SYNTHESIS AND TRANSPLANTATION

Organ transplantation is a medical procedure in which an organ is removed from one body and
placed in the body of a recipient, to replace a damaged or missing organ. The donor and recipient
may be at the same location, or organs may be transported from a donor site to another
location. Organs and/or tissues that are transplanted within the same person's body are
called autografts. Transplants that are recently performed between two subjects of the same
species are called allografts. Allografts can either be from a living or cadaveric source.
Organs that have been successfully transplanted include
the heart, kidneys, liver, lungs, pancreas, intestine, and thymus. Some organs, like the brain,
cannot be transplanted. Tissues include bones, tendons (both referred to as musculoskeletal
grafts), corneae, skin, heart valves, nerves and veins. Worldwide, the kidneys are the most
commonly transplanted organs, followed by the liver and then the heart. Corneae and
musculoskeletal grafts are the most commonly transplanted tissues; these outnumber organ
transplants by more than tenfold.
Organ donors may be living, brain dead, or dead via circulatory death. Tissue may be recovered
from donors who die of circulatory death, as well as of brain death – up to 24 hours past the
cessation of heartbeat. Unlike organs, most tissues (with the exception of corneas) can be
preserved and stored for up to five years, meaning they can be "banked".
Transplantation raises a number of bioethical issues, including the definition of death, when and
how consent should be given for an organ to be transplanted, and payment for organs for
transplantation. Other ethical issues include transplantation tourism and more broadly the socio-
economic context in which organ procurement or transplantation may occur. A particular
problem is organ trafficking.
Transplantation medicine is one of the most challenging and complex areas of modern medicine.
Some of the key areas for medical management are the problems of transplant rejection, during
which the body has an immune response to the transplanted organ, possibly leading to transplant
failure and the need to immediately remove the organ from the recipient. When possible,
transplant rejection can be reduced through serotyping to determine the most appropriate donor-
recipient match and through the use of immunosuppressant drugs.
Types of Transplants
Autographs

Autografts are the transplant of tissue to the same person. Sometimes this is done with surplus
tissue, tissue that can regenerate, or tissues more desperately needed elsewhere (examples
include skin grafts, vein extraction for CABG, etc.). Sometimes an autograft is done to remove
the tissue and then treat it or the person before returning it (examples include stem cell
autograft and storing blood in advance of surgery). In a rotationplasty, a distal joint is used to
replace a more proximal one; typically a foot or ankle joint is used to replace a knee joint. The
person's foot is severed and reversed, the knee removed, and the tibia joined with the femur.

Allograft
An allograft is a transplant of an organ or tissue between two genetically non-identical members
of the same species. Most human tissue and organ transplants are allografts. Due to the genetic
difference between the organ and the recipient, the recipient's immune system will identify the
organ as foreign and attempt to destroy it, causing transplant rejection. The risk of transplant
rejection can be estimated by measuring the Panel reactive antibody level.

Isograft

A subset of allografts in which organs or tissues are transplanted from a donor to a genetically
identical recipient (such as an identical twin). Isografts are differentiated from other types of
transplants because while they are anatomically identical to allografts, they do not trigger
an immune response.

Xenograph

A transplant of organs or tissue from one species to another. An example is porcine heart valve
transplant, which is quite common and successful. Another example is attempted piscine-
primate (fish to non-human primate) transplant of islet (i.e. pancreatic or insular tissue) tissue.
The latter research study was intended to pave the way for potential human use if successful.
However, xenotransplantion is often an extremely dangerous type of transplant because of the
increased risk of non-compatibility, rejection, and disease carried in the tissue. In an opposite
twist, Ganogen Research Institute CEO Eugene Gu is studying how to
transplant human fetal hearts and kidneys into animals for future transplantation into human
patients to address the shortage of donor organs.

Types of donors

Living donor

In living donors, the donor remains alive and donates a renewable tissue, cell, or fluid (e.g.,
blood, skin), or donates an organ or part of an organ in which the remaining organ can regenerate
or take on the workload of the rest of the organ (primarily single kidney donation, partial
donation of liver, lung lobe, small bowel). Regenerative medicine may one day allow for
laboratory-grown organs, using person's own cells via stem cells, or healthy cells extracted from
the failing organs.

Deceased donor

Deceased donors (formerly cadaveric) are people who have been declared brain-dead and whose
organs are kept viable by ventilators or other mechanical mechanisms until they can be excised
for transplantation. Apart from brain-stem dead donors, who have formed the majority of
deceased donors for the last 20 years, there is increasing use of donation-after-circulatory-death-
donors (formerly non-heart-beating donors) to increase the potential pool of donors as demand
for transplants continues to grow. Prior to the recognition of brain death in the 1980s, all
deceased organ donors had died of circulatory death. These organs have inferior outcomes to
organs from a brain-dead donor. For instance, patients who underwent liver transplantation using
donation-after-circulatory- death (DCD) allografts have been shown to have significantly lower
graft survival than those from donation-after-brain-death (DBD) allografts due to biliary
complications and PNF. However, given the scarcity of suitable organs and the number of people
who die waiting, any potentially suitable organ must be considered.

