DNA TECHNOLOGY AND GENOMICS - Part
Mapping entire genomes at the DNA level
DNA variations reflected in RFLPs can serve
as the basis of a detailed map of the entire human genome.
The Human Genome Project was begun in
1990 to determine the complete nucleotide sequence of each human
- An international, publicly funded consortium
has proceeded in three phases: genetic (linkage) mapping, physical
mapping, and DNA sequencing.
The first stage was to construct a linkage map of several thousand
markers spaced throughout the chromosomes.
The next step was converting the relative distances to some physical
measure, usually the number of nucleotides along the DNA.
A physical map is made by cutting the DNA of each chromosome
into identifiable restriction fragments and then determining
the original order of the fragments by making fragments that
overlap and then use probes or automated nucleotide sequencing
of the ends to find the overlaps.
In chromosome walking, the researcher
starts with a known DNA segment (cloned, mapped, and sequenced)
and "walks" along the DNA from that locus, producing
a map of overlapping fragments. Fig.
One common method of sequencing DNA, the Sanger
method, is similar to PCR. Fig.
- Inclusion of special dideoxynucleotides
in the reaction mix ensures that rather than copying the whole
template, fragments of various lengths will be synthesized.
These dideoxynucleotides, marked radioactively or fluorescently,
terminate elongation when they are incorporated randomly into
the growing strand because they lack a 3'-OH to attach the next
The order of these fragments via gel electrophoresis can be interpreted
as the nucleotide sequence.
The public consortium has followed a
hierarchical, three-stage approach for sequencing an entire genome.
in 1992 tried a whole-genome shotgun approach.
- This uses powerful computers to assemble
sequences from random fragments, skipping the first two steps.
In 1995 they reported the complete sequence of a bacterium.
They finished the sequence of Drosophila melanogaster in
In February, 2001, Celera and the public consortium separately
announced sequencing over 90% of the human genome.
Competition and an exchange of information and approaches between
the two groups has hastened progress.
By mid-2001, the genomes of about 50 species had been completely
(or almost completely) sequenced.
Areas with repetitive DNA and certain parts of the chromosomes
of multicellular organisms resist detailed mapping by the usual
DNA sequences, long lists of A's, T's, G's,
and C's, are being collected in computer data banks that are available
to researchers everywhere via the Internet.
The Human Genome Project determined that there are 30,000 to 40,000
human genes. Table
Knowing the sequence does not tell us the function of the genes.
One way to determine their function is to disable
the gene and hope that the consequences provide clues to the gene's
The next step after mapping and sequencing genomes is proteomics,
the systematic study of full protein sets (proteomes) encoded
- One challenge is the sheer number of proteins
in humans and the proteins are extremely varied in structure
and chemical and physical properties.
Because proteins are the molecules that actually carry out cell
activities, we must study them to learn how cells and organisms
These analyses will provide understanding of
the spectrum of genetic variation in humans.
- Because we are all probably descended from
a small population living in Africa 150,000 to 200,000 years
ago, the amount of DNA variation in humans is small.
Most of our diversity is in the form of single nucleotide
polymorphisms (SNPs), single base-pair variations.
In humans, SNPs occur about once in 1,000 bases, meaning that
any two humans are 99.9% identical.
The locations of the human SNP sites will provide useful markers
for studying human evolution and for identifying disease genes
and genes that influence our susceptibility to diseases, toxins
Practical Applications of DNA Technology
Medicine and the pharmaceutical industry
Identification of genes whose mutations are
responsible for genetic diseases could lead to ways to diagnose,
treat, or even prevent these conditions before the onset of symptoms,
even before birth. It is also possible to identify symptomless
Susceptibility to many "nongenetic" diseases, from arthritis
to AIDS, is influenced by a person's genes.
PCR and labeled probes can track down the pathogens responsible
for infectious diseases. PCR can amplify and thus detect HIV DNA
in blood and tissue samples, detecting an otherwise elusive infection.
