The Life Sciences Today

The biological sciences have experienced enormous growth over the last century,

fueled by a stream of discoveries — such as the principles of genetics, the structure of

DNA, and the discovery of gene splicing technologies. These have opened new fields of

inquiry and provided the basis for myriad applications in industry, agriculture, and

medicine. Among the technological breakthroughs in the life sciences, genetic

engineering plays a particularly significant role.

Genetic engineering is a technique that permits the artificial modification and

transfer of the genetic material from one organism to another and from one species to

another. This technology is used throughout the world to alter the protein produced by a

gene and to design organisms with desirable traits for applications ranging from basic

research and development activities to pharmaceutical and industrial uses. During the

last 30 years, these recombinant techniques have spawned a vibrant biotechnology

industry focused largely on the development of new pharmaceuticals to fight disease.6

By 2000 the annual investment in the biotechnology industry peaked at nearly $29

billion, while employment in the biotechnology industry reached 191,000 by 2001.7

In response to the opportunities presented by these developments the resources

devoted to the life sciences have increased dramatically, making further discoveries

possible. The government has funded biological research generously through the

National Institutes of Health and National Science Foundation budgets, with few strings

attached; private foundations and the pharmaceutical industry have also made major

contributions. The number of PhDs awarded each year in the biological and agricultural

sciences has increased steadily; 6,526 were awarded in 2001.8

This ever-expanding research activity has resulted in numerous new

biopharmaceutical products that are transforming medicine. Examples include human

recombinant insulin for the treatment of diabetes, a vaccine against hepatitis B, and

medicines for diabetes, cancer therapy, arthritis, multiple sclerosis, cystic fibrosis, heart

attacks, hemophilia, and sepsis. As knowledge of the human genome increases, it may

even become possible to tailor pharmaceutical products not only to specific diseases but

also to specific individuals. Throughout this process, the time between new discoveries

and their applications has grown ever shorter. One example is the very short time it took

the scientific community to identify the coronavirus as the causal agent of the newly

emerging human disease, severe acute respiratory syndrome (SARS).

Biotechnology research is now a truly global enterprise. While industrialized

countries such as the United States, the United Kingdom, Germany, Israel, and Japan

may be the first to develop advanced research and technologies, other countries have a skill base that will enable broad domestic utilization of biological technologies.9 For

example:

China has an aggressive program in plant biotechnology, and as of 2002

plans to increase funding by 400 percent by 2005. This energetic

investment also exists in the Chinese private sector, and the national

scientific establishment is attempting to lure foreign-trained scientists to

return with lucrative financial packages. India is in the process of tripling

funding to its national biotech center, and is promoting the development

and use of genetically modified crops throughout Asia. Singapore has for

many years made a practice of recruiting foreign scientists. Taiwan is

investing large amounts in biotechnology and is seeking citizens to return

home to build up biotechnology in academia and industry. A Brazilian

coalition recently demonstrated sophisticated domestic use of biological

technologies by successfully sequencing the plant pathogen X. fastidiosa in

2000.10

In addition to the dispersed research enterprise, publications and personnel are

also widely spread. Well over 10,000 journals in the life sciences are published

worldwide. Biological Abstracts, an international database on biology, clinical and

experimental medicine, biochemistry, and biotechnology, provides coverage of over

6,000 active international journals and 14,000 archival titles from over 100 countries;

Medline, the online service of the National Institutes of Medicine, provides abstract

information for more than 4,600 biomedical journals published in the United States and

70 other countries; and PubMed currently provides full-text web access to 4,058 journals.

According to Medline, the total number of scientific articles published in the peerreviewed

biomedical literature has increased from 449,109 in 1998 to 491,620 in 2001.

Given the global nature of the biotechnology research and development enterprise, it is

unrealistic to think that biological technologies and the knowledge base upon which they

rest can somehow be isolated within the borders of a few countries.

The rapid advance of scientific knowledge and applications owes much to a

research culture in which knowledge and biological materials are shared among

scientists and people move freely between universities, government agencies, and

private industry. Large numbers of foreign graduate students and postdoctoral

associates have been an essential ingredient in the success of the biological research

enterprise. The scientific workforce is increasingly international; at the National Institutes

of Health, for example, approximately 50 percent of the technical staff are non-U.S.

citizens. Research results have been widely disseminated, so that even high school

students now routinely perform experiments involving recombinant DNA techniques. In

short, a dynamic national and international research enterprise has evolved, with an

extraordinary record of achievement at multiple centers of excellence. These are values

that should be preserved in any sensible policy for minimizing the risks associated with

the misapplication of the fruits of the biotechnology enterprise.