BIOCHEMISTRY, BIOTECHNOLOGY AND SOCIETY

Socio-economic Aspects on the Use of Cloned Genes in Medicine, Agriculture,

Industry and Ecology

The possibilities and potential benefits of genetic engineering have the very real potential of
“destabilizing both the biological and the social foundations of the human world". In order to
comprehend the scope of the implications of, as well as the potential benefits of genetic
engineering, it is important to understand what genetic engineering is, what scientific evidence
supports and refutes its use and for what purposes, and how various stakeholders debate it as a
construct.

Ultimately, it will be proven that genetic engineering is not an area of scientific practice that
should be advanced without further study. Until approximately 30 years ago, scientists
specializing in genetics directed their attention towards cracking the code of human, animal, and
plant life by analyzing the components of DNA and identifying the characteristics that
correspond to each gene.

As this process developed, the focus of genetic engineering began to shift, transitioning from
“readingto writinggenetic code". Shand, Thomas, and Wetter contend that the values shift that
accompanied this practical shift in study reflected a “scorn [for] nature‟s designs in favor of
made-to-order life forms". One of the earliest examples of genetic manufacturing that was made
public occurred ten years ago, when Scottish scientists announced that they had cloned a sheep
successfully, thus sparking not only this debate, but questions about the ethics of stem cell
research in general.

It was at this point that the argument on the benefits of genetic engineering were contested with
those of a more ethereal ethical and even religious nature. According to the Canadian Medical
Association Journal (613), Dolly‟s “birth" was the shot heard round the world for genetic
science, and researchers began to imagine all of the applications that such new knowledge and
skill could portend, both for human and animal life alike. The majority of the general public
panicked about the possibility of cloning human life, and even the United Nations became
involved in an effort to spearhead a ban on human cloning (Canadian Medical Association
Journal 613).Since Dolly‟s arrival in the world the field of genetic engineering has become
exponentially more complicated.

Genetic manufacturing and the drawbacks or benefits to genetic engineering is one of the most
publicly discussed areas of science and argument outside the field itself, inspiring intense interest
and equally passionate debate. One of the areas in which genetic engineering has been applied
enthusiastically by scientists is in agricultural production and the manufacture of comestibles. At
present, the U.S. Food and Drug Administration does not monitor or regulate genetically
modified foods, although intense debate about the relative merits and dangers of this practice has
compelled the FDA to examine the issue more closely. The agency, which is charged with
protecting the safety of Americans with respect to the food they eat and the medicines they take,
just closed a public debate period in which it reviewed comments of the public on the subject of
genetically engineered and modified food supplies.

Human genetic engineering relies heavily on science and technology. It was developed to help
end the spread of diseases. With the advent of genetic engineering, scientists can now change the
way genomes are constructed to terminate certain diseases that occur as a result of genetic
mutation. Today genetic engineering is used in fighting problems such as cystic fibrosis,
diabetes, and several other diseases. Another deadly disease now being treated with genetic
engineering is the "bubble boy" disease (Severe Combined Immunodeficiency). This is a clear
indication that genetic engineering has the potential to improve the quality of life and allow for
longer life span.
Clearly, one of the greatest benefits of this field is the prospect of helping cure illness and
diseases in unborn children. Having a genetic screening with a fetus can allow for treatment of
the unborn. Overtime this can impact the growing spread of diseases in future generations.
However, these benefits are not without peril. Human genetic engineering is a development that
people are either very passionate about or opposed to completely. This article gives a brief
account on the effect of this principle on the biosphere together with several controversial issues
that accompany the acceptance of this technology. The manuscript has been prepared by using
information from peer reviewed journals indexed in pubmed in the period of 2000 to 2015.

