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Recombinant DNA Technology

Plant tissue produces a complex mixture of chemicals that, if extracted, have many
valuable uses for humans. In the past, extraction and purification were expensive, timeconsuming, and low-yielding processes. But today, plants can be genetically engineered
to secrete proteins and other pharmaceutically desirable chemicals from their roots,
making other extraction methods obsolete.
The term that describes root cell secretion is rhizosecretion. Although secretion is a basic
function of all plant cells, some scientists
believe roots have perfected the process
because they are responsible for
interactions with soil organisms, nutrient
uptake, and allelopathic reactions.
Allelopathy is a form of rhizosecretion
in which roots secrete chemicals that
prevent other plants from growing in
close proximity. It is a plant-plant
interference mechanism believed to be a
response to resource competition.
Researchers at Rutgers University have
genetically engineered two types of
tobacco plants, Nicotiana tabaccum and
N. plumbagnifolia, to continuously
secrete recombinant proteins from their
roots. Recombination is a naturally
occurring process between homologous
Recombinant DNA technology.
DNA molecules of closely related plant
species. However, in genetic engineering, the process is manipulated in vitro between
completely different species. Thus, the desirable gene is selected from one plant and
inserted into a bacterial strand called a plasmid, which serves as a vector to transfer the
gene into another plant. The second plant is the genetically engineered, or
In one experiment, the genetically engineered tobacco plants were grown in a waterbased nutrient solution rather than in soil. This method, known as hydroponics, was
originally practiced in the mid-1800s and reintroduced in 1937. Researchers either
purchased transgenic tobacco plants or transformed the tobacco plants with DNA from
one of three different sources by using a standard Agrobacterium-mediated leaf-disc
transformation method (protocol by R.B. Horsch et. al., illustrated in a 1985 issue of
Science 227). Three proteins were introduced: (1) green fluorescent protein of jellyfish,
(2) human placental alkaline phosphatase, or (3) bacterial xylanase. (Remember that the
suffix -ase denotes an enzyme, all of which are proteins.)

Hydroponics. (Photo
1999 Papi.)

Hydroponic techniques used with tomato plants.

The resulting protein secretion was dependent on whether or not an endoplasmic


reticulum (ER) signal peptide was present. The ER target signals, which were fused to
the recombinant protein sequence, fooled the tobacco plants into shuttling the introduced
genetic information through the plant and out the roots as if it were the plant's own
genetic material. In addition, plants with the ER target signals showed a higher
concentration of protein in the hydroponic medium than in the root cells compared to
plants without the ER target signals. Conversely, plants without ER target signals
maintained higher concentration in the root cells and did not leach the chemicals into the
hydroponic medium.
Because plants secrete few proteins from their roots, isolation of the desirable proteins
was easier, faster, and less expensive from the hydroponic medium than traditional plant
extraction and isolation methods. When the secreted proteins are separated from the
hydroponic medium and analyzed, they are found to retain their own biological activity!
Also, rhizosecretion can run continuously without harming the plant, leading to a higher
yield of pharmaceutically favorable proteins in the transgenic plants.
Nicotiana is a genus in the Solanaceae family. Members of Solanaceae are dicots with
stamen adnate to the fused petals. They have a superior ovary that typically develops
into either a dry fruit, such as a capsule, or a fleshy fruit, such as a berry. The leaves
and/or stems of the plants are commonly covered with prickles. Economically, tobacco
plants have been grown as ornamentals and for the alkaloids they produce. Since 1664,
several types of alkaloids, including nicotine, have been used for their insecticidal
properties. In addition, different cultivars of N. tabaccum have been commonly used in
cigarettes and cigars, ground and flavored for snuff, or mixed with molasses for chewing
tobacco. Thus, through advances in genetic engineering, hydroponics, and rhizosecretion,
scientists may have found yet another, and more beneficial, use for tobacco plants--as
mini drug manufacturers.

Introduction to Cloning and Biotechnology


What is a clone? It's a group of genetically identical molecules, cells, or organisms
derived from a single ancestor.
A clone may be...
naturally produced from a single zygote (as in the case of human identical
(=monozygotic) twins
natural vegetative reproduction of plants ("pupping" - e.g., eye of a potato)
artificially produced in the laboratory by one of several methods

Plant Cloning
Plants can be cloned from single, meristematic cells.
This is commonly done in agriculture.

Animal Cloning
Animals can be cloned in several different ways...
embryo splitting
similar to producing monozygotic twins
a growing embryo is split into individual cells at an early stage, and each
blastomere (embryo cell) is allowed to grow.
nuclear transfer
nucleus of recipient cell (unfertilized ovum) is destroyed (ablated)
nucleus of a donor cell (from a desired adult organism) is implanted
new cell is stimulated to divide as if it were a newly fertilized ovum
this is the method (first successfully used in 1986) that produced Dolly the sheep
in 1997.
Dolly was the first cloned animal that overturned the previously held notion that
embryonic cells could not be stimulated to return to early zygote condition, once
the nucleus reaches a certain stage of specialization during development.

DNA Cloning (The Foundation of Recombinant DNA


Technology)
Clones need not be whole, living organisms. One can also clone DNA itself.
A large number of identical DNA molecules (or fragments of DNA molecules), each of
which has an identical sequence, can be produced by cloning DNA molecules from a
single, ancestral DNA molecule (or fragment).

In the early 1960's and 1970's, genetic and biochemical discoveries and techniques laid
the foundation for later cloning of DNA molecules.
Collectively, these techniques are known as Recombinant DNA Technology
Some of the Goals of the DNA Technologist...
1. Isolation of a particular gene, part of a gene or region of a genome
2. Production of large quantities of a gene product (protein or RNA) for easier study of
those molecules
3. Increased production efficiency for commercially made enzymes and drugs
4. Modification of existing organisms so that they express a particularly desirable trait not
previously encoded in the genome.
5. Correction of genetic defects in complex organisms, including humans.

MAIN CONCEPTS and DEFINITIONS in


RECOMBINANT DNA TECHNOLOGY:
Making and Replicating a desired piece of DNA:
One little piece of DNA by itself can't be studied. You can't determine its base
sequence or its products by looking at it under a microscope!
To effectively study DNA, one must manufacture a large quantity of a DNA segment
of interest, "magnifying" it for easier study with biochemical methods.
To do this, recombinant DNA is made by splicing a DNA fragment of interest into a
small DNA molecule (such as a bacterial plasmid) called a VECTOR.
Once this is done, one can make huge numbers of the desired DNA fragment by
inserting the vector into a very busy piece of DNA in another live cell (such as a
bacterium).
The bacterium "works" for you by allowing the vector to replicate. As the bacteria
multiply, so does your desired DNA!
This magnified sample (your DNA clone) can then be extracted for further study.
A Few Definitions:
The organism from which the DNA of interest is extracted is called the DONOR.
The DNA into which the DNA of interest is inserted (often a bacterial plasmid) is
called a VECTOR.
The organism (or DNA) into which the foreign DNA is inserted is called the
RECIPIENT.
An organism containing an artificially inserted, foreign piece of DNA is said to be
TRANSGENIC (i.e., the recipient becomes transgenic once the new DNA is inserted).

