Biology
HS/Science
Unit: 08 Lesson: 01
Techniques for Studying Genomes Fact Sheets
– Class Set The following is a group of techniques that have been used/are being used by the National Human
Genome Research Institute to map and study the genomes of humans and other organisms. This is
the information provided through their Fact Sheets to help the public better understand their work;
more information is also available at www.genome.gov.
1. Polymerase Chain Reaction (PCR)
What is PCR?
Sometimes called "molecular photocopying," the polymerase chain reaction (PCR) is a fast and
inexpensive technique used to "amplify" - copy - small segments of DNA. Because significant
amounts of a sample of DNA are necessary for molecular and genetic analyses, studies of isolated
pieces of DNA are nearly impossible without PCR amplification.
Often heralded as one of the most important scientific advances in molecular biology, PCR
revolutionized the study of DNA to such an extent that its creator, Kary B. Mullis, was awarded the
Nobel Prize for Chemistry in 1993.
What is PCR used for?
Once amplified, the DNA produced by PCR can be used in many different laboratory procedures. For
example, most mapping techniques in the Human Genome Project (HGP) rely on PCR.
PCR is also valuable in a number of newly emerging laboratory and clinical techniques, including
DNA fingerprinting, detection of bacteria or viruses (particularly AIDS), and diagnosis of genetic
disorders.
How does PCR work?
To amplify a segment of DNA using PCR, the sample is first heated so the DNA denatures, or
separates, into two pieces of single-stranded DNA. Next, an enzyme called "Taq polymerase"
synthesizes - builds - two new strands of DNA using the original strands as templates. This process
results in the duplication of the original DNA, with each of the new molecules containing one old and
one new strand of DNA. Then, each of these strands can be used to create two new copies and so on
and so on. The cycle of denaturing and synthesizing new DNA is repeated as many as 30 or 40
times, leading to more than one billion exact copies of the original DNA segment.
The entire cycling process of PCR is automated and can be completed in just a few hours. It is
directed by a machine called a thermocycler, which is programmed to alter the temperature of the
reaction every few minutes to allow DNA denaturing and synthesis.
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Biology
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Unit: 08 Lesson: 01
2. Gel Electrophoresis
What is gel electrophoresis?
Electrophoresis is a laboratory technique used to separate DNA, RNA, or protein molecules based on
their size and electrical charge. An electric current is used to move molecules to be separated
through a gel. There are pores in the gel that work like a sieve, allowing smaller molecules to move
faster than larger molecules. The conditions used during electrophoresis can be adjusted to separate
molecules in a desired size range.
Using Restriction Enzymes
A restriction enzyme is an enzyme isolated from bacteria that cuts DNA molecules at specific
sequences. After the DNA is cut using restriction enzymes, the DNA fragments are isolated through
the process of gel electrophoresis. The smaller a DNA fragment is, the farther it travels. In this way,
the fragments are separated based on size.
Restriction Fragment Length Polymorphism (RFLP)
Restriction fragment length polymorphism (RFLP) is a type of polymorphism that results from
variation in the DNA sequence recognized by restriction enzymes. These are bacterial enzymes used
by scientists to cut DNA molecules at known locations. RFLPs (pronounced "rif lips") are used as
markers on genetic maps. Gel electrophoresis is used to visualize RFLPs.
How is Gel Electrophoresis used?
Gel electrophoresis is commonly used in DNA fingerprinting, which is a laboratory technique used to
establish a link between biological evidence and a suspect in a criminal investigation. A DNA sample
taken from a crime scene is compared with a DNA sample from a suspect. If the two DNA profiles are
a match, then the evidence came from that suspect. Conversely, if the two DNA profiles do not
match, then the evidence cannot have come from the suspect. DNA fingerprinting is also used to
establish paternity.
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Biology
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Unit: 08 Lesson: 01
3. Spectral Karyotyping (SKY)
What is SKY?
Spectral karyotyping (SKY) is a laboratory technique that
allows scientists to visualize all of the human
chromosomes at one time by "painting" each pair of
chromosomes in a different fluorescent color.
How is SKY used?
