Array-based hybridization and solution which can then be visualized using a

hybridization are two techniques used in detector such as a phosphorimager

molecular biology to study interactions or chemiluminescence.
between molecules, such as DNA and RNA.  Slot blot is similar to a dot blot,
except that the sample is loaded into
ARRAY-BASED HYBRIDIZATION a slot or well on the membrane using
Array-based hybridization involves the use of a vacuum manifold. This allows for
microarrays, which are small chips containing more precise loading of the sample
many thousands of immobilized probes, each and more accurate quantification of
designed to hybridize with a specific target the target sequence.
molecule. The probes are arranged in an array
format, allowing for high-throughput analysis
of many different samples simultaneously.
During the hybridization process, the target
molecules in a sample are labeled with a
fluorescent or radioactive marker, which is Example configuration of a dot blot (left)
detected by a scanner or other imaging device. and a slot blot (right). The target is
This technique is commonly used for gene spotted in duplicate, side by side, on the
expression profiling, Single Nucleotide dot blot. The last two rows of spots
Polymorphism (SNP) genotyping, and contain positive, sensitivity, and negative
genome-wide association studies. control followed by a blank with no target.
The top two rows of the slot blot gel on
1. Dot/Slots Blots the left represent four samples spotted in
 Dot blot and slot blot are two related duplicate, with positive, sensitivity, and
techniques in molecular biology negative control followed by a blank with
used for the detection and no target in the last four samples on the
quantification of specific DNA or right. The bottom two rows represent a
protein sequences in a sample. loading or normalization control that is
 In a dot blot, a small amount of the often useful in expression studies to
sample is spotted onto a membrane, confirm that equal amounts of DNA or
usually made of nitrocellulose or RNA were spotted for each test sample.
nylon. For dot blots, the target is  Dot blot and slot blot are both useful
deposited in a circle or dot. For slot techniques for rapid and
blots, the target is deposited in an high-throughput screening of large
oblong bar. numbers of samples for the presence
 The membrane is then blocked to or absence of specific DNA or
prevent nonspecific binding of protein sequences. They can be used
detection probes and probed with a for a wide range of applications,
labeled DNA or protein probe that is including the detection of viral or
complementary to the target bacterial pathogens, genotyping, and
sequence of interest. The presence of the measurement of protein
the target sequence is detected by the expression levels. However, the main
binding of the labeled probe to the limitation of these techniques is their
target sequence on the membrane, relatively low sensitivity compared
 to other more sensitive detection macroarrays are less sensitive
methods, such as PCR or ELISA. and have lower resolution, but
they have advantages for
2. Genomic Array Technology studying large pieces of DNA
Genomic array technology is a powerful tool or entire genes. Macroarrays
that allows researchers to analyze large can also be less expensive to
amounts of genetic information in a single produce than microarrays,
experiment. It involves the use of microarrays, which can make them more
which are chips that contain thousands or accessible for some researchers.
millions of DNA probes that can detect  Applications of genomic
specific genetic sequences or variations. macroarray technology include
identifying changes in gene
a. Macroarrays expression in response to
 Genomic macroarray different treatments or disease
technology also known as states, studying the activity of
reverse dot blot, is a technique transcription factors and other
that uses DNA microarrays to regulatory proteins, and
detect changes in gene identifying genetic variations
expression, copy number associated with disease.
variations, and other genetic  Overall, genomic macroarray
variations. Macroarrays are technology is a useful tool for
similar to microarrays, but they studying gene expression and
contain larger spots or probes other aspects of gene regulation
that are capable of detecting on a larger scale than
larger pieces of DNA. microarrays.