How is it Done?
To excise a piece of DNA from a donor organism, RESTRICTION ENZYMES are
used. These act somewhat like "enzymatic scissors," slicing through the DNA at specific,
recognized sequences.
Once the DNA is excised, DNA ligase is the "enzymatic glue" used to insert it into
replicating DNA of the host cell.
Note that DNA ligase isn't picky: it can't tell the difference between foreign and host
DNA (who'd figure it would ever have to?), and this enables the creation of hybrid
DNA--DNA from two separate sources (sometimes different species!).
A vector molecule with an insert of foreign DNA is a RECOMBINANT DNA
MOLECULE. DNA made from the combined DNA of two (or more) species is
sometimes called CHIMERIC DNA after the beast of Greek Mythology. (Now why, do
you suppose?)
Vectors are often mixed with bacterial strains which take them up and incorporate
them into their own genomes, a process known as TRANSFORMATION)
Vectors may also be replicated autonomously (without being inserted into the
bacterial DNA) as the bacterium goes about its daily business.
By growing the bacterial strain carrying the desired recombinant DNA vector, one can
grow a large number of the desired DNA fragment. This is the DNA CLONE.
Once a large DNA clone (remember: a clone is a group of things, not a single
individual!) has been grown, the researcher can
characterize the DNA (determine its base sequence)
make RNA from it
make protein from it (after you've made the RNA)
modify the DNA to see what happens when it mutates
reinsert it into a recipient organism for production of products or further study

Restriction Enzymes
First discovered in bacteria, RESTRICTION ENZYMES cut DNA at very specific
DNA base sequences (called RESTRICTION SEQUENCES).
These enzymes are believed to be a bacterial defense against viruses.
Each Restriction Enzyme recognizes and cleaves a very specific sequence of DNA,
like SO.
Restriction sequences are PALINDROMES: they read the same, forward and
backward on the opposite strands.
Cutting with restriction enzymes creates highly reactive "sticky ends" that act as
attachment points for other fragments of DNA with complementary restriction sequences.
By connecting pieces of DNA from two different species (that happen to have the
same restriction sites), we create CHIMERIC DNA.
Note that restriction sites are a "happy accident" of nature. They have nothing to do
with gene function in the organism in which they are found.
In fact, they are a defense mechanism, found primarily in bacteria, which function to

fragment and destroy the DNA of invading bacteriophages (i.e, "bacterium-eating"


viruses) before it can incorporate into the bacterial host's genome to do its dirty work.
Bacterial DNA is immune to the bacteria's own restriction enzymes: in its normal
state a bacterium's own restriction sites are highly methylated (i.e., the bases have many
methyl groups (-CH3 attached), protecting them from the activity of the restriction
enzymes.
Isn't evolution fantastic?
Restriction enzymes are named for the organism from which they were first isolated.
For example
EcoRI is isolated from E. coli strain RY13.
Eco refers to the genus and species (1st letter of genus; 1st two letters of specific
epithet)
R is the strain of E. coli
I (Roman numeral) indicates it was the first enzyme of that type isolated from E.
coli RY13.
BamHI is isolated from Bacillus amyloliquefaciens strain H
Sau3A is isolated from Staphylococcus aureas strain 3A.
And so on.
Each enzyme recognizes and cuts specific DNA sequences. For example, BamHI
recognizes the double stranded sequence:

5'--GGATCC--3'
3'--CCTAGG--5'
Here's how it works. Notice the "sticky" ends mentioned previously.
To summarize...

Most restriction enzymes cut only one specific restriction site


Restriction sites are recognized no matter what the DNA's species.
The number of cuts in an organism's DNA made by a particular restriction
enzyme depends on the number of restriction sites (specific to that restriction
enzyme) in that organism's DNA.
A fragment of DNA produced by a pair of adjacent cuts is called a
RESTRICTION FRAGMENT.
A particular restriction enzyme will typically cut an organism's DNA in to many
pieces, from several thousand to more than a million.
There is a great deal of variation in restriction sites, even within a species
(Everyone in this room has different numbers and locations of restriction sites.
Your restriction site numbers and locations are more similar to those of your close
family members than to unrelated humans.
Although these DNA variations are not phenotypically expressed, the variants can
be considered molecular "alleles," and they can be detected with sequencing
techniques.

This is yet another type of genetic variation of interest to the evolutionary


biologist.

Vectors
A VECTOR is a piece of DAN that carries a fragment of desired DNA into a living
cell for replication.
Vectors can be any type of DNA that has an affinity for living cells:
plasmids (self-replicating, circular bits of DNA found in bacteria) can carry
relatively small segments of desired DNA
Yeast Artificial Chromosome (YAC) - an artificially constructed "plasmid" that
can carry large segments of DNA. It contains
o telomeres
o centromeres
o lots of restriction sequences for easy splicing
YAC can be spliced with many different types of genes/DNA fragments, inserted
into live yeast, and then allowed to replicate as if it were a normal, natural yeast
chromosome.
Because yeast reproduce very quickly, you can get tens of thousands of copies of
your desired DNA fragments in relatively short time.
YAC has been used extensively in the Human Genome Project, to amplify
segments of the human genome for easier study.

Viruses of various types may also be useful as vectors, since they have very
specific affinities for specific tissues in the body.
o This means that specific viruses could be used to deliver genes for
implantation into the cell's genome in specific tissues where those genes
are needed, as in the case of gene therapy.
There are many types of viral vectors in use and under study for future use,
including...
o retroviruses
o adenoviruses
o adeno-associated viruses
o herpes simplex virus
o rhinoviruses
o Human Immunodeficiency Virus (HIV)

Each has its benefits and drawbacks. The search for the perfect vector continues-because the perfect vector probably does not exist. (There's probably no single vector that
will work for every purpose.

The overall object: get a vector that will allow you to clone large amounts of a desired
DNA fragment by inserting it into a rapidly dividing cell.

THE POLYMERASE CHAIN REACTION (PCR)

An alternative to cloning DNA fragments via insertion into vectors, and then
introduction into bacterial or yeast hosts is the POLYMERASE CHAIN
REACTION (PCR).
PCR allows rapid, efficient amplification of DNA sequences of interest.
Let us look at a nice little film...
PCR is probably the most widely used method for making large quantities of a
desired fragment of DNA.

Once you have large quantities of a DNA Clone, what do you do with it? One of the most
important aspects of DNA study is...

DNA SEQUENCING
If you have grown a DNA clone of interest, but do not know the order of its base
pairs, you certainly can't determine what proteins it might code, or what function
it might have.
Therefore, the first important step is to determine the base sequence of your
cloned fragment.
There are many different methods used for DNA sequencing, including

Dideoxy Method
Your assignment is to study the three diagrams in the link and understand the
general workings of this protocol. Don't try to memorize it, but understand the
basic ideas.