Effects on the Environment

Although the positive impacts of this field could be enormous, there are many questions raised
that needs to be answered. New organisms created by genetic engineering could present an
ecological problem. One cannot predict the changes that a genetically engineered species would
make on the environment. The release of a new genetically engineered species would also have
the possibility of causing an imbalance in the ecology of a region just exotic species would do.
An accident or an unknown result could cause several problems. An accident in engineering the
genetics of a virus or bacteria for example could result in a stronger type, which could cause a
serious epidemic when released. This could be fatal in human genetic engineering creating
problems ranging from minor medical problems, to death.
Effects on Human
Looking at the fact that genetic engineering employs viral vector that carries functional gene
inside the human body; the repercussion are still unknown. There are no clues as to where
functional genes are being placed. They may even replace the important genes, instead of
mutated genes. Thus, this may lead to another health condition or disease to human. Also, as
defective genes are replaced with functional gene, then it is expected that there will be a
reduction in genetic diversity and if human beings will have identical genomes, the population as
a whole will be susceptible to virus or any form of diseases.
Genetic engineering could also create unknown side effects or outcomes. Certain changes in a
plant or animal could cause unpredicted allergic reactions in some people which, in its original
form, did not occur. Other changes could result into the toxicity of an organism to humans or
other organisms.
Antibiotic Resistance
Genetic engineering often uses genes for antibiotic resistance as "selectable markers." Early in
the engineering process, these markers help identify cells that have taken up foreign genes.
Although they have no further use, the genes continue to be expressed in plant tissues. Most
genetically engineered plant foods carry fully functioning antibiotic-resistance genes.
The presence of antibiotic-resistance genes in foods could have lethal effects. Therefore, eating
these foods could reduce the effectiveness of antibiotics to fight disease when these antibiotics
are taken with meals. More so, the resistance genes could be transferred to human or animal
pathogens, making them impervious to antibiotics. If transfer were to occur, it could aggravate
the already serious health problem of antibioticresistant disease organisms.
Ethical and Social Issues
"Playing God" has become a strong argument against genetic engineering. Several issues have
also been raised as regards the acceptance of this technology. These concerns range from ethical
issues to lack of knowledge on the effects genetic engineering may have. One major concern is
that once an altered gene is placed in an organism, the process cannot be reversed. Public
reaction to the use of rDNA in genetic engineering has been mixed. The production of medicines
through the use of genetically altered organisms has generally been welcomed. However, critics
of rDNA fear that disease-producing, organisms used in some rDNA experiments might develop
extremely infectious forms that could cause worldwide epidemics.
As more human genes are being used in non-human organisms to create new forms of life that
are genetically partly human, new ethical questions arise. For instance, what percentage of
human genes does an organism have to contain before it is considered human and how many
human genes would a green pepper for example have to contain before it can be eaten without
qualms. Human genes are now being inserted into tomatoes and peppers to make them grow
faster. This suggests that one can now be a vegetarian and a cannibal at the same time. For
meateaters, the same question could be posed about eating pork with human genes. What about
the mice that have been genetically engineered to produce human sperm. The question is „what
psychological effect would it pose on the offspring?
Critics have questioned the safety of genetically engineered bovine somatotropin (BST) to
increase the milk yield of dairy cows (BST) for both the cows that are injected with it and the
humans who drink the resulting milk; owing to the fact that it increases a cow‟s likelihood of
developing mastitis, or infection of the udder, and it also makes cows more susceptible to
infertility and lameness.
Transgenic plants also present controversial issues. Allergens can be transferred from one food
crop to another through genetic engineering. Another concern is that pregnant women eating
genetically modified products may endanger their offspring by harming normal fetal
development and altering gene expression.
In 2002 the National Academy of Sciences released a report calling for a legal ban on human
cloning. The report concluded that the high rate of health problems in cloned animals suggests
that such an effort in humans would be highly dangerous for the mother and developing embryo
and is likely to fail. Beyond safety, the possibility of cloning humans also raises a variety of
social issues like the psychological issues that would result for a cloned child who is the identical
twin of his or her parent.
Another frightening scenario is the destructive use of genetic engineering. Terrorist groups or
armies could develop more powerful biological weaponry. These weapons could be resistant to
medicines, or even targeted at people who carry certain genes. Genetically engineered organisms
used for biological weapons might also reproduce faster, which would create larger quantities in
shorter periods of time, increasing the level of devastation.

In Medicine

Recombinant DNA technology, apart from being an important tool of scientific research, has
also played a vital role in the diagnosis and treatment of various diseases, especially those
belonging to genetic disorders.

Some of the recent advances made possible by recombinant DNA technology are:

1. Isolating proteins in large quantities: many recombinant products are now available, including
follicle stimulating hormone (FSH), Follistim AQ vial, growth hormone, insulin and some other
proteins.

2. Making possible mutation identification: due to this technology, people can be easily tested for
mutated protein presence that can lead to breast cancer, neurofibromatosis, and retinoblastoma.

3. Hereditary diseases carrier diagnosis: tests now available to determine if a person is carrying
the gene for cystic fibrosis, the Tay-Sachs diseases, Huntington‟s disease or Duchenne muscular
dystrophy.

4. Gene transfer from one organism to other: the advanced gene therapy can benefit people with
cystic fibrosis, vascular disease, rheumatoid arthritis and specific types of cancers.

Mammalian Gene Expression in Bacteria

Bacterial genetics can be manipulated to allow for mammalian gene expression systems
established in bacteria. Gene expression is the process by which information from a gene is used
in the synthesis of a functional gene product. These products are often proteins and are produced
after the process of translation. An expression system that is categorized as a genetic engineering
product is a system specifically designed for the production of a gene product of choice. This is
normally a protein, although may also be RNA, such as tRNA or a ribozyme.

The genetically engineered expression system contains the appropriate DNA sequence for the
gene of choice which is engineered into a plasmid that is introduced into a bacteria host. The
molecular machinery that is required to transcribe the DNA is derived from the innate and
naturally occurring machinery in the host. The DNA is then transcribed into mRNA and then
translated into protein products.

In a genetically engineered system, this entire process of gene expression may be induced
depending on the plasmid used. In the broadest sense, mammalian gene expression includes
every living cell but the term is more normally used to refer to expression as a laboratory tool.
An expression system is therefore often artificial in some manner. Viruses and bacteria are an
excellent example of expression systems.

The oldest and most widely used expression systems are cell-based. Expression is often done to a
very high level and therefore referred to as overexpression. There are many ways to introduce
foreign DNA to a cell for expression, and there are many different host cells which may be used
for expression. Each expression system also has distinct advantages and liabilities.

Expression systems are normally referred to by the host and the DNA source or the delivery
mechanism for the genetic material. For example, common bacterial hosts are E.coli and B.
subtilis. With E. coli, DNA is normally introduced in a plasmid expression vector. The
techniques for overexpression in E. coli work by increasing the number of copies of the gene or
increasing the binding strength of the promoter region so as to assist transcription.

