freight unloaded—simply seized
the train—then he put 400 soldiers into those wagons and in two
were mountain guns. The great covers were pulled close and leaving
a guard over the abandoned freight and horses, Harney started on
his journey as a bull-whacker. Not a soldier or officer was permitted
to put his head from under a cover in the day time, and only at night
a few got leave to stretch their legs. All day they sat in those wagon

beds, hot and dusty, playing cards, fighting and chewing tobacco for
pastime.
“There were twenty-six of those wagons and they trailed along as if
they were carrying dead freight; no faster nor slower than the
ordinary freighters, and making camp at the usual places, forming
the usual corral of wagons and herding stock at night. The train
reached Fort Kearny and slowly went across the South Platte to
Julesburg. Occasional Indian signs made Harney have hope.
“The outfit was seventy miles on the way to Laramie when the big
day came, and it came quick. Behind them on the trail the men on
the outside saw a war party—some say there were five hundred
Indians in it. Even if they hadn’t been painted the fact that they
were without women or children would have told the story. The train
made the usual preparations for an Indian attack, throwing the
wagons into a circle, or more of an ellipse, and unhooking the five
lead yokes to each wagon. A front wheel of each wagon touched a
hind wheel of the one in front and the tongues were turned to the
outside. At the front end of the corral an opening about fifteen feet
wide was left, but at the rear the opening into the corral was about
fifty feet wide. That, also, was according to the freighters’ methods;
after a night camp the cattle would be driven into the corral through
the big end to be yoked for the day.
“Harney didn’t have time to drive his oxen into the corral, or else he
didn’t want to. Only the five yoke of leaders were unhooked and
they were then chained to the front wheel of their wagon. The space
in the corral was all clear for the Indians, whose method of attacking
a wagon train was to rush into the corral and do their shooting. They
were a happy lot of braves this day; the war band started for the
train when the corral was forming; they spread out like a fan and
then came together again and started for the big opening as hard as
their war ponies could carry them. A whooping, variegated mob with
no more clothes than the paint gave it fell into the corral and then
real fun began.

“Those soldiers, who had been sweating under canvas for a few
weeks wanted excitement and revenge. The tarpaulins went up and
they shot down into that mess of braves as fast as they could load.
The two mountain guns completed the surprise and the bucks hardly
fired a shot before their ponies were climbing over one another to
get out the way they came. It was the only real Indian panic. When
the last Sioux brave able to ride disappeared across the prairie there
was a big mess to clean up. In those days the Indians needed school
all the year around. However, one old buck, a little chief, seemed to
be impressed. He was near a mountain gun when the fire opened.
‘Harney is the man who shot wagons at us,’ is the way he told about
it years later.
* * * * *
Charles S. Stroble, “Mountain Charley,” known as the cowboy painter,
was adopted by the Ute Indians at the age of nineteen. I have often
heard him tell the following experience:
“It was the most marvelous instance of daredevil bravery I ever
witnessed. It happened in 1866 when I was living with the Utes west
of the range in Middle Park, Colorado. They had adopted me a year
or so before when I was twenty years of age. My name in Ute was
Paghaghet, which means ‘long-haired.’
“It was at this time that the old feud between the Utes and
Arapahoes was at its height. Our scouts found the Arapahoes coming
in from North Park in the endeavor to surprise some of the Utes’
hunting parties. Our runners having come in and informed us, we
soon collected a war party and started north to intercept our
enemies.
“I was with the scouting party which went in advance, and I was the
only white man in the entire tribe. We found the sign left by their
scouts, and then concealed ourselves until our war party could come
up. As soon as reinforcements arrived we deployed on either side of
a gulch or canon, with our horses hidden away among the rocks and
timber in charge of horse-holders.