2. Biotechnology
2.1 The Science
Biotechnology may be defined as the use of living organisms or their sub-cellular
components to develop useful products, processes or services. Essentially, it is a group of
technologies which involve the application of many biological systems that have evolved
with the development of life itself. In essence, the definition illustrates the depth and
breadth of this science which encompasses a wide range of disciplines, including the life

sciences, agriculture, environmental science, medicine, chemistry, veterinary medicine,


engineering and computer science.
The discovery of antibiotics in the 1940s and the need to develop process fermentations
capable of large-scale antibiotic production during the World War II years led to striking
new developments in fermentation technology and to the coining of the term
biotechnology. In the 1970s, biotechnology went into a remarkable new phase of
expansion stimulated by discoveries in molecular genetics. These discoveries led to the
extraordinarily powerful technology of genetic engineering and drew renewed attention
to the whole field of biotechnology.
OECD Definition (1982)
Biotechnology means the application of scientific and engineering principles to the
processing of materials by biological agents to provide goods and services. These
principles cover a wide range of disciplines but rely heavily on microbiology,
biochemistry, genetics, biochemical and chemical engineering.
Although the term is relatively recent, man has used biotechnology for thousands of
years. For most of human history, plants and animals have been selectively bred to
improve particular traits, such as yield, disease resistance and hardiness. The making of
bread, wine and beer by microbial fermentation processes are age-old activities,
documented in our historical development even as far back as Egyptian times.
Archaeological evidence suggests that the early Romans recovered copper leached by
bacteria from natural copper sulphide deposits. The first recorded, large-scale bio-mining
operation was initiated in the early 1700s in Rio Tinto, Spain.4
Biological processes have been used for many years, long before the role of
microorganisms in these processes was understood. Appreciation of the enormous
diversity of microorganisms and simultaneous development of the science of genetics
greatly expanded the potential of traditional biotechnology and led to the development of
what has been termed modern or new biotechnology.
The discovery of the double helix structure of DNA (deoxyribonucleic acid) by Watson
and Crick in 1953 resulted in an understanding of gene function, expression and control.
Isolation of highly specific microbial enzymes, known as restriction endonucleases, by
Hamilton Smith in 1970 and of ligase or stitching enzymes opened up the possibility of
controlled gene manipulation and inter-species gene transfer. These discoveries
revolutionised the potential of biotechnology in the health-care, food and other sectors.
The umbrella of modern biotechnology encompasses a broad array of technologies, both
traditional and new. However, the term biotechnology has, to the general public,
become synonymous with genetic engineering. Genetic engineering, in turn, encompasses
recombinant DNA technology, genetic modification, gene technology and/or gene
manipulation. Genetic engineering makes it possible to cut DNA into its fundamental
functional units, the genes, and to splice (recombine) those genes into other DNA

molecules. Thus, it is now possible to enhance the ability of an organism to produce a


particular product, to prevent it producing a product, or to enable it to produce an entirely
new product. While maintaining all or most of its original properties, a genetically
engineered (GE) or genetically modified (GM) organism can do something it had not
done before or ceases something which it did before.
The microorganism which was commonly used as a host for genes from other
organisms at the onset of molecular genetics was the bacterium, Escherichia coli (E. coli).
There was no great deliberation, at the time, on the most suitable microbe of choice. E.
coli was a common research laboratory organism that grew easily, was able to incorporate
foreign DNA vehicles (in the form of phages and plasmids), and a considerable amount
of information was available on its genetic make-up.
The ability to manipulate living organisms at the genetic level is one of the principal tools
of modern biotechnology. Although the aim of traditional biotechnology, such as selective
breeding, was to develop new traits or enhance existing functions (or to add or enhance a
particular trait), new biotechnology (or genetic engineering) allows sophisticated
manipulation of the genes in plants and animals which encode for particular
characteristics in a more direct, precise manner. Genetic engineering is capable of
providing an organism with a specifically chosen, designed and desirable new ability or
property. In making such a specific manipulation, the outcome becomes much more
predictable, precise and controlled than was feasible with traditional biotechnology
techniques. This level of control is a tremendous asset in the application of modern
biotechnology to sustainable development and improvement of the quality of life.
2.1.1 The Stages in Recombinant DNA Technology
The essential stages of recombinant DNA technology are briefly outlined below and
diagrammatically represented for a microbial species in Figure 1. The stages involved
include:
1. identifying, in a donor species, the gene that directs the production of the desired
substance
2. isolating the gene from the donor using restriction enzymes
3. splicing of the gene into a circular piece of DNA, called a vector, which can
replicate in the host
4. transferring the recombined DNA into a bacterium or other suitable host.
The overall process is referred to as cloning since the genetically-engineered organism
becomes the parent of a population of identical organisms, which are literally clones of
each other in the sense that they are genetically identical. Plants, animals and microorganisms, which have been subjected to genetic manipulation artificially using these
techniques, are referred to as genetically modified organisms (GMOs). Species that have
artificially received a gene or genes from a different genus (plant, animal, microbial) are
described as transgenic.

Figure 1: An Example of DNA Cloning Processes

2.2 The Application


The potential of biotechnology, together with the new sciences required for its current
and future development and application, are illustrated in Figure 2. Chapter 4 provides
some examples of the current applications of traditional and modern biotechnology in a
number of industrial, agricultural, healthcare and environmental sectors.
Figure 2: Overview of Scientific Disciplines Involved in Recombinant DNA Technology

Genetic engineering using recombinant DNA techniques is currently widely used in the
pharmaceutical industry and in human medicine. Drugs produced by biotechnological
processes are used to treat invasive fungal infections, pulmonary embolisms, kidney
transplant rejection, infertility, growth hormone deficiency, diabetes, AIDS and other
serious disorders. In the future, drug therapy will become more personalised through the
use of genomics and pharmacogenomics. Many of the products we eat, wear and use are
made using the tools of modern biotechnology. Using genetic engineering, scientists are
able to enhance the nutritional content, texture, colour, flavour, growing season, yield,
disease resistance and other properties of production crops.
Transgenic techniques are being applied to farmed animals to produce pharmaceuticals
and nutraceuticals, and to improve the growth, fitness and other qualities of agriculturally
important mammals, poultry and fish.
The public are generally unaware of the extent of application of traditional
biotechnological processes in food production, industrial processes, waste and
wastewater treatment, bioremediation, renewable energy generation and biomining.
These applications will undoubtedly continue but are likely to be improved by using
recombinant DNA technology, for example, in the more informed, more specific and
more controlled use of microbes or microbial enzymes. Used efficiently, after appropriate
risk assessment and with effective and enforced regulation, biotechnology has enormous
potential to improve the quality of life and to enhance our capacity to conserve and
protect the environment.
2.3 The Economics
Biotechnology is of strategic importance to the Irish economy in order to sustain current
economic growth and to enhance the ability of Ireland to become a knowledge-based
economy.5
Table 1: Biotechnology in Perspective (millions) (1999)

The current market value of biotechnology companies world-wide is estimated to be in


the region of $500 billion. Ernst & Young estimate that in 1999 the European
entrepreneurial life sciences included over 1350 companies and employed nearly 54,000
people6. The table below shows the state of the market in 1999, as reported by Ernst &
Young.7