Mammalian Proteins and Products

Genetic engineering enables scientists to create plants, animals, and microorganisms by

manipulating genes.

The first successful products of genetic engineering were protein drugs like insulin, which is
used to treat diabetes, and growth hormone somatotropin. These proteins are made in large
quantities by genetically engineered bacteria or yeast in large “bioreactors.” Some drugs are also
made in transgenic plants, such as tobacco.

Other human proteins that are used as drugs require biological modifications that only the cells
of mammals, such as cows, goats, and sheep, can provide. For these drugs, production in
transgenic animals is a good option. Using farm animals for drug production has many
advantages because they are reproducible, have flexible production, are easily maintained, and
have a great delivery method (e.g. milk).
Recombinant DNA technology not only allows therapeutic proteins to be produced on a large
scale but using the same methodology protein molecules may be purposefully engineered.
Genetic modifications introduced to a protein have many advantages over chemical
modifications. Genetically engineered entities are biocompatible and biodegradable. The changes
are introduced in 100% of the molecules with the exclusion of rare errors in gene transcription or
translation. The preparations do not contain residual amounts of harsh chemicals used in the
conjugation process. Bacterial expression systems, due to their simplicity, are often not able to
produce a recombinant human protein identical to the naturally occurring wild type. Bacteria did
not develop sophisticated mechanisms for performing post-translational modifications that are
present in higher organisms. As a consequence, an increasing number of protein therapeutics is
expressed in mammalian cells. However the low cost and simplicity of cultivating bacteria is an
unbeatable advantage over any other expression system and therefore E. coli is always a
preferable choice both on a lab scale and in industry.

Many mammalian proteins are produced by genetic engineering. These include, in particular, an
assortment of hormones and proteins for blood clotting and other blood processes. For example,
tissue plasminogen activator (TPA) is a blood protein that scavenges and dissolves blood clots
that may form in the final stages of the healing process. TPA is primarily used in heart patients or
others suffering from poor circulation to prevent the development of clots that can be life-
threatening. Heart disease is a leading cause of death in many developed countries, especially in
the United States, so microbially produced TPA is in high demand.

In contrast to TPA, the blood clotting factors VII, VIII, and IX are critically important for the
formation of blood clots. Hemophiliacs suffer from a deficiency of one or more clotting factors
and can therefore be treated with microbially produced clotting factors. In the past hemophiliacs
have been treated with clotting factor extracts from pooled human blood, some of which was
contaminated with viruses such as HIV and hepatitis C, putting hemophiliacs at high risk for
contracting these diseases. Recombinant clotting factors have eliminated this problem.

In Agriculture

Seed Culture

Seed culture is the type of tissue culture that is primarily used for plants such as orchids. For this
method, explants (tissue from the plant) are obtained from an in-vitro derived plant and
introduced in to an artificial environment, where they get to proliferate. In the event that a plant
material is used directly for this process, then it has to be sterilized to prevent tissue damage and
ensure optimum regeneration.

Embryo Culture

Embryo culture is the type of tissue culture that involves the isolation of an embryo from a given
organism for in vitro growth.
*Note, the term embryo culture is used to refer to sexually produced zygotic embryo culture.

Embryo culture may involve the use of a mature of immature embryo. Whereas mature embryos
for culture are essentially obtained from ripe seeds, immature embryo (embryo rescue) involves
the use of immature embryos from unripe/hybrid seeds that failed to germinate. In doing so, the
embryo is ultimately able to produce a viable plant.

For embryo culture, the ovule, seed or fruit from which the embryo is to be obtained is sterilized,
and therefore the embryo does not have to be sterilized again. Salt sucrose may be used to
provide the embryo with nutrients. The culture is enriched with organic or inorganic compounds,
inorganic salts as well as growth regulators.

Callus Culture

Callus - This is the term used to refer to unspecialized, unorganized and a dividing mass of cells.
A callus is produced when explants (cells) are cultured in an appropriate medium - A good
example of this is the tumor tissue that grows out of the wounds of differentiated tissues/organs.

In practice, callus culture involves the growth of a callus (composed of differentiated and non-
differentiated cells), which is the followed by a procedure that induces organ differentiation.

For this type of tissue culture, the culture is often sustained on a gel medium, which is composed
of agar and a mixture of given macro and micronutrients depending on the type of cells.
Different types of basal salt mixtures such as murashige and skoog medium are also used in
addition to vitamins to enhance growth.

Organ Culture

Organ culture is a type of tissue culture that involves isolating an organ for in vitro growth. Here,
any organ plant can be used as an explant for the culture process (Shoot, root, leaf, and flower).

With organ culture, or as is with their various tissue components, the method is used for preserve
their structure or functions, which allows the organ to still resemble and retain the characteristics
they would have in vivo. Here, new growth (differentiated structures) continues given that the
organ retains its physiological features. As such, an organ helps provide information on patterns
of growth, differentiation as well as development.

There are number of methods that can be used for organ culture. These include;

 Plasma clot method - Here, the method involves the use of a clot that is composed of
plasma and chick embryo extract (or any other extract) in a watch glass. This method is
particularly used for the purposes of studying morphogenesis in embryonic organ
 rudiments and more recently for studying the actions of various hormones, vitamins and
carcinogens of adult mammalian tissues.

