Polymerase Chain Reaction (PCR)
PCR is simply repetitive DNA replication over a limited, primer defined, region of a suitable template. It provides a way of amplifying a short segment of DNA without going through the cloning procedures described above. The region defined by the primers is amplified to such an extent that it can be easily isolated for further study. The reaction exploits the fact that a DNA duplex, in a low-salt buffer, will melt (i.e., separate into two single strands) at 167°F (75°C), but will reanneal (rehybridize) at 98.6°F (37°C).
The reaction is initiated by melting the template, in the presence of primers and polymerase in a suitable buffer, cooling quickly to 98.6°F (37°C), and allowing sufficient time for the polymerase to replicate both strands of the template. The temperature is then increased to 167°F (75°C) to melt the newly formed duplexes and then cooled to 98.6°F (37°C). At the lower temperature more primer will anneal to initiate another round of replication. The heating-cooling cycle is repeated 20 to 30 times, after which the reaction products are fractionated on an agarose gel, and the region containing the amplified fragment is cut out of the gel and purified for further study. The DNA polymerase used in these reactions is isolated from thermophilic bacteria that can withstand temperatures of 158°F (70°C) to 176°F (80°C). PCR applications are nearly limitless. It is used to amplify DNA from samples containing at times no more than a few cells. It is being used in the development of ultrafast DNA sequencers, identification of tissue samples in criminal investigations, amplification of ancient DNA obtained from fossils, and the identification of genes that are turned on or off during embryonic development or during cellular transformation (cancer formation).
GENE THERAPY
An illness is often due to invading microbes that destroy or damage cells and organs in our body. Cholera, smallpox, measles, diphtheria, AIDS, and the common cold are all examples of what is called an infectious disease. Such diseases may be treated with a drug that will in some cases remove the microbe from the body, thus curing the disease. Unfortunately, most diseases are not of the infectious kind. In such cases there are no microbes to fight, no drugs to apply. Instead, physicians are faced with a far more difficult problem, for this type of disease is an ailment that damages a gene. Gene therapy attempts to cure these diseases by replacing, or supplementing, the damaged gene.
When a gene is damaged, it usually is caused by a point mutation, a change that affects a single nucleotide. Sickle-cell anemia, a disease affecting red blood cells, was the first genetic disorder of this kind to be described. The mutation occurs in a gene that codes for the IE (beta) chain of hemoglobin, converting the codon GAG to GTG, which substitutes the amino acid valine at position 6, for glutamic acid. This single amino-acid substitution is enough to cripple the hemoglobin molecule, making it impossible for it to carry enough oxygen to meet the demands of a normal adult. Scientists have identified several thousand genetic disorders that are known to be responsible for diseases such as breast cancer, colon cancer, hemophilia, and two neurological disorders, Alzheimer’s disease and Parkinson’s disease.
Gene therapy is made possible by recombinant DNA technology (biotechnology). Central to this technology is the use of viruses to clone specific pieces of DNA. That is, the DNA is inserted into a viral chromosome and is amplified as the virus multiplies. Viruses are parasites that specialize in infecting bacterial and animal cells. Consequently, scientists realized that a therapeutic gene could be inserted into a patient’s cells by first introducing it into a virus and then letting the virus carry it into the affected cells. In this context the virus is referred to as gene therapy delivery vehicle or vector (in recombinant technology it is referred to as a cloning vector).
Commonly used viruses are the retrovirus and the adenovirus. A retrovirus gets its name from the fact that it has an RNA genome that is copied into DNA after it infects a cell. Corona viruses (which cause the common cold) and the AIDS virus are common examples of retroviruses. The adenovirus (from "adenoid," a gland from which the virus was first isolated) normally infects the upper respiratory tract, causing colds and flulike symptoms. This virus, unlike the retrovirus, has a DNA genome. Artificial vectors, called liposomes, have also been used that consist of a phospholipid vesicle (bubble), containing the therapeutic gene.
Vectors used in gene therapy. Adenoviruses have a DNA genome, contained in a crystalline protein capsid, and normally infect cells of the upper respiratory tract, causing colds and flulike symptoms. The protein filaments are used to infect cells. Retroviruses have an RNA genome that is converted to DNA when a cell is infected. The capsid is enclosed in a phospholipid envelope, studded with proteins that are used to infect cells. The AIDS virus is a common example of a retrovirus. Artificial vectors have also been used, consisting of a phospholipid bilayer enclosing the therapeutic gene.