World-wide, the pharmaceuticals, chemicals, agri-food and medical device sectors, which
have contributed significantly to industrial development in Ireland in the past 20 years,
and account for a high proportion of Irish high technology jobs and exports are being
revolutionised by biotechnology. In Ireland, over 76,000 people are employed in
biotechnology-related sectors pharmaceuticals and chemicals (23,000) and food and
beverages (53,000)8. Turnover in 1997 in these sectors was 21 billion of which food
accounted for 15 billion. Exports were worth 16 billion with food accounting for 10
billions9.
Developments in biotechnology will have a profound effect on a number of sectors that
are vital to the future development of the Irish economy. While employment in the food
and beverages sector is unlikely to grow significantly, substantial growth is expected in
the pharmaceuticals and chemicals sector, with an approximate doubling of permanent
employment expected by 2010.10 A recent study by Forfs and Enterprise Ireland
concluded that there was potential for 6,200 jobs in Irish-owned biotechnology
companies with associated revenues of 490 million by 2010. From a wider perspective,
it is estimated that there will be up to 40,000 jobs in the rest of the economy that will
have a biotechnology component (in many cases this will be significant).
Irelands biotechnology capability is thus a major issue. If companies based in Ireland do
not capitalise on the potential and deal with the possible risks arising from biotechnology
developments, not only will the significant potential of high quality new jobs be at risk,
but there would also be a significant threat to a high proportion of the existing job base.
To enable Ireland to:

develop world class research capability in strategic technologies for the future
competitiveness of indigenous industry

facilitate the undertaking of R&D in this country by multinational companies


attract more high tech companies to Ireland in the future

it is necessary to have well focussed and significant investment in upgrading the


technological infrastructure of the economy. This includes significant investment in
biotechnology.
The Technology Foresight Ireland Reports, published in 1999 by ICSTI and Forfs,
recommended that the Government establish a major fund to develop Ireland as a centre
for world class research excellence in strategic niches of Biotechnology and ICT
(Information and Communication Technologies). As part of its response the Government
approved a Technology Foresight Fund of over 711 million for investment in research,
initially in the niche areas of ICT and Biotechnology in the years 2000-2006. Science
Foundation Ireland (SFI) is responsible for the management, allocation, disbursement and
evaluation of expenditure of the Technology Foresight Fund.

Recombinant DNA
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GloFish are a type of zebrafish with recombinant DNA. Genes for fluorescent proteins
have been inserted into their genome to produce their fluorescent colors.
Recombinant DNA is a form of artificial DNA that is engineered through the
combination or insertion of one or more DNA strands, thereby combining DNA
sequences that would not normally occur together.[1] In terms of genetic modification,
recombinant DNA is produced through the addition of relevant DNA into an existing
organismal genome, such as the plasmid of bacteria, to code for or alter different traits for
a specific purpose, such as immunity.[1] It differs from genetic recombination, in that it
does not occur through processes within the cell or ribosome, but is exclusively
engineered.[1]
The Recombinant DNA technique was engineered by Stanley Norman Cohen and Herbert
Boyer in 1973. They published their findings in a 1974 paper entitled "Construction of
Biologically Functional Bacterial Plasmids in vitro", which described a technique to
isolate and amplify genes or DNA segments and insert them into another cell with
precision, creating a transgenic bacterium. Recombinant DNA technology was made
possible by the discovery of restriction endonucleases by Werner Arber, Daniel Nathans,
and Hamilton Smith, for which they received the 1978 Nobel Prize in Medicine.

Introduction
Because of the importance of DNA in the replication of new structures and characteristics
of living organisms, it has widespread importance in recapitulating via viral or non-viral
vectors, both desirable and undesirable characteristics of a species to achieve
characteristic change or to counteract effects caused by genetic or imposed disorders that
have effects upon cellular or organismal processes.[2] Through the use of recombinant
DNA, genes that are identified as important can be amplified and isolated for use in other
species or applications, where there may be some form of genetic illness or discrepancy,
and provides a different approach to complex biological problem solving.[2]

Applications and methods


Cloning and relation to plasmids
Main article: Cloning

A simple example of how a desired gene is inserted into a plasmid. In this example, the
gene specified in the white color becomes useless as the new gene is added.
The use of cloning is interrelated with Recombinant DNA in classical biology, as the term
"clone" refers to a cell or organism derived from a parental organism,[1] with modern
biology referring to the term as a collection of cells derived from the same cell that
remain identical.[1] In the classical instance, the use of recombinant DNA provides the
initial cell from which the host organism is then expected to recapitulate when it
undergoes further cell division, with bacteria remaining a prime example due to the use of
viral vectors in medicine that contain recombinant DNA inserted into a structure known
as a plasmid.[1]
Plasmids are extrachromosomal self-replicating circular forms of DNA present in most
bacteria, such as Escherichia coli (E. Coli), containing genes related to catabolism and
metabolic activity,[1] and allowing the carrier bacterium to survive and reproduce in
conditions present within other species and environments. These genes represent
characteristics of resistance to bacteriophages and antibiotics[1] and some heavy metals,
but can also be fairly easily removed or separated from the plasmid by restriction
endonucleases,[1], which regularly produce "sticky ends" and allow the attachment of a
selected segment of DNA, which codes for more "reparative" substances, such as peptide
hormone medications including insulin, growth hormone, and oxytocin. In the
introduction of useful genes into the plasmid, the bacteria are then used as a viral vector,
which are encouraged to reproduce so as to recapitulate the altered DNA within other
cells it infects, and increase the amount of cells with the recombinant DNA present within
them.

The use of plasmids is also key within gene therapy, where their related viruses are used
as cloning vectors or carriers, which are means of transporting and passing on genes in
recombinant DNA through viral reproduction throughout an organism.[1] Plasmids contain
three common features -- a replicator, selectable marker and a cloning site.[1] The
replicator or "ori"[1] refers to the origin of replication with regard to location and bacteria
where replication begins. The marker refers to a gene that usually contains resistance to
an antibiotic, but may also refer to a gene that is attached alongside the desired one, such
as that which confers luminescence to allow identification of successfully recombined
DNA.[1] The cloning site is a sequence of nucleotides representing one or more positions
where cleavage by restriction endonucleases occurs.[1] Most eukaryotes do not maintain
canonical plasmids; yeast is a notable exception.[3] In addition, the Ti plasmid of the
bacterium Agrobacterium tumefaciens can be used to integrate foreign DNA into the
genomes of many plants. Other methods of introducing or creating recombinant DNA in
eukaryotes include homologous recombination and transfection with modified viruses.