Gene therapy. - A normal allele is inserted into somatic
cells of a tissue affected by a genetic disorder.
- For gene therapy of somatic cells to be permanent,
the cells that receive the normal allele must be ones that multiply
throughout the patient's life.
Bone marrow cells, which include the stem cells that give
rise to blood and immune system cells, are prime candidates for
gene therapy. Fig.
A normal allele could be inserted by a viral vector into some
bone marrow cells removed from the patient.
The returned modified cells will multiply throughout the patient's
life and express the normal gene, providing missing proteins.
Effective gene therapy has not been successful
yet. Even when genes are successfully and safely transferred and
expressed in their new host, their activity typically diminishes
after a short period.
- Most current gene therapy is used to fight
major killers such as heart disease and cancer.
The most successful are those in which a limited activity period
is not only sufficient but desirable.
Some success has been reported in stimulated new heart blood
vessels in pigs after gene therapy.
Gene therapy raises some difficult ethical
and social questions.
- Is tampering with human genes, even for those
with life-threatening diseases, wrong?
Will it lead to the practice of eugenics, a deliberate
effort to control the genetic makeup of human populations?
Should we treat human germ-line cells to correct the defect in
Should we interfere with evolution in this way?
DNA technology has been used to create many
useful pharmaceuticals, mostly proteins. By transferring
the gene for a protein into a host that is easily grown in culture,
one can produce large quantities of normally rare proteins.
The first practical applications were the production of mammalian
hormones in bacteria.
- Human insulin,
produced by bacteria, is superior for the control of diabetes
than the older treatment of pig or cattle insulin.
Human growth hormone benefits children with hypopituitarism,
a form of dwarfism.
Tissue plasminogen activator (TPA) helps dissolve blood
clots and reduce the risk of future heart attacks.
New pharmaceutical products are responsible
for novel ways of fighting diseases that do not respond to traditional
- Can use genetically engineered proteins that
either block or mimic surface receptors on cell membranes.
One experimental drug mimics a receptor protein that HIV bonds
to when entering white blood cells, but HIV binds to the drug
instead and fails to enter the blood cells.
A vaccine is a harmless variant or derivative
of a pathogen that stimulates the immune system.
- Traditional vaccines are either particles
of virulent viruses that have been inactivated by chemical or
physical means or active virus particles of a nonpathogenic strain.
Recombinant DNA techniques can generate large amounts of a specific
protein molecule normally found on the pathogen's surface.
If this protein triggers an immune response against the intact
pathogen, then it can be used as a vaccine.
Alternatively, genetic engineering can modify the genome of the
pathogen to attenuate it which usually triggers a greater response
by the immune system. These may be safer than the natural mutants
Forensic, environmental, and agricultural
- In violent crimes, blood, semen, or traces
of other tissues may be left at the scene or on the clothes or
other possessions of the victim or assailant.
If enough tissue is available, forensic laboratories can determine
blood type or tissue type by using antibodies for specific cell
These tests require relatively large amounts of fresh tissue
and can only exclude a suspect.
- DNA testing can identify the guilty individual
with a much higher degree of certainty, because the DNA sequence
of every person is unique (except for identical twins).
RFPL analysis by Southern blotting can detect similarities
and differences in DNA samples and requires only tiny amount
of blood or other tissue.
Radioactive probes mark electrophoresis bands that contain certain
Even as few as five markers from an individual can be used to
create a DNA fingerprint. Fig.
- DNA fingerprinting can be used to settle
conclusively a question of paternity or to identify the remains
PCR is often used to amplify the DNA before electrophoresis,
especially if the DNA is poor or in minute quantities.
Forensic DNA tests focus on only about five tiny regions of the
genome. The probability that two people will have identical DNA
fingerprints in these highly variable regions is typically between
one in 100,000 and one in a billion depending on the number of
Genetic engineering is being applied to environmental
- Genetically engineered microbes can extract
heavy metals from their environments and incorporate the metals
into recoverable compounds - important both in mining materials
and cleaning up highly toxic mining wastes.