MOLECULAR BIOLOGY

The great achievements of molecular biology and genetics over the last 50 years

have produced advances in agriculture and industrial processes and have revolutionized

the practice of medicine. The very technologies that fueled these benefits to society,

however, pose a potential risk as well — the possibility that these technologies could

also be used to create the next generation of biological weapons. Biotechnology

represents a “dual use” dilemma in which the same technologies can be used

legitimately for human betterment and misused for bioterrorism.

Events over the 1990s focused growing attention on this balance of risks and

benefits, part of a larger concern about the proliferation of weapons of mass destruction

(WMD) — chemical, nuclear, or biological. In early 1992, President Yeltsin

acknowledged that, despite being an original signatory and State party to the Biological

and Toxin Weapons Convention (BWC), the Soviet Union had maintained a major

clandestine biological weapons program into the early 1990s.1 Yeltsin ordered the

program shut down, but concerns about other possible secret programs remained.

Policymakers in the United States became increasingly concerned that so-called ”rogue

states” would turn to WMD to counter the overwhelming U.S. conventional military

superiority. Secretary of Defense Les Aspin launched the “Defense Counterproliferation

Initiative” in December 1993 to develop additional means to address these threats.

Official statements continue to cite at least a dozen countries believed to have or to be

pursuing a biological weapons capability.2 U.S. and British concerns about Iraq’s

reported biological and other WMD programs were a primary reason for launching

preemptive military action to find and destroy these weapons capabilities.3 The terrorist

attacks of September 11, 2001 and the subsequent anthrax letters accelerated already

existing concerns that terrorists would seek WMD capabilities as well. President Bush, in

a speech at West Point in 2002, said: “The gravest danger to freedom lies at the perilous

crossroads of radicalism and technology. When the spread of chemical and biological

and nuclear weapons, along with ballistic missile technology — when that occurs, even

weak states and small groups could attain a catastrophic power to strike great nations.”4

States, groups, and individuals are pursuing a biological weapons capability — and the

means for them to do so are widely available.

Biological weapons have long been stigmatized as “indiscriminant agents of

unnecessary suffering, [whose] use … contradict(s) the universal principles of war.”5 As

discussed below, since November 1969 the U.S. programs linked to biological weapons

have been restricted to research and development on defensive measures only. Thus

few biologists in the United States today have knowledge of our country’s past offensive

weapons programs or of the concerns of the national security branches of government.

In this respect the life sciences community is in a different situation from that of the

physics community, which in large part has been continuously involved in governmentsponsored

weapons research programs since at least World War II. The scientific

community and the government jointly face a double challenge: (1) to establish a

working relationship with the national security branches of government, and (2) to help

craft a system that will minimize the risk of wrongful use of biological agents or

technology without damaging the scientific infrastructure that has made biological

research so vital to the health of the nation.

Techniques of molecular biology

Since the late 1950s and early 1960s, molecular biologists have learned to characterize, isolate, and manipulate the molecular components of cells and organisms. These components include DNA, the repository of genetic information; RNA, a close relative of DNA whose functions range from serving as a temporary working copy of DNA to actual structural and enzymatic functions as well as a functional and structural part of the translational apparatus; and proteins, the major structural and enzymatic type of molecule in cells.

For more extensive list on protein methods, see protein methods.

For more extensive list on nucleic acid methods, see nucleic acid methods.

[edit]Expression cloning

Main article: Expression cloning

One of the most basic techniques of molecular biology to study protein function is expression cloning. In this technique, DNA coding for a protein of interest is cloned (using PCR and/or restriction enzymes) into a plasmid (known as an expression vector). This plasmid may have special promoter elements to drive production of the protein of interest, and may also have antibiotic resistance markers to help follow the plasmid.

This plasmid can be inserted into either bacterial or animal cells. Introducing DNA into bacterial cells can be done by transformation (via uptake of naked DNA), conjugation (via cell-cell contact) or by transduction (via viral vector). Introducing DNA into eukaryotic cells, such as animal cells, by physical or chemical means is called transfection. Several different transfection techniques are available, such as calcium phosphate transfection,electroporation, microinjection and liposome transfection. DNA can also be introduced into eukaryotic cells using viruses or bacteria as carriers, the latter is sometimes called bactofection and in particular uses Agrobacterium tumefaciens. The plasmid may be integrated into the genome, resulting in a stable transfection, or may remain independent of the genome, called transient transfection.