[edit] Chimeric plasmids

An example of chimeric plasmid formation from two "blunt ends" via the enzyme, T4
Ligase.
When recombinant DNA is then further altered or changed to host additional strands of
DNA, the molecule formed is referred to as "chimeric" DNA molecule,[1] with reference
to the mythological chimera, which consisted as a composite of several animals.[1] The
presence of chimeric plasmid molecules is somewhat regular in occurrence, as,
throughout the lifetime of an organism[1], the propagation by vectors ensures the presence
of hundreds of thousands of organismal and bacterial cells that all contain copies of the
original chimeric DNA.[1]
In the production of chimeric plasmids, the processes involved can be somewhat
uncertain[1], as the intended outcome of the addition of foreign DNA may not always be
achieved and may result in the formation of unusable plasmids. Initially, the plasmid
structure is linearised[1] to allow the addition by bonding of complementary foreign DNA
strands to single-stranded "overhangs"[1] or "sticky ends" present at the ends of the DNA
molecule from staggered, or "S-shaped" cleavages produced by restriction endonucleases.
[1]

A common vector used for the donation of plasmids originally was the bacterium
Escherichia coli and, later, the EcoRI derivative[2], which was used for its versatility[2]
with addition of new DNA by "relaxed" replication when inhibited by chloramphenicol
and spectinomycin, later being replaced by the pBR322 plasmid.[2]In the case of EcoRI,
the plasmid can anneal with the presence of foreign DNA via the route of sticky-end
ligation, or with "blunt ends" via blunt-end ligation, in the presence of the phage T4 ligase
[2]
, which forms covalent links between 3-carbon OH and 5-carbon PO4 groups present on
blunt ends.[2] Both sticky-end, or overhang ligation and blunt-end ligation can occur
between foreign DNA segments, and cleaved ends of the original plasmid depending
upon the restriction endonuclease used for cleavage.[2]

Synthetic insulin production using recombinant DNA


Until the 1920s, there was no known way to produce insulin because the hormone was
not officially identified until 1921. Once identified, the production problem was quickly
solved when it was found that insulin from the pancreas of a cow, pig or even some
species of fish could be used successfully in humans. This method was the primary
solution for type 1 diabetes mellitus for decades, and manufacturing methods had steadily
improved the purity of the hormone which was made from animal pancreases. However,
proponents of the genetic engineering technology continued to raise what they claimed
was a looming problem with traditional methods of insulin production: a supposed
shortage of supply in the not-too-distant future. But in the 1987 book Invisible Frontiers:
The Race to Synthesize a Human Gene[4], author Stephen S. Hall wrote that the supposed
shortage is now known to be an assumption based on mistaken facts. He wrote:
To hear some tell it, there was never a supply problem with pig pancreases in the first
place. "The whole thing was rubbish," insists Paul Haycook, research director at SquibbNovo. "There was never a shortage of pig pancreases, and there never will be." Haycook
blames the scare on a miscalculation by an official who had prepared projections for the
Food and Drug Administration a mistake based, ironically, on a mistake in an Eli Lilly
training brochure which confused kilograms with pounds. Instead of projecting an insulin
shortage by 1982, a revised FDA report predicted adequate insulin supplies through the
year 2006. In any event, there is never likely to be a shortage caused by a scarcity of
pancreases.[4]
Scientists and entrepreneurs were very eager to prove they could devise another way to
synthesize the hormone, in part, because of competition from other researchers and also
because of the promise for the fame and fortune that its so-called "discovery" could bring
them. Insulin was part of a wider vision to introduce biotechnology medicines, and was
chosen specifically because it is a simple hormone and was therefore relatively easy to
copy. However, the motive was never to improve the lives of people with diabetes, but to
prove that the technology worked. Insulin was chosen as the ideal candidate because it is
a relatively simple protein, it was so widely used that if researchers could prove that
biosynthetic "human" insulin was safe and effective, then the technology would be
accepted as such, and it would open the flood gates for many other products to be made
this way, along with millions of dollars.

That was exactly what happened. One of the biggest breakthroughs in recombinant DNA
technology happened in the manufacture of biosynthetic "human" insulin, which was the
first medicine made via recombinant DNA technology ever to be approved by the FDA.
Henry I. Miller was an early advocate of biotechnology and drugs, and continues to be so
as a fellow at the Hoover Institute even though he no longer works for the FDA. Miller
began work for the FDA as head of a special department created to establish new
procedures for approving drugs created through biotechnology. In his book "To America's
Health: A Model for Reform of the Food and Drug Administration" (Hoover Institution
Press, 2000), Miller states that he pushed for rapid approval of biosynthetic insulin from
his boss, who was not comfortable approving it on such short notice, especially when it
had been tested on so few people. Amazingly, Miller admits that he actually waited for
his boss to go on vacation, and then took the approval to his boss' boss, who then
approved the drug[5].
As far as technical details, the specific gene sequence, or oligonucleotide, that codes for
insulin production in humans was introduced to a sample colony of E. coli (the bacteria
found in feces). Only about 1 out of 106 bacteria picks up the sequence. However, this is
not really a problem, because the lifecycle is only about 30 minutes for E. coli. This
means that in a 24-hour period, there may be billions of E. coli that are coded with the
DNA sequences needed to induce insulin production.[6]
However, a sampling of initial reaction showed that Humulin was greeted more as a
technological rather than a medical breakthrough, and that this sentiment was building
even before the drug reached pharmacies. As early as 1980, the British magazine New
Scientist reported, "Other big chemical manufacturers predict that Eli Lilly's massive $40
million investment in two plants to make insulin - may be a classic example of backing a
loser."[citation needed]
The Economist concluded: "The first bug-built drug for human use may turn out to be a
commercial flop. But the way has now been cleared-and remarkably quickly, too - for
biotechnologists with interesting new products to clear the regulatory hurdles and run
away with the prizes."[citation needed]
Ultimately, widespread consumer adoption of biosynthetic "human" insulin did not occur
until the manufacturers removed highly-purified animal insulin from the market.

Recombinant DNA technology

Here is how recombinant technology works:


1. Recombinant technology begins with the isolation of a gene of interest. The gene
is then inserted into a vector and cloned. A vector is a piece of DNA that is
capable of independent growth; commonly used vectors are bacterial plasmids
and viral phages. The gene of interest (foreign DNA) is integrated into the
plasmid or phage, and this is referred to as recombinant DNA.

2. Before introducing the vector containing the foreign DNA into host cells to
express the protein, it must be cloned. Cloning is necessary to produce numerous
copies of the DNA since the initial supply is inadequate to insert into host cells.
3. Once the vector is isolated in large quantities, it can be introduced into the desired
host cells such as mammalian, yeast, or special bacterial cells. The host cells will
then synthesize the foreign protein from the recombinant DNA. When the cells
are grown in vast quantities, the foreign or recombinant protein can be isolated
and purified in large amounts.

Other uses for recombinant DNA


Recombinant DNA technology is not only an important tool in scientific research, but it
has also impacted the diagnosis and treatment of diseases and genetic disorders in many
areas of medicine. It has enabled many advances, including:

Isolation of large quantities of pure protein


In addition to the follicle-stimulating hormone (FSH) used in Follistim AQ
Cartridge and Follistim AQ Vial, insulin, growth hormone and other proteins are
now available as recombinant products. Physicians no longer have to rely on
biological products (e.g. urine-derived FSH), that don't possess the same level of
purity and consistency of recombinant products to treat their patients.

Identification of mutations
People may be tested for the presence of mutated proteins that may be associated
with breast cancer, retino-blastoma, and neurofibromatosis.
Diagnosis of affected and carrier states for hereditary diseases
Tests exist to determine if people are carriers of the cystic fibrosis gene, the
Huntingtons disease gene, the Tay-Sachs disease gene, or the Duchenne muscular
dystrophy gene.
Transferring of genes from one organism to another
People suffering from cystic fibrosis, rheumatoid arthritis, vascular disease, and
certain cancers may now benefit from the progress made in gene therapy.
Mapping of human genes on chromosomes
Scientists are able to link mutations and disease states to specific sites on
chromosomes.

Recombinant DNA Technology in the Synthesis


of Human Insulin

The nature and purpose of synthesising human insulin.