Gene therapy vectors are prepared by cutting the viral chromosome and the therapeutic gene with the same restriction enzyme, after which the two are joined together with a DNA ligase. This recombinant chromosome is packaged into viral particles to form the final vector. The vector may be introduced into cultured cells suffering from a genetic defect and then returned to the patient from whom they were derived (ex vivo delivery). Alternatively, the vector may be injected directly into the patient’s circulatory system (in vivo delivery). The ex vivo procedure is used when the genetic defect appears in white blood cells, or stem cells that may be harvested from the patient and grown in culture. The in vivo procedure is used when the genetic defect appears in an organ, such as the liver, brain, or pancreas. This is the most common form of gene therapy, but it is also potentially hazardous because the vector, being free in the circulatory system, may infect a wide range of cells, thus activating an immune response that could lead to widespread tissue and organ damage.
The first gene therapy trial, conducted in 1990, used ex vivo delivery. This trial cured a young patient named Ashi deSilva of an immune deficiency (adenosine deaminase deficiency) that affects white blood cells. Other trials since then have either been ineffective or were devastating failures. Such a case occurred in 1999, when Jesse Gelsinger, an 18-year-old patient suffering from a liver disease, died while participating in a gene therapy trial. His death was caused by multiorgan failure brought on by the viral vector. In 2002 two children being treated for another form of immune deficiency developed vector-induced leukemia (cancer of the white blood cells). Subsequent studies, concluded in 2009, appear to have resolved these problems. Gene therapy holds great promise as a medical therapy. In the United States alone, there are currently more than 900 trials in progress to treat a variety of genetic disorders.
Vector preparation and delivery. A viral chromosome and a therapeutic gene are cut with the same restriction enzyme, and the two are joined together, after which, the recombinant chromosome is packaged into viral particles to form the vector. The vector may be introduced into cultured cells and then returned to the patient from whom they were derived (ex vivo delivery), or the vector may be injected directly into the patient’s circulatory system (in vivo delivery).
THE HUMAN GENOME PROJECT
Sequencing the entire human genome is an idea that grew over a period of 20 years, beginning in the early 1980s. At that time the DNA-sequencing method invented by the British biochemist Fred Sanger, then at the University of Cambridge, was but a few years old and had only been used to sequence viral or mitochondrial genomes. Indeed, one of the first genomes to be sequenced was that of bacteriophage G4, a virus that infects the bacterium Escherichia coli (E. coli). The G4 genome consists of 5,577 nucleotide pairs (or base pairs, abbreviated bp) and was sequenced in Dr. Sanger’s laboratory in 1979. By 1982 the Sanger protocol was used by others to sequence the genome of the animal virus SV40 (5,224 bp), the human mitochondrion (16,569 bp), and bacteriophage lambda (48,502 bp). Besides providing invaluable data, these projects demonstrated the feasibility of sequencing very large genomes.
The possibility of sequencing the entire human genome was first discussed at scientific meetings organized by the U.S. Department of Energy (DOE) between 1984 and 1986. A committee appointed by the U.S. National Research Council endorsed the idea in 1988 but recommended a broader program to include the sequencing of the genes of humans, bacteria, yeast, worms, flies, and mice. They also called for the establishment of research programs devoted to the ethical, legal, and social issues raised by human genome research. The program was formally launched in late 1990 as a consortium consisting of coordinated sequencing projects in the United States, Britain, France, Germany, Japan, and China. At about the same time, the Human Genome Organization (HUGO) was founded to provide a forum for international coordination of genomic research.
By 1995 the consortium had established a strategy, called hierarchical shotgun sequencing, which they applied to the human genome as well as to the other organisms mentioned. With this strategy, genomic DNA is cut into one-megabase (Mb) fragments (i.e., each fragment consists of 1 million bases) that are cloned into bacterial artificial chromosomes (BACs) to form a library of DNA fragments. The BAC fragments are partially characterized, then organized into an overlapping assembly called a contig. Clones are selected from the contigs for shotgun sequencing. That is, each shotgun clone is digested into small 1,000 bp fragments, sequenced, and then assembled into the final sequence with the aid of computers. Organizing the initial BAC fragments into contigs greatly simplifies the final assembly stage.
