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 chromosome.

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. 20.11.

One common method of sequencing DNA, the Sanger method, is similar to PCR. Fig. 20.12.

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 nucleotide.
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. Fig. 20.13

Celera Genomics 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 2000.
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 methods.

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 20.1.

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 normal function.

The next step after mapping and sequencing genomes is proteomics, the systematic study of full protein sets (proteomes) encoded by genomes.

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 function.

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 or drugs.

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 carriers.
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. 20.16.
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 future generations?
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 drug treatments.

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 traditionally used.

Forensic, environmental, and agricultural applications

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 surface proteins.
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 RFLP markers.
Even as few as five markers from an individual can be used to create a DNA fingerprint. Fig. 20.17.
DNA fingerprinting can be used to settle conclusively a question of paternity or to identify the remains of individuals.

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 markers.

Genetic engineering is being applied to environmental work.

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 animals.
Transgenic organisms have been developed for faster growth, larger muscles. Fig. 20.18
Other transgenic organisms are pharmaceutical "factories" - producing large amounts of an otherwise rare substance for medical use.

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 mother.
If development is successful, the result is a transgenic animal, containing genes from a "third" parent, even from another species.

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. 20.19
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 cells.
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 modified seeds.

These plants may receive genes for resistance to weed-killing herbicides or to infectious microbes and pest insects.
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. 20.20
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. 54.18

Only certain bacteria can "fix" nitrogen.
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 themselves.

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 ethical questions

Recombinant DNA technology may create hazardous new pathogens.

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 debated.
This is required by exporters in a Biosafety Protocol.
GM crops might somehow be hazardous to human health or cause ecological harm.
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 else's genes?
How should that information be used?
Should a person's genome be a factor in suitability for a job or eligibility for life insurance?