 Raft method - For this method, the explant is placed on a raft of lens paper/rayon acetate and
floated on a serum in a watch glass.
 Agar gel method - The medium used for this method is composed of a salt solution, serum as
well as the embryo extract or a mixture of various amino acids and vitamin with 1 percent
agar. The explant has to be subcultured every 5 to 7 days. The method is largely used for the
study of developmental aspects of normal organs and tumors.
 Grid method - Grid method, as the name suggests involves the use of perforated stainless steel
sheet, on which the tissue of interest is placed before being placed in a culture chamber
containing fluid medium.

Protoplast Culture

*Protoplast -cells without cell walls. A protoplast is the term used to refer to cell (fungi, bacteria,
plant cells etc) in which the cell wall has been removed, which is why they are also referred to as
naked cells.Protoplasts may be cultured in the following ways;

 Hanging-drop cultures
 Micro culture chambers
 Soft agars matrix

Once a protoplast has regenerated a cell wall, then it goes through the process of cell division to
form a callus, which may then be subcultured for continued growth.

Protoplast culture is an important method that provides numerous cells (single cells) that can be
used for various studies. These include;

 Protoplast culture regenerated into a whole plant

 Development of hybrids
 Cell cloning
 Genetic transformations
 Membrane studies

In protoplast culture, a number of phases can be observed. These include;

 Development of a cell wall

 Cell division
 Continuous growth or regeneration to a whole plant
 For plants, some of the special requirements include;

 Less amounts of iron and zinc and no ammonium

 Higher concentration of calcium
 High auxin/kinetic ratio for cell division and high kinetin/auxin ration for regeneration
 Glucose and vitamins

Some of the other types of tissue culture include;

 Single cell culture

 Suspension culture
 Anther culture
 Pollen culture
 Somatic Embryogenesis

Applications

1. Commercial production of plants used as landscape, potting and florist subject which uses
meristem and shoot culture.

2. To conserve endangered plant species to avoid extinction.

3. To screen cells rather than plants for specific characters such as herbicide
resistance/tolerance.

4. Use of meristem tip cultures to produce clean plant material from virus stock such as
potatoes.

5. To produce disease free plants due to its production in sterile environment

6. For chromosome doubling and induction of polyploidy for example doubled haploid,
tetraploids, and other forms of polyploids. This is usually achieved by application of antimitotic
agents such as Colchicine or Oryzalin.

In Industry

Biodegradable Plastic Industry:

Biodegradable plastics like polyhydroxylbutyrate (PHB) can be obtained commercially by
fermentation with the bacterium Alcaligenes eutrophus. But its production cost is very high.
Recently three genes, viz., phbA, phbB and phbC encoding the enzymes 3- ketothiolase,
acetoacetyl-CoA reductase and PHB synthase have been isolated from A. eutrophus.

These genes were introduced into a model plant Arabidopsis which produced PHB globules
exclusively in their chloroplasts without affecting plant‟s growth and development. The amount
of PHB in the leaves of this transgenic plant was up to 10 mg/g fresh weight. Industry has
already started to explore the production of biodegradable plastics from other transgenic tree
plants like poplar.

Application in Oil Industry:

Plants store oil in their seeds (e.g., ground nut, mustard, rapeseed, sunflower, sesamum, soya
bean etc.) or in fruits (e.g., olive, avocado, oil palm etc.). Such vegetable oils are used either as
food or in industrial purposes. According to various requirements the fatty acid quality and yield
can be improved by using genetic engineering technology.

Fuel Industry:
In recent years, ethanol has found its use as an important chemical feedstock and as a fuel
supplement. Ethanol is generally produced by fermentation of some sugar (starch, cellulose) rich
products with the help of yeast, Saccharomyces cerevisiae or sometimes with Kluyveromyces
fragilis.

At present E. coli and Klebsiella planticola carrying genes from Z. mobilis have been developed
which could utilize glucose and xylose as the substrate to give maximum yield of ethanol.

In Ecology

Bio-Hydrometallurgy:
Bio-hydrometallurgy is a new branch of metallurgy that amalgamate metallurgy and biotechno-
logy together. Extraction of metals from ores through conventional metallurgy involves smelting
ores at high temperature, a process which is very much polluting and energy intensive approach.

However, bio-metallurgy is an approach which is environment friendly and can be used to

extract metals from low grade ores in contrast to high grade ores used in conventional metallurgy
which are gradually becoming exhausted.

In bio-metallurgy, different bacterial species are being used for the extraction of metals from
ores. For example, Thiobacillus ferrooxidans and related thiobacilli are used for copper
extraction. Recently, genetically engineered strains of thiobacilli have been developed to
increase the speed of extraction.

Such bacterial strains have increased resistance to arsenite and arsenate (which do not allow
bacterial growth) and increased recovery of gold from arsenopyrite-pyrite ores.

Bio-Mineralisation:
It has been shown recently that some bacteria which are normal constituents of soil can deposit
valuable metals like gold, silver etc. in soil. It is suggested that negatively charged polymer on
the outside of the bacteria, attracts the positively charged gold particles in the soil. Thus, the
metal particles clump together as grains that eventually form gold nuggets.