Sequencing of the human genome was divided into two stages. The first stage, completed in 2001, was a rough draft that covered about 80 percent of the genome with an estimated size of more than 3 billion bases (also expressed as 3 gigabases, or 3 Gb). The final draft, completed in April 2003, covers the entire genome and refines the data for areas of the genome that were difficult to sequence. It also filled in many gaps that occurred in the rough draft. The final draft of the human genome gives us a great deal of information that may be divided into three categories: gene content, gene origins, and gene organization.
Gene content
Analysis of the final draft has shown that the human genome consists of 3.2 Gb of DNA that encodes about 30,000 genes (estimates range between 25,000 to 32,000). The estimated number of genes is surprisingly low; many scientists had believed the human genome contained 100,000 genes. By comparison, the fruit fly has 13,338 genes and the simple roundworm, Caenorhabditis elegans (C. elegans), has 18,266. The genome data suggests that human complexity, as compared to the fruit fly or the worm, is not simply due to the absolute number of genes but involves the complexity of the proteins that are encoded by those genes. In general, human proteins tend to be much more complex than those of lower organisms. Data from the final draft and other sources provides a detailed overview of the functional profile of human cellular proteins.
Gene Origins
Fully one-half of human genes originated as transposable elements, also known as jumping genes (these will be discussed at length in a following section). Equally surprising is the fact that 220 of our genes were obtained by horizontal transfer from bacteria, rather than ancestral, or vertical, inheritance. In other words, humans obtained these genes directly from bacteria, probably during episodes of infection, in a kind of natural gene therapy, or gene swapping. Scientists know this to be the case because while these genes occur in bacteria, they are not present in yeast, fruit flies, or any other eukaryotes that have been tested.
The function of most of the horizontally transferred genes is unclear, although a few may code for basic metabolic enzymes. A notable exception is a gene that codes for an enzyme called mono-amine oxidase (MAO). Monoamines are neurotransmitters, such as dopamine, norepinephrine, and serotonin, which are needed for neural signaling in the human central nervous system. Monoamine oxidase plays a crucial role in the turnover of these neurotransmit-ters. How MAO, obtained from bacteria, could have developed such an important role in human physiology is a great mystery.
Gene Organization
In prokaryotes, genes are simply arranged in tandem along the chromosome, with little if any DNA separating one gene from the other. Each gene is transcribed into messenger RNA (mRNA), which is translated into protein. Indeed, in prokaryotes, which have no nucleus, translation often begins even before transcription is complete. In eukaryotes, as one might expect, gene organization is more complex. Data from the genome project shows clearly that eukaryote genes are split into subunits, called exons, and that each exon is separated by a length of DNA, called an intron. A gene, consisting of introns and exons, is separated from other genes by long stretches of noncoding DNA called intervening sequences.
Eukaryote genes are transcribed into a primary RNA molecule that includes exon and intron sequences. The primary transcript never leaves the nucleus and is never translated into protein. Nuclear enzymes remove the introns from the primary transcript, after which the exons are joined together to form the mature mRNA. Thus only the exons carry the necessary code to produce a protein.
Understanding clinical trials
Clinical trials are conducted in four phases and are always preceded by research conducted on experimental animals such as mice, rats, or monkeys. The format for preclinical research is informal; it is conducted in a variety of research labs around the world, with the results being published in scientific journals. Formal approval from a governmental regulatory body is not required.
Phase I clinical Trial
Pending the outcome of the preclinical research, investigators may apply for permission to try the experiments on human subjects. Applications in the United States are made to the Food and Drug Administration (FDA), the National Institutes of Health (NIH), and the Recombinant DNA Advisory Committee (RAC). RAC was set up by NIH to monitor any research, including clinical trials, dealing with cloning, recombinant DNA, or gene therapy. Phase I trials are conducted on a small number of adult volunteers, usually between two and 20, who have given informed consent. That is, the investigators explain the procedure, the possible outcomes, and especially, the dangers associated with the procedure before the subjects sign a consent form. The purpose of the Phase I trial is to determine the overall effect the treatment has on humans. A treatment that works well in monkeys or mice may not work at all on humans. Similarly, a treatment that appears safe in lab animals may be toxic, even deadly, when given to humans. Since most clinical trials are testing a new drug of some kind, the first priority is to determine a safe dosage for humans. Consequently, subjects in the Phase I trial are given a range of doses, all of which, even the high dose, are less than the highest dose given to experimental animals. If the results from the Phase I trial are promising, the investigators may apply for permission to proceed to Phase II.
