The Basics of DNA Microarrays

The Human Genome Project has created a massive amount of DNA sequence information. To
take full advantage of this latest information, scientists have developed new techniques and
tools for conducting research. DNA microarrays, which are also called DNA arrays or gene chips,
are an example of a tool that uses genome sequence information to analyze the structure and
function of tens of thousands of genes at a time.

How Do Arrays Work?

DNA arrays come in many varieties. Whether they are created by scientists or produced
commercially by one of several companies, arrays depend on the same basic principle:
Complementary sequences of nucleotides stick to, or “hybridize” to, one another. For example,
a DNA molecule with the sequence -A-T-T-G-C- will hybridize to another with the sequence -T-
A-A-C-G- to form double-stranded DNA.

For the past 25 years, scientists have been using hybridization as a standard technique to
detect specific DNA or RNA sequences. A single-stranded DNA molecule with a known sequence
is labeled with a radioactive isotope or fluorescent dye and then used as a “probe” to detect a
fragment of DNA or messenger RNA (mRNA, the molecule that is produced when a gene is
turned on or “expressed”) with the complementary sequence. For example, if a researcher
wants to know whether gene A is expressed in a particular tissue, the researcher would make a
radio-labeled DNA probe by using a small piece of gene A, isolate mRNA from the tissue of
interest, bind the mRNA to a solid medium (such as a nylon filter), and then hybridize the
probe to the filter. If gene A is expressed in the tissue, the researchers will see a radioactive
signal on the filter. This procedure is known as a Northern blot. Imagine the power of being
able to do thousands of these experiments at a time.

DNA microarrays use the same DNA probe detection method but on a much larger scale. Instead
of detecting one gene or one mRNA at a time, microarrays allow thousands of specific DNA or
RNA sequences to be detected simultaneously on a glass or plastic slide about 1.5 centimeters
square (about the size of your thumb). Each microarray is made up of many bits of single-
stranded DNA fragments arranged in a grid pattern on the glass or plastic surface. When sample
DNA or RNA is applied to the array, any sequences in the sample that find a match will bind to a
specific spot on the array. A computer then determines the amount of sample bound to each
spot on the microarray.
An Array of Applications

Gene Expression Arrays

DNA arrays are commonly used to study gene expression. In this type of study, mRNA is
extracted from a sample (for example, blood cells or tumor tissue), converted to
complementary DNA (cDNA), which is easier to work with than RNA, and tagged with a
fluorescent label. In a typical microarray experiment, cDNA from one sample (sample A) is
labeled with red dye and cDNA from another (sample B) with green dye. The fluorescent red
and green cDNA samples are then applied to a microarray that contains DNA fragments
corresponding to thousands of genes. (For certain organisms whose genomes have been
sequenced, such as the roundworm Caenorhabditis elegans, there are arrays that represent all
the genes in the genome.) If a DNA sequence is present both on the array and on one or both
samples, the sequences bind, and a fluorescent signal sticks to a specific spot on the array. The
signals are picked up using a “reader” or “scanner” that consists of lasers, a special
microscope, and a camera, which work together to create a digital image of the array. Special
computer programs then calculate the red to green fluorescence ratio in each spot. The
calculated ratio for each spot on the array reflects the relative expression of a given gene in
the two samples. (For example, a red signal indicates that a particular gene is expressed in
sample A but not sample B; a green signal that the gene is expressed in sample B but not
sample A; and a yellow signal that the gene is expressed at roughly equal levels in both
samples. No signal means that the gene is not expressed in either sample.) The result of a gene
expression experiment is referred to as a gene expression “profile” or “signature.”

Expression arrays can be used to answer basic biology problems. For example, by comparing
the expression profile of a cell that is in a resting state to the profile of one that is dividing,
scientists can determine which genes are turned on during cell division. Microarrays also have
medical applications. For example, by comparing the expression profile of a cancer cell with
that of a normal cell, scientists can use microarrays to diagnose different cancers. Better
diagnosis can lead to more-informed treatment choices.
Eric Lander’s group was the first to show that gene expression arrays could be used to
distinguish between two types of leukemia, acute lymphoblastic leukemia (ALL) and acute
myeloid leukemia (AML). In a paper published in 1999 in the journal Science, the scientists used
expression results from 6,800 human genes measured on arrays to accurately predict whether a
patient had AML or ALL. Until then, doctors distinguished between the two cancers by using a
battery of expensive tests that could cost critical time. Since that study, a number of studies
have shown similar results with different types of cancers.

DNA Sequence Arrays

Other types of arrays can be used to measure genetic variations among individuals. For
example, single nucleotide variations, or SNPs (pronounced “snips”), which occur throughout
the genome, can be detected by using genotyping arrays or SNP chips. These types of arrays
carry all the possible variations of one gene or several genes in a grid pattern. A DNA sample is
extracted, multiple copies of the gene or genes of interest are generated using polymerase
chain reaction (PCR), and the sample is then applied to the chip. The spots that light up
correspond to the particular gene variants the individual has.