Since Banting and Best discovered the hormone, insulin in 1921.(1)
diabetic patients, whose elevated sugar levels (see fig. 1) are due
to impaired insulin production, have been treated with insulin derived
from the pancreas glands of abattoir animals. The hormone, produced
and secreted by the beta cells of the pancreas' islets of Langerhans,(2)
regulates the use and storage of food, particularly carbohydrates.

Fig. 1
Fluctuations in diabetic person's blood glucose levels, compared with healthy individuals
. Source: Hillson,R. - Diabetes: A beyond basics guide, pg.16.

Although bovine and porcine insulin are similar to human insulin, their
composition is slightly different. Consequently, a number of patients'
immune systems produce antibodies against it, neutralising its actions and
resulting in inflammatory responses at injection sites. Added to these
adverse effects of bovine and porcine insulin, were fears of long term
complications ensuing from the regular injection of a foreign substance,(3)
as well as a projected decline in the production of animal derived insulin.(4)
These factors led researchers to consider synthesising Humulin by inserting

the insulin gene into a suitable vector, the E. coli bacterial cell, to produce
an insulin that is chemically identical to its naturally produced counterpart.
This has been achieved using Recombinant DNA technology. This
method (see fig. 2) is a more reliable and sustainable(5) method than
extracting and purifying the abattoir by-product.

Fig. 2
An overview of the recombination process. Source: Novo - Nordisk promotional
brochure,pg 6.

Understanding the genetics involved.

The structure of insulin.


Chemically, insulin is a small, simple protein. It consists of 51
amino acid, 30 of which constitute one polypeptide chain, and 21 of
which comprise a second chain. The two chains (see fig. 3) are linked
by a disulfide bond.(6)

Fig. 3
Source: Chance, R. and Frank B. - Research, development, production and safety
of Biosynthetic Human Insulin.

Inside the Double Helix.


The genetic code for insulin is found in the DNA at the top of the short
arm of the eleventh chromosome. It contains 153 nitrogen bases (63 in
he A chain and 90 in the B chain).DNA Deoxyribolnucleic Acid),
which makes up the chromosome, consists of two long intertwined helices,
constructed from a chain of nucleotides, each composed of a sugar
deoxyribose, a phosphate and nitrogen base. There are four different
nitrogen bases, adenine, thymine, cytosine and guanine.(7)
The synthesis of a particular protein such as insulin is determined by

the sequence in which these bases are repeated (see fig. 4).

Fig. 4
DNA strand with the specific nucleotide sequence for Insulin chain B. Source:
Based on the diagram in Watson, J.D., Gilman, M., Witkovski, J., Zoller, M. - Recombinant DNA, pg 22.

Insulin synthesis from the genetic code.


The double strand of the eleventh chromosome of DNA divides in
two, exposing unpaired nitrogen bases which are specific to
insulin production (see fig. 5).

Fig. 5
Unravelling strand of the DNA of chromosome 11, with the exposed nucleotides coding
for the B chain of Insulin. Source: Based on the diagram in Watso
, J.D., Gilman, M., Witkovski, J., Zoller, M. - Recombinant DNA, pg 22.

Using one of the exposed DNA strands (see fig.6) as a template,


messenger RNA forms in the process of transcription (see fig. 7).

Fig 6
A single strand of DNA coding for Insulin chain B. Source: Novo-Nordisk promotional
brochure, pg 13.

Fig. 7
The (m) RNA strand. Source: Novo-Nordisk promotional brochure, pg 13.

The role of the mRNA strand, on which the nitrogen base thymine is replaced by uracil, is to carry genetic
information, such as that pertaining to insulin,from the nucleus into the cytoplasm, where it attaches to a
ribosome (see fig. 8).

Fig. 8
Process of translation at the Ribosome. Source: Novo-Nordisk promotional brochure, pg 13.

The nitrogen bases on the mRNA are grouped into threes, known as codons
. Transfer RNA (tRNA) molecules, three unpaired nitrogen bases bound to
a specific amino acid, collectively known as an anti-codon (see fig.9)
pair with complementary bases (the codons) on the mRNA.

Fig. 9
Source: Novo-Nordisk promotional brochure, pg 13.

The reading of the mRNA by the tRNA at the ribosome is known as translation. A specific chain of amino
acids is formed by the tRNA following the code determined by the mRNA. The base sequence of the mRNA
has been translated into an amino acid sequence which link together to form specific proteins such as insulin

The Vector (Gram negative E. coli).


A weakened strain of the common bacterium, Escherrichia coli (E. coli) (see fig. 10), an inhabitant of the
human digestive tract, is the 'factory' used in the genetic engineering of insulin.

Fig. 10
The insulin is introduced into an E. coli cell such as this. Source: Novo-Nordisk promotional brochure, pg 16

When the bacterium reproduces, the insulin gene is replicated along with the plasmid,(8) a circular section of
DNA (see fig. 11). E. coli produces enzymes that rapidly degrade foreign proteins such as insulin. By using
mutant strains that lack these enzymes, the problem is avoided.(9)

Fig. 11
Electron micrograph of the Vector's plasmid. Source: Watson, J.D., Gilman, M., Witkovski,
J., Zoller, M. - Recombinant DNA, pg 73.

In E. coli, B-galactosidase is the enzyme that controls the transcription of


the genes. To make the bacteria produce insulin, the insulin gene needs
to be tied to this enzyme.

Inside the genetic engineer's toolbox.


Restriction enzymes, naturally produced by bacteria, act like biological
scalpels(10) (see fig.12), only recognising particular stretches of nucleotides,
such as the one that codes for insulin.(11)

Fig 12
An analogous look at Restriction enzymes. Source: CSIRO Research of Australia No. 8.

This makes it possible to sever certain nitrogen base pairs and remove the
section of insulin coding DNA from one organism's chromosome so that it
can manufacture insulin (See fig. 13). DNA ligase is an enzyme which
rves as a genetic glue, welding the sticky ends of exposed nucleotides
together.

Fig. 13
Source: Watson, J.D., Gilman, M., Witkovski., Zoller, M. - Recombinant DNA, pg 78.

Manufacturing Humulin.
The first step is to chemically synthesise the DNA chains that carry the specific nucleotide sequences
characterising the A and B polypeptide chains of insulin (see fig. 14).

Fig. 14
Human insulin structure. Amino acid RNA to DNA conversion. Source: Genetic Engineering

Activities, pg 176.

The required DNA sequence can be determined because the amino acid
compositions of both chains have been charted. Sixty three nucleotides
are required for synthesising the A chain and ninety for the B chain, plus a
codon at the end of each chain,signalling the termination of protein
synthesis. An anti-codon, incorporating the amino acid, methionine, is
then placed at the beginning of each chain which allows the removal of
the insulin protein from the bacterial cell's amino acids. The synthetic A
and B chain 'genes' (see fig. 15) are then separately inserted into the
gene for a bacterial enzyme, B-galactosidase, which is carried in the
vector's plasmid. At this stage, it is crucial to ensure that the codons of
the synthetic gene are compatible with those of the B-galactosidase.

Fig. 15
Source: Watson, J.D., Gilman, M.,Witkovski., Zoller, M. - Recombinant DNA, pg 456.