The process of gold deposition on the negatively charged polymers can continue even after the
death of the bacteria. Naturally occurring bacteria involved in such biomineralisation include
Bacillus cereus, podomicrobium-like budding bacteria and Chlorella vulgaris.

It is suggested that genes responsible for mineralization in a variety of microbes can be isolated,
cloned and transferred into E. coli, which may then help in metal deposition more efficiently.

What is a Patent?

A patent is a type of intellectual property. All properties can be understood as collections rights
to control a particular thing. Tangible properties give the property holder rights to control
tangible things, such as cars or land. Intellectual properties, on the other hand, give the property
holder rights to control intangible things, such as inventions, poems, or computer programs.
Tangible things have a particular location in space and time; intangible things do not. The main
types of intellectual property are patents, copyrights, trademarks, and trade secrets.

A patent is a private right granted by the government to someone who creates an invention. The
patent gives the inventor the right to exclude others from making, using, or commercializing the
invention. A patent may be awarded to more than one person. Today, most patents are awarded
to groups of researchers, who are listed as coinventors. Once a patent is granted, the rights may
be transferred, licensed, or assigned to other parties. Academic and industrial researchers usually
assign their patent rights to their employer and receive a share of royalties. The employer then
becomes the patent holder. Patent holders may also grant licenses to other parties in exchange for
royalties or a fee. For example, a biotechnology company with a patent on a gene therapy
technique could grant individuals or companies licenses to use the technique.

In the United States, a patent holder has the right to refrain from making,using, or licensing
his/her invention. There, a patent confers rights to make, use, or commercialize a thing, but
implies no corresponding obligations. As a result, some companies in the United States use
patents to block technological development and gain an advantage over competitors. Some
European countries, however, have compulsory licensing, which requires the patent holder to
make, use, or commercialize his/her invention or license others to do so.

The term of patent in the United States and countries that belong to the European Union lasts 20
years from the time the inventor submits his application. A patent is not renewable. Once the
patent expires, the invention becomes part of the public domain and anyone can make, use, or
commercialize the invention without permission from the inventor.

In the pharmaceutical industry the average interval between discovery of a new drug and its final
approval by the relevant regulatory agency is 10 years, which includes the time required to
conduct clinical research, product development, as well as regulatory review. Thus, most
pharmaceutical companies can expect that they will have about 10 years to recoup the money
they have invested in a new drug before the patent expires. Once the patent expires, the name of
the drug may still have trademark protection, but other companies can manufacture and market a
generic version of the drug without obtaining permission from the company.

The main policy rationale for patent laws is that they promote the progress of science,
technology, and industry by providing financial incentives for inventors, entrepreneurs, and
investors. By granting property rights over inventions, the patent system gives inventors and
research sponsors the opportunity to profit from their investments of time and money in research
and development. Additionally, society benefits because the patent application becomes part of
the public domain once the patent is granted, which gives other researchers the opportunity to
learn from the invention and use the knowledge contained in the application. The agreement to
grant patents rights in exchange for public disclosure is known as the patent bargain. The public
benefits from this bargain because it encourages inventors share information instead of
attempting to protect it through trade secrecy.

How Does One Obtain a Patent?

To obtain a patent, one must submit a patent application to the patent office. In the United States,
the Patent and Trademark Office (PTO) examines patent applications. The application must
provide a description of the invention that would allow someone trained in the relevant practical
art to make and use the invention. One or more individuals may be listed as inventors on the
patent application. The application need not include a sample or model of the invention; a written
description will suffice. The application will contain information about the invention,
background references, data, as well as one or more claims pertaining to the invention. The
claims stated on the patent application will determine the scope of the patent rights.

If the PTO rejects a patent application, the inventor may submit a revised application. The
process of submission/revision/resubmission, otherwise known as prosecuting a patent, may
continue for months or even years. If the PTO rejects the patent, the applicant may appeal the
decision to a federal court. If the PTO accepts the patent, a competitor may still file a lawsuit
challenging the PTO‟s decision. The PTO will award a patent to an inventor only if he/she
provides evidence that his/her invention satisfies all of the following conditions (European Union
countries have similar requirements):
 1. Originality. The invention is new and original – it has not been previously
disclosed in the prior art. The rationale for this condition is that the public does
not benefit when the patent office grants a patent on something that has already
been invented. Thus, if someone else has already patented the same invention, this
would qualify as a prior disclosure. Also, disclosure could occur if a significant
part of the invention has been published or used in public.
2. Nonobviousness. The invention is not obvious to someone who is trained in the
relevant practical art. Prior disclosure of parts of the invention or similar
inventions in the literature can undermine nonobviousness claims. The
justification for this requirement is that the public does not benefit from granting
obvious inventions.
3. Usefulness. The invention has some definite, practical utility. The utility of the
invention should not be merely hypothetical, abstract, or contrived. A patent is not
a fishing license. The rationale for this condition is self-explanatory: the public
does not benefit from useless patents. In the late 1990s, the PTO raised the bar for
proving the utility of patents on DNA in response to concerns that it was granting
patents on DNA sequences when the inventors did not even know the biological
functions of those sequences.