In either case, DNA coding for a protein of interest is now inside a cell, and the protein can now be expressed. A variety of systems, such as inducible promoters and specific cell-signaling factors, are available to help express the protein of interest at high levels. Large quantities of a protein can then be extracted from the bacterial or eukaryotic cell. The protein can be tested for enzymatic activity under a variety of situations, the protein may be crystallized so its tertiary structure can be studied, or, in the pharmaceutical industry, the activity of new drugs against the protein can be studied.

[edit]Polymerase chain reaction (PCR)

Main article: Polymerase chain reaction

The polymerase chain reaction is an extremely versatile technique for copying DNA. In brief, PCR allows a single DNA sequence to be copied (millions of times), or altered in predetermined ways. For example, PCR can be used to introduce restriction enzyme sites, or to mutate (change) particular bases of DNA, the latter is a method referred to as "Quick change". PCR can also be used to determine whether a particular DNA fragment is found in a cDNA library. PCR has many variations, like reverse transcription PCR (RT-PCR) for amplification of RNA, and, more recently, real-time PCR (QPCR) which allow for quantitative measurement of DNA or RNA molecules.

[edit]Gel electrophoresis

Main article: Gel electrophoresis

Gel electrophoresis is one of the principal tools of molecular biology. The basic principle is that DNA, RNA, and proteins can all be separated by means of an electric field. In agarose gel electrophoresis, DNA and RNA can be separated on the basis of size by running the DNA through an agarose gel. Proteins can be separated on the basis of size by using an SDS-PAGE gel, or on the basis of size and their electric charge by using what is known as a 2D gel electrophoresis.

[edit]Southern blotting

Main article: Southern blot

Named after its inventor, biologist Edwin Southern, the Southern blot is a method for probing for the presence of a specific DNA sequence within a DNA sample. DNA samples before or after restriction enzyme digestion are separated by gel electrophoresis and then transferred to a membrane by blotting via capillary action. The membrane is then exposed to a labeled DNA probe that has a complement base sequence to the sequence on the DNA of interest. Most original protocols used radioactive labels, however non-radioactive alternatives are now available. Southern blotting is less commonly used in laboratory science due to the capacity of other techniques, such as PCR, to detect specific DNA sequences from DNA samples. These blots are still used for some applications, however, such as measuring transgene copy number intransgenic mice, or in the engineering of gene knockout embryonic stem cell lines.

[edit]Northern blotting

Main article: northern blot

The northern blot is used to study the expression patterns a specific type of RNA molecule as relative comparison among of a set of different samples of RNA. It is essentially a combination of denaturing RNA gel electrophoresis, and a blot. In this process RNA is separated based on size and is then transferred to a membrane that is then probed with a labeled complement of a sequence of interest. The results may be visualized through a variety of ways depending on the label used; however, most result in the revelation of bands representing the sizes of the RNA detected in sample. The intensity of these bands is related to the amount of the target RNA in the samples analyzed. The procedure is commonly used to study when and how much gene expression is occurring by measuring how much of that RNA is present in different samples. It is one of the most basic tools for determining at what time, and under what conditions, certain genes are expressed in living tissues.

[edit]Western blotting

Main article: western blot

Antibodies to most proteins can be created by injecting small amounts of the protein into an animal such as a mouse, rabbit, sheep, or donkey (polyclonal antibodies)or produced in cell culture (monoclonal antibodies). These antibodies can be used for a variety of analytical and preparative techniques.

In western blotting, proteins are first separated by size, in a thin gel sandwiched between two glass plates in a technique known as SDS-PAGE(sodium dodecyl sulfate polyacrylamide gel electrophoresis). The proteins in the gel are then transferred to a PVDF, nitrocellulose, nylon or other support membrane. This membrane can then be probed with solutions of antibodies. Antibodies that specifically bind to the protein of interest can then be visualized by a variety of techniques, including colored products, chemiluminescence, or autoradiography. Often, the antibodies are labeled with an enzymes. When a chemiluminescent substrate is exposed to the enzyme it allows detection. Using western blotting techniques allows not only detection but also quantitative analysis.

Analogous methods to western blotting can be used to directly stain specific proteins in live cells or tissue sections. However, theseimmunostaining methods, such as FISH, are used more often in cell biology research.

[edit]Blotting jokes

The terms "western" and "northern" are molecular biology jokes that play on the term southern blot. The first blots were with DNA, and since they were done by Ed Southern, they came to be known as Southerns. Patricia Thomas, inventor of the RNA blot, which became known as a "northern", actually didn't use the term. ^[2]. To carry the joke further, one can find references in the literature to "southwesterns" (protein-DNA interactions), "northwesterns" (protein-RNA interactions) and "farwesterns" (protein-protein interactions).