“We had not waited long when we sighted the advance of the
Arapahoes down below us in the gulch. We were unnoticed, because
we left no tracks in the gulch and had deployed some distance
below.
“When the main body of the enemy had passed our place of
concealment we opened fire on them from each side of the gulch,
and they, not knowing our numbers, were panic-stricken. They
wheeled and came tumbling back up the gulch in great confusion,
and all the time subjected to our fire. To be sure, they were
returning the fire wherever they caught sight of us, but we had by
far the best of them and peppered them hotly.
“The Utes got about eight scalps, as the Arapahoes, although they
carried their wounded with them in their flight, were in too big a
hurry to look after the dead.
“My Indian brother, Paah, or ‘Black Tailed Deer,” and Wangbich, the
‘Antelope,’ were with me behind some sheltering rocks, and on each
side of me. As the Arapahoes were scurrying away through the
canon below we noticed particularly one fine-looking young buck,
wearing a splendid war bonnet, which flaunted bravely in the breeze.
This fellow was singled out by Paah. At the crack of his rifle the
Arapahoe threw out his arms and fell backward from his pony and
the pony galloped away.
“Paah, elated at the success of his shot, dropped his rifle and
plunged down the steep side of the canon, which ran up here at an
angle of about forty-five degrees, the other Indians passing all the
time and letting loose at him a fusillade of rifle shots and flights of
arrows. At length Paah got to his dead Arapahoe, planted his foot on
the back of the man’s neck, grasped both scalplock and side braids,
gave them a turn on his wrist and with the aid of his knife secured
the full scalp.
“Then seizing the war bonnet, he came tearing up the side of the
gulch, his trophies in one hand and his knife held dagger wise in the
other, to assist him in making the steep ascent.

“The arrows and bullets flew thickly about him, but, marvelous to
tell, he arrived on the little flat space back of us without a scratch.
Waving his bloody spoils above his head he essayed to give the Ute
yell of victory, but he was so exhausted that he was only able to let
out a funny squeak as he fell prostrate to avoid the shots that were
now pouring in our direction. Wangbich and I covered him the best
we could by emptying our six-shooters at the Arapahoes, and he
finally succeeded in crawling to shelter.
“On the return of our war expedition to the principal village we
celebrated our victory in royal style. The Utes from other villages
kept pouring in, and there was dancing afternoon and night for
many days. This chief village was located under some high rocks on
the Grand river, near a hot spring. The principal feature of the
celebration was a scalp parade, a gorgeous affair in which all kinds
of silvered ornaments, feathered and beaded costumes were worn. I
afterward painted this splendid scene as it appeared to me and the
picture is now hanging in the Iroquois club in Chicago.”
* * * * *
“Possibly my experience in the bullwhacking days across the plains,”
says George P. Marvin, “does not materially differ from that of other
men who piloted six yoke of cattle hitched to eighty hundred of
freight across the desert. Yet there were many incidents connected
with life upon the plains that have never been written.
“There was scarcely a day passed but something occurred that
would furnish material upon which the writer of romance could build
an interesting book of adventures.
“In the freighting days of the early ’60’s, the overland trail up the
Platte River was a broad road 200 or more feet in width. This was
reached from various Missouri River points, as a great trunk line of
railroad is now supplied by feeders. From Leavenworth, Atchison and
St. Joe, those freighters who went the northern route crossed the
Blue River at Marysville, Kansas, Oketo and other points, and
traveled up the Little Blue, crossing over the divide and striking the

big road at Dogtown, ten miles east of Fort Kearney. From Nebraska
City, which was the principal freighting point upon the river from ’64
until the construction of the Union Pacific railroad. What was known
as the Steam Wagon road was the great trail. This feeder struck the
Platte at a point about forty miles east of Kearney. It derived its
name from an attempt to draw freight wagons over it by the use of
steam, after the manner of the traction engine of today.
“My first trip across the plains was over this route, which crossed the
Big Blue a few miles above the present town of Crete, Nebraska. At
the Blue crossing we were ‘organized,’ a detachment of soldiers
being there for that purpose, and no party of less than thirty men
was permitted to pass. Under this organization, which was military in
its character, we were required to remain together, to obey the
orders of our ‘captain,’ and to use all possible precaution against the
loss of our scalps and the freight and cattle in our care.
“The daily routine of the freighter’s life was to get up at the first
peep of dawn, yoke up and if possible get ‘strung out’ ahead of
other trains, for there was a continuous stretch of white covered
wagons as far as the eye could reach.
“With the first approach of day, the night herder would come to
camp and call the wagon boss. He would get up, pound upon each
wagon and call the men to ‘turn out,’ and would then mount his
saddle mule and go out and assist in driving in the cattle.
“The corral was made by arranging the wagons in a circular form,
the front wheel of one wagon interlocking with the hind wheel of the
one in front of it. Thus two half circles were formed with a gap at
either end. Into this corral the cattle were driven and the night
herder watched one gap and the wagon boss the other, while the
men yoked up.
“The first step in the direction of yoking up was to take your lead
yoke upon your shoulder and hunt up your off leader. Having found
your steer you put the bow around his neck and with the yoke
fastened to him, lead him to the wagon, where he was fastened to

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