Many diseases are associated with particular
chromosomal abnormalities. For example, chromosomes
in cancer cells frequently exhibit aberrations called
translocations, in which a piece of one chromosome
breaks off and attaches to the end of another
chromosome. Identifying such chromosomal
abnormalities and determining their role in disease is an important step in developing new methods
for diagnosing many genetic disorders.
Traditional karyotyping allows scientists to view the full set of human chromosomes in black and
white, a technique that is useful for observing the number, size, and shape of the chromosomes.
Interpreting these karyotypes, however, requires an expert who might need hours to examine a single
chromosome. By using SKY, even non-experts can easily see instances where a chromosome,
painted in one color, has a small piece of a different chromosome, painted in another color, attached
to it.
How does SKY work?
SKY involves the preparation of a large collection of short sequences of single-stranded DNA called
probes. Each of the individual probes in this DNA "library" is complementary to a unique region of one
chromosome. Together, all of the probes make up a collection of DNA that is complementary to all of
the chromosomes within the human genome.
Each probe is labeled with a fluorescent color that is designated for a specific chromosome. For
example, probes that are complementary to chromosome 1 are labeled with yellow molecules, while
those that are complementary to chromosome 2 are labeled with red molecules and so on.
When these probes are mixed with the chromosomes from a human cell, the probes hybridize - bind to the DNA in the chromosomes. As they hybridize, the fluorescent probes essentially paint the full
set of chromosomes in a rainbow of colors. Scientists can then use computers to analyze the painted
chromosomes to determine whether any of them exhibits translocations or other structural
abnormalities.
Last Updated: October 13, 2011
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Biology
HS/Science
Unit: 08 Lesson: 01
4. Fluorescence In Situ Hybridization (FISH)
What is FISH?
Fluorescence in situ hybridization (FISH)
provides researchers with a way to visualize and
map the genetic material in an individual's cells,
including specific genes or portions of genes.
This is important for understanding a variety of
chromosomal abnormalities and other genetic
mutations. Unlike most other techniques used to
study chromosomes, FISH does not have to be
performed on cells that are actively dividing. This
makes it a very versatile procedure.
How does FISH work?
FISH is useful, for example, to help a researcher
identify where a particular gene falls within an individual's chromosomes. The first step is to prepare
short sequences of single-stranded DNA that match a portion of the gene the researcher is looking
for. These are called probes. The next step is to label these probes by attaching one of a number of
colors of fluorescent dye.
DNA is composed of two strands of complementary molecules that bind to each other like chemical
magnets. Since the researchers' probes are single-stranded, they are able to bind to the
complementary strand of DNA, wherever it may reside on a person's chromosomes. When a probe
binds to a chromosome, its fluorescent tag provides a way for researchers to see its location.
How is FISH used?
Scientists use three different types of FISH probes, each of which has a different application:
1. Locus specific probes bind to a particular region of a chromosome. This type of probe is useful
when scientists have isolated a small portion of a gene and want to determine on which
chromosome the gene is located.
2. Alphoid or centromeric repeat probes are generated from repetitive sequences found in the
middle of each chromosome. Researchers use these probes to determine whether an individual
has the correct number of chromosomes. These probes can also be used in combination with
"locus specific probes" to determine whether an individual is missing genetic material from a
particular chromosome.
3. Whole chromosome probes are actually collections of smaller probes, each of which binds to a
different sequence along the length of a given chromosome. Using multiple probes, labeled with a
mixture of different fluorescent dyes, scientists are able to label each chromosome in its own
unique color. The resulting full-color map of the chromosome is known as a spectral karyotype.
Whole chromosome probes are particularly useful for examining chromosomal abnormalities, for
example, when a piece of one chromosome is attached to the end of another chromosome.
Last Updated: October 13, 2011
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Biology
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Unit: 08 Lesson: 01
5. Knockout Mice
What is a knockout mouse?
A knockout mouse is a laboratory mouse in which researchers have
inactivated, or "knocked out," an existing gene by replacing it or
disrupting it with an artificial piece of DNA. The loss of gene activity
often causes changes in a mouse's phenotype, which includes
appearance, behavior, and other observable physical and
biochemical characteristics.
How are knockout mice used?
Knocking out the activity of a gene provides valuable clues about what that gene normally does.
Humans share many genes with mice. Consequently, observing the characteristics of knockout mice
gives researchers information that can be used to better understand how a similar gene may cause or
contribute to disease in humans.