Human Genome Project

The Human Genome Project was a 13-year-long, publicly funded project initiated in 1990 with
the objective of determining the DNA sequence of the entire euchromatic human genome within
15 years. In its early days, the Human Genome Project was met with skepticism by many people,
including scientists and nonscientists alike. One prominent question was whether the huge cost
of the project would outweigh the potential benefits.

Today, however, the overwhelming success of the Human Genome Project is readily apparent.
Not only did the completion of this project usher in a new era in medicine, but it also led to
significant advances in the types of technology used to sequence DNA.

Phases of the Human Genome Project

Based on the insights gained from the yeast and worm studies, the Human Genome Project
employed a two-phase approach to tackle the human genome sequence (IHGSC, 2001). The first
phase, called the shotgun phase, divided human chromosomes into DNA segments of an
appropriate size, which were then further subdivided into smaller, overlapping DNA fragments
that were sequenced. The Human Genome Project relied upon the physical map of the human
genome established earlier, which served as a platform for generating and analyzing the massive
amounts of DNA sequence data that emerged from the shotgun phase. Next, the second phase of
the project, called the finishing phase, involved filling in gaps and resolving DNA sequences in
ambiguous areas not obtained during the shotgun phase. Figure 1 shows the exponential increase
in DNA sequence information deposited in the High-Throughput Genomic Sequences (HTGS)
division of GenBank by the end of the shotgun phase. Indeed, the shotgun phase yielded 90% of
the human genome sequence in draft form.
The shotgun phase of the Human Genome Project itself consisted of three steps:

1. Obtaining a DNA clone to sequence

2. Sequencing the DNA clone
3. Assembling sequence data from multiple clones to determine overlap and establish a
contiguous sequence

When possible, the DNA fragments within the library vectors were mapped to chromosomal
regions by screening for sequence-tagged sites (STSs), which are DNA fragments, usually less
than 500 base pairs in length, of known sequence and chromosomal location that can
be amplified using polymerase chain reaction (PCR). Library clones were also digested with
the restriction enzyme HindIII, and the sizes of the resulting DNA fragments were determined
using agarose gel electrophoresis. Each library clone exhibited a DNA fragment "fingerprint,"
which could be compared to that of all other library clones in order to identify overlapping
clones. Fluorescence in situ hybridization (FISH) was also used to map library clones to
specific chromosomal regions. Collectively, the STS, DNA fingerprint, and FISH data allowed
the IHGSC to generate contigs, which consisted of multiple overlapping bacterial artificial
chromosome (BAC) library clones spanning each of the 24 different human chromosomes (i.e.,
22 autosomes and the X and Y chromosomes).

Next, individual BAC clones selected for DNA sequence analysis were further fragmented, and
the smaller genomic DNA fragments were subcloned into vectors to generate a BAC-derived
shotgun library. The inserts were sequenced using primers matching the vector sequence
flanking the genomic DNA insert, and overlapping shotgun clones were used to generate a DNA
sequence spanning the entire BAC clone. A summary of this step is shown in Figure 3. The
members of the IHGSC agreed that each center would obtain an average of fourfold sequence
coverage, with no clone having less than threefold coverage. The term "shotgun" comes from the
fact that the original BAC clone was randomly fragmented and sequenced, and the raw DNA
sequence data was then subjected to computational analyses to generate an ordered set of DNA

sequences that spanned the BAC clone.

Celera: Shooting at Random and Organizing Later

Before the IHGSC had completed the first phase of the Human Genome Project, a
private biotechnology company called Celera Genomics also entered the race to sequence the
human genome. Led by Dr. Craig Venter, Celera proclaimed that it would sequence the entire
human genome within three years. As outlined in Figure 4, Celera used two independent data
sets together with two distinct computational approaches to determine the sequence of the human
genome (Venter et al., 2001). The first data set was generated by Celera and consisted of 27.27
million DNA sequence reads, each with an average length of 543 base pairs, derived from five
different individuals. The second data set was obtained from the publicly funded Human
Genome Project and was derived from the BAC contigs (called bactigs); here, Celera "shredded"
the Human Genome Project DNA sequence into 550-base-pair sequence reads representing a
total of 16.05 million sequence reads. The company then used a whole-genome assembly method

and a regional chromosome assembly method to sequence the human genome.

In contrast, with the regional chromosome assembly approach (also called the
compartmentalized shotgun assembly method), Celera organized its own data and the Human
Genome Project sequence data into the largest possible chromosomal segments, followed by
shotgun assembly of the sequence data within each segment (Venter et al., 2001); this approach
was similar to the hierarchical shotgun approach used by the IHGSC. The first step of the
regional assembly approach involved separating Celera reads that matched Human Genome
Project reads from those that were distinct from the public sequence data. Of the 27.27 million
Celera reads, 21.38 million matched a Human Genome Project bactig, and 5.89 million did not
match the public sequence data. These reads were assembled into Celera-specific or Human
Genome Project-specific scaffolds, which were then combined and analyzed using whole-gene
assembly algorithms. The resulting bactig data were again "shredded" to permit unbiased
assembly of the combined sequence data.

Celera's whole-genome and regional chromosome assembly methods were independent of each
other, permitting direct comparison of the data. Celera found that the regional chromosome
assembly method was slightly more consistent than the whole-genome assembly method. Using
these complementary approaches, Celera generated data that was in strong agreement with that
of the IHGSC.