[edit]Arrays

Main article: DNA microarray

A DNA array is a collection of spots attached to a solid support such as a microscope slide where each spot contains one or more single-stranded DNA oligonucleotide fragment. Arrays make it possible to put down a large quantity of very small (100 micrometre diameter) spots on a single slide. Each spot has a DNA fragment molecule that is complementary to a single DNA sequence (similar to Southern blotting). A variation of this technique allows the gene expression of an organism at a particular stage in development to be qualified (expression profiling). In this technique the RNA in a tissue is isolated and converted to labeled cDNA. This cDNA is then hybridized to the fragments on the array and visualization of the hybridization can be done. Since multiple arrays can be made with the exact same position of fragments they are particularly useful for comparing the gene expression of two different tissues, such as a healthy and cancerous tissue. Also, one can measure what genes are expressed and how that expression changes with time or with other factors. For instance, the common baker's yeast,Saccharomyces cerevisiae, contains about 7000 genes; with a microarray, one can measure qualitatively how each gene is expressed, and how that expression changes, for example, with a change in temperature. There are many different ways to fabricate microarrays; the most common are silicon chips, microscope slides with spots of ~ 100 micrometre diameter, custom arrays, and arrays with larger spots on porous membranes (macroarrays). There can be anywhere from 100 spots to more than 10,000 on a given array.

Arrays can also be made with molecules other than DNA. For example, an antibody array can be used to determine what proteins or bacteriaare present in a blood sample.

[edit]Allele Specific Oligonucleotide

Allele specific oligonucleotide (ASO) is a technique that allows detection of single base mutations without the need for PCR or gel electrophoresis. Short (20-25 nucleotides in length), labeled probes are exposed to the non-fragmented target DNA. Hybridization occurs with high specificity due to the short length of the probes and even a single base change will hinder hybridization. The target DNA is then washed and the labeled probes that didn't hybridize are removed. The target DNA is then analyzed for the presence of the probe via radioactivity or fluorescence. In this experiment, as in most molecular biology techniques, a control must be used to ensure successful experimentation.

[edit]Abandoned technology

As new procedures and technology become available, the older technology is rapidly abandoned. A good example is methods for determining the size of DNA molecules. Prior to gel electrophoresis (agarose or polyacrylamide) DNA was sized with rate sedimentation in sucrose gradients, a slow and labor intensive technology requiring expensive instrumentation; prior to sucrose gradients, viscometry was used.

Aside from their historical interest, it is worth knowing about older technology as it may be useful to solve a particular problem.

Molecular biology

Molecular biology is the study of biology at a molecular level. The field overlaps with other areas of biology and chemistry, particularlygenetics and biochemistry. Molecular biology chiefly concerns itself with understanding the interactions between the various systems of a cell, including the interactions between DNA, RNA and protein biosynthesis and learning how these interactions are regulated.

Writing in Nature, William Astbury described molecular biology as:

"[...]not so much a technique as an approach, an approach from the viewpoint of the so-called basic sciences with the leading idea of searching below the large-scale manifestations of classical biology for the corresponding molecular plan. It is concerned particularly with the forms of biological molecules and[...]is predominantly three-dimensional and structural—which does not mean, however, that it is merely a refinement of morphology. It must at the same time inquire into genesis and function." ^[1]

Central dogma of molecular biology

The central dogma of molecular biology was first enunciated by Francis Crick in 1958^[1]and re-stated in a Nature paper published in 1970:^[2]

The central dogma of molecular biology deals with the detailed residue-by-residue transfer of sequential information. It states that information cannot be transferred back from protein to either protein or nucleic acid.

In other words, 'once information gets into protein, it can't flow back to nucleic acid.'

The dogma is a framework for understanding the transfer of sequence information between sequential information-carrying biopolymers, in the most common or general case, in livingorganisms. There are 3 major classes of such biopolymers: DNA and RNA (both nucleic acids), and protein. There are 3×3 = 9 conceivable direct transfers of information that can occur between these. The dogma classes these into 3 groups of 3: 3 general transfers (believed to occur normally in most cells), 3 special transfers (known to occur, but only under specific conditions in case of some viruses or in a laboratory), and 3 unknown transfers (believed to never occur). The general transfers describe the normal flow of biological information: DNA can be copied to DNA (DNA replication), DNA information can be copied into mRNA, (transcription), and proteins can be synthesized using the information in mRNA as a template (translation).^[2]