Macroarrays are used to study b. Microarrays
gene expression, transcription  Genomic microarray
factors, and other aspects of technology is a powerful
gene regulation. technique that allows
 Macroarrays are created by researchers to study the
printing DNA probes onto a expression of thousands or
substrate such as a glass slide or millions of genes in a single
nylon membrane. The probes experiment.
can be specific sequences or
they can be whole genes. The
target DNA is labeled with a
fluorescent or radioactive tag
and hybridized to the probes on
the macroarray. The resulting
signal is detected and analyzed
 Microarrays consist of a glass
to determine the level of gene
slide or other substrate that is
expression or the presence of
coated with thousands or
genetic variations.
millions of tiny spots or probes
 Compared to microarrays,
that are capable of detecting
 specific genetic sequences or  (To perform a high-density
variations. oligonucleotide array
 To perform a microarray experiment, researchers extract
experiment, researchers extract RNA or DNA from a sample
RNA from a sample of cells or and convert it into cDNA. The
tissue and convert it to cDNA is then labeled with a
Complimentary DNA (cDNA) fluorescent tag and hybridized
using reverse transcriptase. The to the probes on the array. The
cDNA is labeled with a resulting signal is detected and
fluorescent tag and hybridized analyzed to determine the
to the probes on the microarray. expression level of each gene or
The resulting signal is detected the presence of specific genetic
and analyzed to determine the variations.)
level of gene expression for  High-density oligonucleotide
each gene represented on the arrays have many applications
microarray. in genetics research. They can
c. High-Density Oligonucleotide be used to study gene
Arrays expression in response to
 High-density oligonucleotide different environmental
arrays, also known as conditions or disease states, to
high-density gene chips, are a identify genetic variations
type of microarray technology associated with disease, and to
that are capable of detecting perform high-throughput
gene expression levels for tens genotyping. They are also
of thousands of genes in a commonly used in
single experiment. They are a pharmacogenomics to identify
powerful tool for studying gene genetic variants that affect drug
expression, genotyping, and metabolism and efficacy.
genetic variations in a  Overall, high-density
high-throughput manner. oligonucleotide arrays are a
 High-density oligonucleotide powerful tool for studying gene
arrays consist of small spots or expression and genetic
probes that are synthesized variations in a high-throughput
directly onto a substrate such as manner.
a glass slide or silicon chip. d. Microelectronic Arrays
Each probe consists of a short,  Microelectronic arrays are a
single-stranded piece of DNA type of microarray technology
that is complementary to a that use microelectronic devices
specific gene or genomic region. to detect and analyze biological
The probes are arranged in a samples. Unlike traditional
grid pattern on the substrate, microarrays, which use
with each spot corresponding to fluorescence or other chemical
a different gene or genomic methods to detect signals,
region. microelectronic arrays use
 electrical signals to detect and applications in medicine,
analyze biological molecules. biotechnology, and other fields,
 Microelectronic arrays are and are likely to continue to be
typically composed of a an important tool in biological
substrate material, such as research for many years to
silicon or glass, that is coated come.)
with a layer of conducting e. Bead Array Technology
material, such as gold or  Bead array technology is a type
platinum. Microelectrodes are of assay platform used in
then patterned onto the biological research and
substrate, with each electrode diagnostics that enables the
designed to detect a specific detection and quantification of
biological molecule or cellular multiple biomolecules in a
signal. single sample. The technology
 To perform a microelectronic involves attaching
array experiment, the biological biomolecules, such as DNA or
sample is introduced onto the proteins, to small beads or
array and allowed to bind to the microspheres, which are then
microelectrodes. The resulting arrayed on a solid support, such
electrical signals are then as a glass slide or a microplate.
detected and analyzed to  In this technology, the target
determine the presence and molecules in a sample, such as
quantity of the biological DNA or proteins, are labeled
molecules or cellular signals. with a fluorescent or
 Microelectronic arrays have colorimetric tag, which allows
many applications in their detection and
biotechnology and medical quantification by measuring the
research. They are commonly signal emitted from each bead.
used in biosensors for rapid and Each bead is uniquely labeled
accurate detection of biological with a different color, allowing
molecules such as DNA, the detection of multiple targets
proteins, and antibodies. They simultaneously in a single
are also used in cellular sample.
electrophysiology to study the  Bead array technology has
electrical signals generated by applications in various fields of
cells, and in drug discovery to research and diagnostics,
screen potential drug candidates including genomics, proteomics,
for their effects on cellular and clinical diagnostics. It
signaling pathways. enables high-throughput
 (Overall, microelectronic arrays screening of multiple targets in
are a powerful tool for detecting a single sample, providing a
and analyzing biological rapid and cost-effective method
molecules and cellular signals. for detecting and quantifying
They have many important biomolecules.