In February 2001, drafts of the human genome sequence were published simultaneously by both
groups in two separate articles (IHGSC, 2001; Venter et al., 2001). Due to technical advances
in DNA sequencing methods and a productive level of synergy between the two groups, they tied
at the finish line, and both projects were completed ahead of schedule.

A Quick Lesson in DNA Sequencing

As previously mentioned, the IHGSC and Celera used different approaches to determine the
sequence of the human genome. However, they used the same general method for the DNA
sequencing step (Hood & Galas, 2003). This method uses DNA polymerase, the same enzyme
used in DNA replication, to produce DNA sequence information. As shown in Figure 6a, DNA
polymerase binds to a single-stranded DNA template and adds DNA bases to the 3′ end of the
complementary DNA strand it synthesizes. DNA polymerase requires an existing primer with a
free 3′ end to which it adds new DNA bases in a 5′ to 3′ manner, and it moves along the template
strand in a 3′ to 5′ direction.

Researchers from both the IHGSC and Celera combined the DNA template they were interested
in sequencing with DNA polymerase, a single-stranded DNA primer, free deoxynucleotide bases
(dATP, dCTP, dGTP, and dTTP), and a sparse mixture of fluorescently labeled
dideoxynucleotide bases (ddATP, ddCTP, ddGTP, and ddTTP) that were each labeled with a
different color and would terminate new DNA strand synthesis once incorporated into the end of
a growing DNA strand. The mixture was first heated to denature the template DNA strand; this
was followed by a cooling step to allow the DNA primer to anneal. Following primer annealing,
the polymerase synthesized a complementary DNA strand. The template would grow in length
until a dideoxynucleotide base (ddNTP) was incorporated; the conditions were such that this
occurred at random along the length of the newly synthesized DNA strands. In the end, the
researchers were left with a mixture of newly synthesized DNA strands that differed in length by
a single nucleotide, and that were labeled at their 3′ end with the color of the ddNTP-associated
dye molecule.

In order to determine the sequence of the newly synthesized, color-coded DNA strands,
researchers needed a way to separate them based on their size, which differed by only one DNA
nucleotide. To accomplish this, they electrophoresed the DNA through a gel matrix that
permitted single-base differences in size to be easily distinguished. Small fragments run more
quickly through the gel, and larger fragments run more slowly (Figure 6c). By putting the entire
mixture into a single well of the gel, a laser can be used to scan the DNA bands as they move
through the gel and determine their color; this data can be used to generate a sequence trace (also
called an electropherogram), showing the color and signal intensity of each DNA band that
passes through the gel (Figure 6d). The color of each band represents the final 3′ base
incorporated at that position, and by reading from the bottom to the top of the gel, one can
determine the sequence of the newly synthesized DNA strand from the 5′ to the 3′ end.

From Rough Draft to Final Form

As stated earlier, after the completion of the draft phase of the Human Genome Project, the
IHGSC pursued the second phase of the project: the finishing phase (IHGSC, 2004). During this
phase, the researchers filled in gaps and resolved DNA sequences in ambiguous areas that were
not solved during the shotgun phase. The finishing phase yielded 99% of the human genome in
final form. The final form of the human genome contained 2.85 billion nucleotides, with a
predicted error rate of 1 event per 100,000 bases sequenced. Furthermore, the IHGSC reduced
the number of gaps by 400-fold; only 341 gaps out of 147,821 gaps remained. The remaining
gaps were associated with technically challenging chromosomal regions. Although the earlier
draft publications had predicted as many as 40,000 protein-encoding genes, the finishing phase
reduced this estimate to between 20,000 and 25,000 protein-encoding genes. Future challenges
identified by the IHGSC during this phase included the identification of polymorphisms as a
platform for understanding genetic links to human disease, the identification of functional
elements within the genome (genes, proteins, elements involved in gene regulation, and
structural elements), and the identification of gene and protein "modules" that act in concert with
one another.

From Digital Information to Molecular Medicine

One particularly striking finding of the Human Genome Project research is that the human
nucleotide sequence is nearly identical (99.9%) between any two individuals. However, a single
nucleotide change in a single gene can be responsible for causing human disease. Because of
this, our knowledge of the human genome sequence has also contributed immensely to our
understanding of the molecular mechanisms underlying a multitude of human diseases.
Furthermore, a merging of cytogenetic approaches with the human genome sequence will
continue to propel our understanding of human disease to an entirely new level. Thus, although it
was met with skepticism at its inception, the Human Genome Project will certainly be heralded
as one of the most important scientific endeavors of our time.

Unfortunately, the initial hope of accelerating the discovery of new treatments for disease was
not necessarily accomplished by the Human Genome Project. With the sequence of the human
genome in hand, we have learned that it requires more than just knowledge of the order of the
base pairs in our genome to cure human disease. Current efforts are therefore focused on
understanding the protein products that are encoded by our genes. When a gene is mutated, the
corresponding protein is most often defective. The emerging field of proteomics aims to
understand how protein function and expression are altered in human disease states.
Furthermore, investigators are also turning their attention to the expansive regions of our genome
devoid of traditional protein-encoding genes. We have already started to reap the benefits of our
knowledge of the human genome, and future data-mining efforts will most certainly uncover
many more exciting and unexpected links to human disease.