In addition to the normal microbes that participate in sewage
treatment, new microbes that can degrade other harmful compounds
are being engineered.
DNA technology to improve agricultural productivity.
To make vaccines and growth hormones for farm
Transgenic organisms have been developed for faster growth,
larger muscles. Fig.
Other transgenic organisms are pharmaceutical "factories"
- producing large amounts of an otherwise rare substance for medical
- To develop a transgenic organism, scientists
remove ova from a female and fertilize them in vitro.
The desired genes from another organism are cloned and then
inserted into the nuclei of the eggs.
Some cells will integrate the foreign DNA into their genomes
and are able to express its protein.
The engineered eggs are then surgically implanted in a surrogate
If development is successful, the result is a transgenic animal,
containing genes from a "third" parent, even from another
A number of transgenic crop plants have genes
for desirable traits, such as delayed ripening and resistance
to spoilage and disease.
- The Ti plasmid, from the soil bacterium
Agrobacterium tumefaciens, is often used to introduce
new genes into plant cells.
The Ti plasmid normally integrates a segment of its DNA into
its host plant and induces tumors. Fig.
Foreign genes can be inserted into the Ti plasmid (a version
that does not cause disease) using recombinant DNA techniques.
The recombinant plasmid can be put back into Agrobacterium,
which then infects plant cells, or introduced directly into plant
The Ti plasmid can only be used as a vector to transfer genes
to dicots (plants with two seed leaves).
Monocots, including corn and wheat, cannot
be infected by Agrobacterium (or the Ti plasmid).
Electroporation and DNA guns are used to introduce
DNA into these plants.
In the past few years, roughly half of the
soybeans and corn in America have been grown from genetically
- These plants may receive genes for resistance
to weed-killing herbicides or to infectious microbes and pest
Scientists are using gene transfer to improve the nutritional
value of crop plants.
For example, a transgenic rice plant has been developed that
produces yellow grains containing beta-carotene. Fig.
Humans use beta-carotene to make vitamin A.
Currently, 70% of children under the age of 5 in Southeast Asia
are deficient in vitamin A, leading to vision impairment and
increased disease rates.
An important potential use of DNA technology
focuses on nitrogen-fixation. Fig.
- Only certain bacteria can "fix"
In areas with nitrogen-deficient soils, expensive fertilizers
must be added for crops to grow.
Nitrogen fertilizers also contribute to water pollution.
DNA technology offers ways to increase bacterial nitrogen fixation
and eventually, perhaps, to engineer crop plants to fix nitrogen
DNA technology has lead to new alliances between
the pharmaceutical industry and agriculture.
- Plants can be engineered to produce human
proteins for medical use and viral proteins for use as vaccines.
Several such "pharm" products are in clinical trials,
including vaccines for hepatitis B and an antibody that blocks
the bacteria that cause tooth decay.
DNA technology raises important safety and
Recombinant DNA technology may create hazardous
- Today, most public concern centers on genetically
modified (GM) organisms used in agriculture.
Genetically modified animals are still not part of our food supply,
but GM crop plants are.
In Europe, there is pending new legislation regarding GM crops
and bans on the import of all GM foodstuffs.
In the United States and other countries where the GM revolution
had proceeded more quietly, the labeling of GM foods is now being
- This is required by exporters in a Biosafety
GM crops might somehow be hazardous to human health or cause
Transgenic plants may pass their new genes to close relatives
in nearby wild areas through pollen transfer.
Transference of genes for resistance to herbicides, diseases,
or insect pests may lead to the development of wild "superweeds"
that would be difficult to control.
As with all new technologies, developments
in DNA technology have ethical overtones.
- Who should have the right to examine someone
How should that information be used?
Should a person's genome be a factor in suitability for a job
or eligibility for life insurance?