 there are low amounts of target RNA. One
There are many different types of genomic version of the method is called RNase
array technology, each designed to answer protection, or S1 mapping, for the S1
specific questions about genetic information. single-strand–specific nuclease. In S1 mapping,
For example, gene expression arrays can be the labeled probe is hybridized to the target
used to study how genes are turned on or off in sample in solution. After digestion of excess
response to different environmental conditions probe by a single-strand–specific nuclease, the
or disease states. Comparative genomic resulting labeled, double-stranded fragments
hybridization (CGH) arrays can be used to are resolved by polyacrylamide gel
detect copy number variations (CNVs) in DNA electrophoresis (Fig. 5.28).
samples, which can be important in the
diagnosis and treatment of cancer and other
genetic disorders.
Single nucleotide polymorphism (SNP)
arrays are used to study genetic variation
between individuals, and can be used to
identify genes associated with disease.
Methylation arrays are used to study the
epigenetic modifications of DNA that regulate
gene expression, which can be important in
understanding the development of cancer and
other diseases. Chromatin
Immunoprecipitation (ChIP) -on-chip arrays
are used to identify the genomic regions bound
by specific proteins, which can be important in
understanding gene regulation and protein
function.
Overall, genomic array technology has
revolutionized the study of genomics by
allowing researchers to analyze large amounts
of genetic data in a single experiment. It has Target RNAs are hybridized to a labeled RNA
many important applications in medicine, or DNA carrying the complementary sequence
agriculture, and other fields, and is likely to to the target. After digestion by a
continue to be an important tool in genetic singlestrand-specific nuclease, only the target:
research for many years to come. probe double-stranded hybrid remains. The
hybrid can be visualized by the label on the
SOLUTION HYBRIDIZATION probe after electrophoresis.
In solution hybridization, neither the probe nor
the target is immobilized. Probes and targets S1 mapping is useful for determining the start
bind in solution, followed by resolution of the point or termination point of transcripts.
bound products. Nuclease protection assays are also used to
detect and quantify specific RNA targets from
Solution hybridization has been used to complex RNA mixtures. This technique is now
measure mRNA expression, especially when performed using commercial reagent sets. The
procedure is more sensitive than northern
blotting because no target can be lost during This assay has been used to identify trans
electrophoresis and blotting. It is more factors that bind to cis-acting elements that
applicable to RNA expression analysis due to control gene regulation. Solution hybridization
limited sensitivity with double-stranded DNA can also be used to detect sequence changes in
targets. DNA or mutational analysis. Hybridization
methods offer the advantage of direct analysis
Another variation of solution hybridization is of nucleic acids at the sequence level without
the capture of DNA probe:RNA target hybrids cloning of target sequences. The significance
on a solid support or beads rather than by of hybridization methodology to clinical
electrophoresis. For these “ sandwich ” -type applications is the direct discovery of
assays, two probes are used, both of which molecular genetic information from routine
hybridize to the target RNA. One probe, the specimen types. Widely varying modifications
capture probe, is biotinylated and will bind of the basic blotting methods have been and
specifically to streptavidin immobilized on a will be developed for clinical and research
plate or on magnetic beads. The other probe, applications. Although amplification methods,
called the detection probe, is detected by a specifically the PCR, have replaced many
monoclonal antibody directed against blotting procedures, some hybridization
RNA:DNA hybrids or a covalently attached methods are still used in routine clinical
digoxigenin molecule used to generate a analysis.
chromogenic or chemiluminescent signal.
In general, array-based hybridization is more
Solution hybridization has also been applied to suited for large-scale studies of gene
the analysis of protein–protein interactions and expression or genetic variation, while solution
to nucleic acid – binding proteins using a gel hybridization is better for smaller-scale
mobility shift assay. After mixing the labeled applications requiring high sensitivity and
DNA or protein with the test material, such as specificity.
a cell lysate, a change in mobility, usually a
shift to slower migration, indicates binding of
a component in the test material to the probe
protein or nucleic acid (Fig.
5.29).

Gel mobility shift assay showing

protein-protein or protein – DNA interaction.
The labeled test substrate is mixed with the
probe in solution and then analyzed on a
polyacrylamide gel. If the test protein binds the
probe protein or DNA, the protein will shift up
in the gel assay.