Phase II clinical Trial
Having established the general protocol, or procedure, the investigators now try to replicate the encouraging results from Phase I but with a much larger number of subjects (100-300). Only with a large number of subjects is it possible to prove the treatment has an effect. In addition, dangerous side effects may have been missed in Phase I because of a small sample size. The results from Phase II will determine how safe the procedure is and whether it works or not. If the statistics show the treatment is effective and toxicity is low, the investigators may apply for permission to proceed to Phase III.
Phase iii clinical Trial
Based on Phase II results, the procedure may look very promising, but before it can be used as a routine treatment, it must be tested on thousands of patients at a variety of research centers. This is the expensive part of bringing a new drug or therapy to market, costing millions, sometimes billions, of dollars. It is for this reason that Phase III clinical trials invariably have the financial backing of large pharmaceutical or biotechnology companies. If the results of the Phase II trial are confirmed in Phase III, the FDA will approve the use of the drug for routine treatment. The use of the drug or treatment now passes into an informal Phase IV trial.
Phase iv clinical Trial
Even though the treatment has gained formal approval, its performance is monitored for very long-term effects, sometimes stretching on for 10 to 20 years. In this way the FDA retains the power to recall the drug long after it has become a part of standard medical procedure. It can happen that in the long term, the drug costs more than an alternative, in which case health insurance providers may refuse to cover the cost of the treatment.
Gene and protein nomenclature
Scientists who were, in effect, probing around in the dark have discovered many genes and their encoded proteins. Once discovered, the new genes or proteins had to be named. Sometimes it turns out, after further study, that the function observed in the original study is a minor aspect of the gene’s role in the cell. It is for this reason that gene and protein names sometimes seem absurd and poorly chosen.
In 2003 an International Committee on Standardized Genetic Nomenclature agreed to unify the rules and guidelines for gene and protein names for the mouse and rat. Similar committees have attempted to standardize gene-naming conventions for human, frog, zebrafish, and yeast genes. In general, the gene name is expected to be brief and to begin with a lowercase letter unless it is a person’s name. The gene symbols are acronyms taken from the gene name and are expected to be three to five characters long and not more than 10. The symbols must be written with Roman letters and Arabic numbers. The same symbol is used for orthologs (i.e. the same gene) among different species, such as human, mouse, or rat. Thus the gene sonic hedgehog is symbolized as shh, and the gene myelocytomatosis is symbolized as myc.
Unfortunately, the various committees were unable to agree on a common presentation for the gene and protein symbols. A human gene symbol, for example, is italicized, uppercase letters, and the protein is uppercase and not italicized. A frog gene symbol is lowercase, and the protein is uppercase, while neither is italicized. Thus the myc gene and its protein, for example, are written as MYC and MYC in humans, myc and MYC in frogs, and Myc and Myc in mice and rats. The latter convention, Myc and Myc, is used throughout the New Biology set, regardless of the species.
Weights and measures
The following table presents some common weights, measures, and conversions that appear in this topic and other volumes of the New Biology set.
|
quantity |
equivalent |
|
Length |
1 meter (m) = 100 centimeters (cm) = 1.094 yards = 39.37 inches 1 kilometer (km) = 1,000 m = 0.62 miles 1 foot = 30.48 cm 1 inch = 1/12 foot = 2.54 cm 1 cm = 0.394 inch = 10-2 (or 0.01) m 1 millimeter (mm) = 10-3 m 1 micrometer (pm) = 10-6 m 1 nanometer (nm) = 10-9 m 1 angstrom (A) = 10-10 m |
|
Mass |
1 gram (g) = 0.0035 ounce 1 pound = 16 ounces = 453.6 grams 1 kilogram (kg) = 2.2 pounds (lb) 1 milligram (mg) = 10-3 g 1 microgram (pg) = 10-6 g |
|
Volume |
1 liter (l) = 1.06 quarts (US) = 0.264 gallon (US) 1 quart (US) = 32 fluid ounces = 0.95 liter 1 milliliter (ml) = 10-3 liter = 1 cubic centimeter (cc) |
|
Temperature |
°C = 5/9 (°F – 32) °F = (9/5 x °C) + 32 |
|
Energy |
Calorie = the amount of heat needed to raise the temperature of 1 gram of water by 1°C. Kilocalorie = 1,000 calories. Used to describe the energy content of foods. |