Like the expression arrays, these types of arrays provide basic information, such as the range
of variation in human genomes. But they can also have clinical applications. By looking at
several SNPs at once, researchers can identify “SNP signatures” associated with a specific
disease or a response to a drug. Individuals at risk for a particular disease could then be tested
for the telltale signature.

A newer technique called microarray comparative genomic hybridization (microarray CGH) has
been developed to identify large regions of DNA that are either missing (deletions) or present
in more copies (amplifications). Rearrangements in the DNA, such as deletions and
amplifications, are often involved in diseases such as cancer. Other types of arrays are able to
detect DNA sequences that bind to proteins or sequences that are chemically modified in the
genome, such as by methylation or acetylation.

Protein Arrays

A different but conceptually similar approach is being applied directly to proteins. Scientists
have developed microarrays that can be used either to identify and quantify thousands of
different proteins at once or to find associations between different kinds of proteins and
between proteins and other molecules. These types of arrays are collectively referred to as
protein arrays.

Protein arrays that are used to identify proteins typically consist of many antibodies arrayed on
a glass or plastic slide. Each antibody can bind to a different target protein. When a mixture of
proteins—for example, in a blood sample—is applied to the array, the proteins recognized by
the different antibodies will bind to the array. Bound proteins can be detected either by adding
a second antibody tagged with a fluorescent molecule or by chemically labeling all the proteins
in the blood sample before adding the sample to the array. Each bound protein can therefore
be detected as a signal on the array, and the intensity of the signal roughly represents the
amount of protein in the blood sample. This type of array, like a gene expression array, can be
used to generate “signatures” for different cell types and tissues.

Another type of protein microarray is used to glean insights into the function of different
proteins by looking at the molecules they bind to. For this application, the proteins
themselves, rather than their antibodies, are arrayed on a slide. Stuart Schreiber’s group was
one of the first to show that more than 10,000 different proteins could be “stuck” to a single
glass microscope slide and still retain their biological activity. In a typical experiment,
thousands of proteins that are found in a cell are bound to an array. Then a particular type of
molecule—for example, a fat molecule (or lipid)—is fluorescently labeled and applied to the
protein array. The spots on the array that light up with a fluorescent signal correspond to
proteins that associate with lipids. This knowledge provides important insights into the function
of many proteins at once.

Protein binding information can also be obtained by using a small molecule microarray, in which
thousands of different small synthetic molecules are arrayed on a slide. An advantage of this
type of array is that small molecules tend to be more stable and rugged, which simplifies
storage and handling requirements and makes the process suitable for mass production. By
letting a single protein species react to this type of array, one can identify different small
molecules that bind to proteins. Some of these small molecules may be candidates for a
reagent that interferes with the functions of a protein, or they may even lead to new drug
discovery.
Any chromosome other than a sex chromosome. Humans have 22 pairs of autosomes.
Cytogenic Map

The visual appearance of a chromosome when stained and examined under a microscope.
Particularly important are visually distinct regions, called light and dark bands, which give each
of the chromosomes a unique appearance. This feature allows a person's chromosomes to be
studied in a clinical test known as a karyotype, which allows scientists to look for chromosomal
alterations.
DNA Replication
The process by which the DNA double helix unwinds and makes an exact copy of itself.
EXON
The region of a gene that contains the code for producing the gene's protein. Each exon codes
for a specific portion of the complete protein. In some species (including humans), a gene's
exons are separated by long regions of DNA (called introns or sometimes "junk DNA") that have
no apparent function.

fluorescence in situ hybridization (FISH)

A process which vividly paints chromosomes or portions of chromosomes with fluorescent

molecules. This technique is useful for identifying chromosomal abnormalities and gene
mapping.
Gene Expression
Proteins are made from instructions encoded DNA
Marker
Also known as a genetic marker, a segment of DNA with an identifiable physical location on a
chromosome whose inheritance can be followed. A marker can be a gene, or it can be some
section of DNA with no known function. Because DNA segments that lie near each other on a
chromosome tend to be inherited together, markers are often used as indirect ways of tracking
the inheritance pattern of genes that have not yet been identified, but whose approximate
locations are known.
Noncoding DNA

The strand of DNA that does not carry the information necessary to make a protein. The non-
coding strand is the mirror image of the coding strand and is also known as the antisense
strand.
Polymerase Chain Reaction - PCR
A fast, inexpensive technique for making an unlimited number of copies of any piece of DNA.
Sometimes called "molecular photocopying," PCR has had an immense impact on biology and
medicine, especially genetic research.