The recombinant plasmids are then introduced into E. coli cells. Practical use of Recombinant DNA
technology in the synthesis of human insulin requires millions of copies of the bacteria whose plasmid has
been combined with the insulin gene in order to yield insulin. The insulin gene is expressed as it replicates

with the B-galactosidase in the cell undergoing mitosis (see fig. 16).

Fig. 16
The process of mitosis. Source: Novo-Nordisk promotional brochure, pg 11.

The protein which is formed, consists partly of B-galactosidase, joined to either the A or B chain of insulin
(see fig.17). The A and B chains are then extracted from the B-galactosidase fragment and purified.

Fig. 17

Source: Watson, J.D., Gilman, M., Witkovski, J., Zoller, M. - Recombinant DNA, pg 456.

The two chains are mixed and reconnected in a reaction that forms the disulfide cross bridges, resulting in
pure Humulin - synthetic human insulin (see fig. 18).

Fig. 18
Human insulin molecule. Source: Source: Watson, J.D., Gilman, M., Witkovski, J., Zoller, M. - Recombinant DNA, pg 456.

Biological implications of genetically engineered Recombinant human insulin.

Human insulin is the only animal protein to have been made in bacteria in such a way that its structure is
absolutely identical to that of the natural molecule. This reduces the possibility of complications resulting
from antibody production. In chemical and pharmacological studies, commercially available Recombinant
DNA human insulin has proven indistinguishable from pancreatic human insulin.(12) Initially the major
difficulty encountered was the contamination of the final product by the host cells, increasing the risk of
contamination in the fermentation broth. This danger was eradicated by the introduction of purification
processes. When the final insulin product is subjected to a battery of tests, including the finest radio-immuno
assay techniques,(13) no impurities can be detected.(14) The entire procedure is now performed using yeast cel
as a growth medium, as they secrete an almost complete human insulin molecule with perfect three
dimensional structure. This minimises the need for complex and costly purification procedures.

The issue of hypoglycaemic complications in the administration of human insulin.

Since porcine insulin was phased out, and the majority of insulin dependent patients are now treated with
genetically engineered recombinant human insulin, doctors and patients have become concerned about the
increase in the number of hypoglycaemic episodes experienced.(15) Although hypoglycaemia can be expected
occasionally with any type of insulin, some people with diabetes claim that they are less cognisant of attacks
of hypoglycaemia since switching from animal derived insulin to Recombinant DNA human insulin.(16) In a
British study, published in the 'Lancet", hypoglycaemia was induced in patients using either pork or human
insulin, The researchers found "no significant difference in the frequency of signs of hypoglycaemia betwee

users of the two different types of insulin."(17)

An anecdotal report from a British patient who had been insulin dependent for thirty years, stated that she
began experiencing recurring, unheralded hypoglycaemia only after substituting Recombinant DNA human
insulin for animal derived insulin. After switching back to pork insulin to ease her mind, she hadn't
experienced any unannounced hypoglycaemia. Eli Lilly and Co., a manufacturer of human insulin, noted tha
a third of people with diabetes, who have been insulin dependent for over ten years, "lose their
hypoglycaemic warning signals, regardless of the type of insulin they are taking."(18)

Dr Simon P. Wolff of the University College of London said in an issue of Nature , "As far as I can make ou
there's no fault (with the human insulin)." He concluded, "I do think we need to have a study to examine the
possible risk."(19)

Although the production of human insulin is unarguable welcomed by the majority of insulin dependent
patients, the existence of a minority of diabetics who are unhappy with the product cannot be ignored.
Although not a new drug, the insulin derived from this new method of production must continue to be studie
and evaluated, to ensure that all its users have the opportunity to enjoy a complication free existence.

Recombinant DNA and Gene Cloning


Recombinant DNA is DNA that has been created artificially. DNA from two or more
sources is incorporated into a single recombinant molecule.

Making Recombinant DNA (rDNA): An Overview

Treat DNA from both sources with the same restriction endonuclease (BamHI in
this case).
BamHI cuts the same site on both molecules
5' GGATCC 3'
3' CCTAGG 5'

The ends of the cut have an overhanging piece of single-stranded DNA.


These are called "sticky ends" because they are able to base pair with any DNA
molecule containing the complementary sticky end.
In this case, both DNA preparations have complementary sticky ends and thus can
pair with each other when mixed.
DNA ligase covalently links the two into a molecule of recombinant DNA.

To be useful, the recombinant molecule must be replicated many times to provide


material for analysis, sequencing, etc. Producing many identical copies of the same
recombinant molecule is called cloning. Cloning can be done in vitro, by a process called
the polymerase chain reaction (PCR). Here, however, we shall examine how cloning is
done in vivo.
Cloning in vivo can be done in
unicellular microbes like E. coli
unicellular eukaryotes like yeast and
in mammalian cells grown in tissue culture.
In every case, the recombinant DNA must be taken up by the cell in a form in which it
can be replicated and expressed. This is achieved by incorporating the DNA in a vector.
A number of viruses (both bacterial and of mammalian cells) can serve as vectors. But

here let us examine an example of cloning


using E. coli as the host and a plasmid as
the vector.

Plasmids
Plasmids are molecules of DNA that are
found in bacteria separate from the
bacterial chromosome.
They:

are small (a few thousand base


pairs)
usually carry only one or a few
genes
are circular
have a single origin of replication

Electron micrograph of an E. coli cell


ruptured to release its DNA. The tangle is a
portion of a single DNA molecule containing
over 4.6 million base pairs encoding
approximately 4,300 genes. The small
circlets are plasmids. (Courtesy of
Huntington Potter and David Dressler,
Harvard Medical School.)

Plasmids are replicated by the same machinery that replicates the bacterial chromosome.
Some plasmids are copied at about the same rate as the chromosome, so a single cell is
apt to have only a single copy of the plasmid. Other plasmids are copied at a high rate
and a single cell may have 50 or more of them.
Genes on plasmids with high numbers of copies are usually expressed at high levels. In
nature, these genes often encode proteins (e.g., enzymes) that protect the bacterium from
one or more antibiotics.
Plasmids enter the bacterial cell with relative ease. This occurs in nature and may account
for the rapid spread of antibiotic resistance in hospitals and elsewhere. Plasmids can be
deliberately introduced into bacteria in the laboratory transforming the cell with the
incoming genes.

An Example
(courtesy of David Miklos and Greg Freyer of the Cold
Spring Harbor Laboratory, who used these plasmids as
the basis of a laboratory introduction to recombinant
DNA technology that every serious biology student
high school or college should experience!)

pAMP

4539 base pairs


a single replication origin

a gene (ampr)conferring resistance to the antibiotic ampicillin (a relative of


penicillin)
a single occurrence of the sequence
5' GGATCC 3'
3' CCTAGG 5'
that, as we saw above, is cut by the restriction enzyme BamHI

a single occurrence of the sequence


5' AAGCTT 3'
3' TTCGAA 5'
that is cut by the restriction enzyme HindIII

Treatment of pAMP with a mixture of BamHI and HindIII produces:


a fragment of 3755 base pairs carrying both the ampr gene and the replication
origin
a fragment of 784 base pairs
both fragments have sticky ends

pKAN

4207 base pairs


a single replication origin
a gene (kanr) conferring resistance to the antibiotic kanamycin.
a single site cut by BamHI
a single site cut by HindIII

Treatment of pKAN with a mixture of BamHI and HindIII produces:


a fragment of 2332 base pairs
a fragment of 1875 base pairs with the kanr gene (but no origin of replication)
both fragments have sticky ends
These fragments can be visualized by subjecting the digestion mixtures to electrophoresis
in an agarose gel. Because of its negatively-charged phosphate groups, DNA migrates
toward the positive electrode (anode) when a direct current is applied. The smaller the
fragment, the farther it migrates in the gel.