Examples of research in which knockout mice have been useful include studying and modeling
different kinds of cancer, obesity, heart disease, diabetes, arthritis, substance abuse, anxiety, aging,
and Parkinson disease. Knockout mice also offer a biological context in which drugs and other
therapies can be developed and tested.
Many of these mouse models are named after the gene that has been inactivated. For example, the
p53 knockout mouse is named after the p53 gene, which codes for a protein that normally
suppresses the growth of tumors by arresting cell division. Humans born with mutations that
inactivate the p53 gene suffer from Li-Fraumeni syndrome, a condition that dramatically increases the
risk of developing bone cancers, breast cancer, and blood cancers at an early age.
Knockout mice offer one of the most powerful means, now available, for studying gene function in a
living animal. Such studies will accelerate efforts to translate newfound knowledge of the human and
mouse genomes into better strategies for diagnosing, treating, and preventing human disease.
How are knockout mice made?
Researchers begin by harvesting embryonic stem (ES) cells from early-stage mouse embryos four
days after fertilization. ES cells are used because they are able to differentiate into nearly any type of
adult cell, which means that if a gene is knocked out in an ES cell, the effects can be observed in any
tissue in an adult mouse. In addition, ES cells grown in the lab can be used to make knockout mice
as long as 10 years after they were harvested.
To produce knockout mice, researchers use one of two methods to insert artificial DNA into the
chromosomes contained in the nuclei of ES cells. Both methods are carried out in vitro, that is, in
cultured cells grown in laboratory conditions.
In the first strategy, called gene targeting, or homologous recombination, researchers specifically
manipulate a gene in the nucleus of an ES cell. Typically, this is done by introducing an artificial piece
of DNA that shares identical, or homologous, sequence to the gene. This homologous sequence
flanks the existing gene's DNA sequence both upstream and downstream of the gene's location on
the chromosome. The cells own nuclear machinery automatically recognizes the identical stretches of
sequence and swaps out the existing gene or portion of a gene with the artificial piece of DNA.
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Biology
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Unit: 08 Lesson: 01
Because the artificial DNA is inactive, bearing only a genetic tag, or "reporter gene," designed for use
in tracking, the swap eliminates, or "knocks out," the function of the existing gene.
In the second strategy, called gene trapping, researchers again manipulate a gene in an ES cell.
However, instead of directly targeting a gene of interest, a random process is used. A piece of
artificial DNA, containing a reporter gene, is designed to insert randomly into any gene. The inserted
piece of artificial DNA prevents the cell's RNA "splicing" machinery from working properly, thus
preventing the existing gene from producing its designated protein and knocking out its function. As in
the first strategy, researchers can track the activity of the artificial reporter gene to figure out the
existing gene's normal pattern of activity in mouse tissues.
For both gene targeting and gene trapping, the vehicle used to ferry the artificial DNA into ES cells
often consists of a modified viral vector or a linear fragment of bacterial DNA. After the artificial DNA
is inserted, the genetically altered ES cells are grown in a lab dish for several days and injected into
early-stage mouse embryos. The embryos are implanted into the uterus of a female mouse and
allowed to develop into mouse pups.
The resulting mouse pups have some tissues in which a gene has been knocked out - those derived
from the altered ES cells. However, they also have some normal tissues derived from the non-altered
embryos into which the altered ES cells were injected. Consequently, they are not complete knockout
mice. It is necessary to crossbreed such mice to produce lines of mice in which both copies of the
gene (one on each chromosome) are knocked out in all tissues. Researchers refer to such mice as
homozygous knockouts.
Last Updated: October 13, 2011
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Biology
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Unit: 08 Lesson: 01
6. Cloning
What is cloning?
The term cloning describes a number of different processes that can be used to produce genetically
identical copies of a biological entity. The copied material, which has the same genetic makeup as the
original, is referred to as a clone.
Researchers have cloned a wide range of biological materials, including genes, cells, tissues, and
even entire organisms, such as a sheep.
Do clones ever occur naturally?
Yes. In nature, some plants and single-celled organisms, such as bacteria, produce genetically
identical offspring through a process called asexual reproduction. In asexual reproduction, a new
individual is generated from a copy of a single cell from the parent organism.
Natural clones, also known as identical twins, occur in humans and other mammals. These twins are
produced when a fertilized egg splits, creating two or more embryos that carry almost identical DNA.
Identical twins have nearly the same genetic makeup as each other, but they are genetically different
from either parent.
What are the types of artificial cloning?
There are three different types of artificial cloning: gene cloning, reproductive cloning, and therapeutic
cloning.
Gene cloning produces copies of genes or segments of DNA. Reproductive cloning produces copies
of whole animals. Therapeutic cloning produces embryonic stem cells for experiments aimed at
creating tissues to replace injured or diseased tissues.
Gene cloning, also known as DNA cloning, is a very different process from reproductive and
therapeutic cloning. Reproductive and therapeutic cloning share many of the same techniques, but
are done for different purposes.
What sort of cloning research is going on at NHGRI?
Gene cloning is the most common type of cloning done by researchers at the National Human
Genome Research Institute (NHGRI). NHGRI researchers have not cloned any mammals, and
NHGRI does not clone humans.
How are genes cloned?
Researchers routinely use cloning techniques to make copies of genes that they wish to study. The
procedure consists of inserting a gene from one organism, often referred to as "foreign DNA," into the
genetic material of a carrier called a vector. Examples of vectors include bacteria, yeast cells, viruses,
or plasmids, which are small DNA circles carried by bacteria. After the gene is inserted, the vector is
placed in laboratory conditions that prompt it to multiply, resulting in the gene being copied many
times over.
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Biology
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Unit: 08 Lesson: 01
How are animals cloned?
The technique used to clone whole animals, such as sheep, is referred to as reproductive cloning. In
reproductive cloning, researchers remove a mature somatic cell, such as a skin cell or an udder cell,
from an animal that they wish to copy. They then transfer the DNA of the donor animal's somatic cell
into an egg cell, or oocyte, that has had its own DNA-containing nucleus removed.
Researchers can add the DNA from the somatic cell to the empty egg in two different ways. In the
first method, they remove the DNA-containing nucleus of the somatic cell and inject it into the empty
egg. In the second approach, they use an electrical current to fuse the entire somatic cell with the
empty egg. In both processes, the egg is allowed to develop into an early-stage embryo in the testtube and then is implanted into the womb of an adult female animal. Ultimately, the adult female gives
birth to an animal that has the same genetic make up as the animal that donated the somatic cell.
This young animal is referred to as a clone. Reproductive cloning may require the use of a surrogate
mother to allow development of the cloned embryo, as was the case for the most famous cloned
organism, Dolly the sheep. Reproductive cloning may require the use of a surrogate mother to allow
development of the cloned embryo, as was the case for the most famous cloned organism, Dolly the
sheep.
What is therapeutic cloning?
Therapeutic cloning involves creating a cloned embryo for the sole purpose of producing embryonic
stem cells with the same DNA as the donor cell. These stem cells can be used in experiments aimed
at understanding disease and developing new treatments for disease. To date, there is no evidence
that human embryos have been produced for therapeutic cloning.
The richest source of embryonic stem cells is tissue formed during the first five days after the egg has
started to divide. At this stage of development, called the blastocyst, the embryo consists of a cluster
of about 100 cells that can become any cell type. Stem cells are harvested from cloned embryos at
this stage of development, resulting in destruction of the embryo while it is still in the test tube.
In November 2007, using a new cloning method that removes the egg's nucleus without dyes or
ultraviolet light, researchers produced the first primate embryonic stem cells. The work involved
transferring the nucleus of a skin cell from a male rhesus monkey into the nucleus-free egg of a
female rhesus monkey. These embryonic stem cells did not develop into a whole monkey, and
researchers said their work was aimed at therapeutic applications. However, the research shows that,
with some adjustments, the techniques used to make whole copies of other animals may also work in
primates.
What are the potential uses of therapeutic cloning?
Researchers hope to use embryonic stem cells, which have the unique ability to generate virtually all
types of cells in an organism, to grow tissues in the laboratory that can be used to grow healthy tissue
to replace injured or diseased tissues. In addition, it may be possible to learn more about the
molecular causes of disease by studying embryonic stem cell lines from cloned embryos derived from
the cells of animals or humans with different diseases. (Last Reviewed: June 12, 2012)
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