Ligation Possibilities
If you remove the two restriction enzymes and provide the conditions for DNA ligase to
do its work, the pieces of these plasmids can rejoin (thanks to the complementarity of
their sticky ends).

Mixing the pKAN and pAMP fragments provides several (at least
10) possibilities of rejoined molecules. Some of these will not
produce functional plasmids (molecules with two or with no
replication origin cannot function).

One interesting possibility is the joining of

the 3755-bp pAMP fragment (with ampr and a replication origin) with the
1875-bp pKAN fragment (with kanr)

Sealed with DNA ligase, these molecules are functioning plasmids that are capable of
conferring resistance to both ampicillin and kanamycin. They are molecules of
recombinant DNA.
Because the replication origin, which enables the molecule to function as a plasmid, was
contributed by pAMP, pAMP is called the vector.

Transforming E. coli
Treatment of E. coli with the mixture of religated molecules will produce some colonies
that are able to grow in the presence of both ampicillin and kanamycin.
A suspension of E. coli is treated with the mixture of religated DNA molecules.
The suspension is spread on the surface of agar containing both ampicillin and
kanamycin.

The next day, a few cells resistant to both


antibiotics will have grown into visible
colonies containing billions of transformed
cells.
Each colony represents a clone of
transformed cells.

However, E. coli can be simultaneously transformed by more than one plasmid, so we


must demonstrate that the transformed cells have acquired the recombinant plasmid.
Electrophoresis of the DNA from doubly-resistant colonies (clones) tells the story.

Plasmid DNA from cells that acquired their resistance from a recombinant
plasmid only show only the 3755-bp and 1875-bp bands (Clone 1, lane 3).
Clone 2 (Lane 4) was simultaneous transformed by religated pAMP and pKAN.
(We cannot tell if it took up the recombinant molecule as well.)
Clone 3 (Lane 5) was transformed by the recombinant molecule as well as by an
intact pKAN.

Cloning other Genes


The recombinant vector described above could itself be a useful tool for cloning other
genes. Let us assume that within its kanamycin resistance gene (kanr) there is a single
occurrence of the sequence
5' GAATTC 3'
3' CTTAAG 5'
This is cut by the restriction enzyme EcoRI, producing sticky ends.
If we treat any other sample of DNA, e.g., from human cells, with EcoRI, fragments with
the same sticky ends will be formed. Mixed with EcoRI-treated plasmid and DNA ligase,
a small number of the human molecules will become incorporated into the plasmid which
can then be used to transform E. coli.
But how to detect those clones of E. coli that have been transformed by a plasmid
carrying a piece of human DNA?
The key is that the EcoRI site is within the kanr gene, so when a piece of human DNA is
inserted there, the gene's function is destroyed.

All E. coli cells transformed by the


vector, whether it carries human DNA
or not, can grow in the presence of
ampicillin. But E. coli cells
transformed by a plasmid carrying
human DNA will be unable to grow in
the presence of kanamycin.
So,

Spread a suspension of treated


E. coli on agar containing
ampicillin only
grow overnight
with a sterile toothpick transfer
a small amount of each colony
to an identified spot on agar
containing kanamycin
(do the same with another

ampicillin plate)
Incubate overnight

All those clones that continue to grow on ampicillin but fail to grow on kanamycin (here,
clones 2, 5, and 8) have been transformed with a piece of human DNA.

Some recombinant DNA products being used in human


therapy
Using procedures like this, many human genes have been cloned in E. coli or in yeast.
This has made it possible for the first time to produce unlimited amounts of human
proteins in vitro. Cultured cells (E. coli, yeast, mammalian cells) transformed with a
human gene are being used to manufacture more than 100 products for human therapy.
Some examples:

insulin for diabetics


factor VIII for males suffering from hemophilia A
factor IX for hemophilia B
human growth hormone (HGH)
erythropoietin (EPO) for treating anemia
several types of interferons
several interleukins
granulocyte-macrophage colony-stimulating factor (GM-CSF) for stimulating
the bone marrow after a bone marrow transplant
granulocyte colony-stimulating factor (G-CSF) for stimulating neutrophil
production, e.g., after chemotherapy and for mobilizing hematopoietic stem cells
from the bone marrow into the blood.

tissue plasminogen activator (TPA) for dissolving blood clots


adenosine deaminase (ADA) for treating some forms of severe combined
immunodeficiency (SCID)
parathyroid hormone
several monoclonal antibodies
hepatitis B surface antigen (HBsAg) to vaccinate against the hepatitis B virus
C1 inhibitor (C1INH) used to treat hereditary angioneurotic edema (HANE)

Many more examples are in the pipeline.


Welcome&Next Search
24 February 2008Recombinant DNA
Recombinant DNA refers to a collection of techniques for creating (and analyzing) DNA
molecules that contain DNA from two unrelated organisms. One of the DNA molecules is
typically a bacterial or viral DNA that is capable of accepting another DNA molecule;
this is called a vector DNA. The other DNA molecule is from an organism of interest,
which could be anything from a bacterium to a whale, or a human. Combining these two
DNA molecules allows for the replication of many copies of a specific DNA. These
copies of DNA can be studied in detail, used to produce valuable proteins, or used for
gene therapy or other applications.
The development of recombinant DNA tools and techniques in the early 1970s led to
much concern about developing genetically modified organisms with unanticipated and
potentially dangerous properties. This concern led to a proposal for a voluntary
moratorium on recombinant DNA research in 1974, and to a meeting in 1975 at the
Asilomar Conference Center in California. Participants at the Asilomar Conference
agreed to a set of safety standards for recombinant DNA work, including the use of
disabled bacteria that were unable to survive outside the laboratory. This conference
helped satisfy the public about the safety of recombinant DNA research, and led to a
rapid expansion of the use of these powerful new technologies.
Overview of Recombination Techniques

The basic technique of recombinant DNA involves digesting a vector DNA with a
restriction enzyme, which is a molecular scissors that cuts DNA at specific sites. A DNA
molecule from the organism of interest is also digested, in a separate tube, with the same
restriction enzyme. The two DNAs are then mixed together and joined, this time using an
enzyme called DNA ligase, to make an intact, double-stranded DNA molecule. This
construct is then put into Escherichia coli cells, where the resulting DNA is copied
billions of times. This novel DNA molecule is then isolated from the E. coli cells and
analyzed to make sure that the correct construct was produced. This DNA can then be
sequenced, used to generate protein from E. coli or another host, or for many other
purposes.

There are many variations on this basic method of producing recombinant DNA
molecules. For example, sometimes researchers are interested in isolating a whole
collection of DNAs from an organism. In this case, they digest the whole genome with
restriction enzyme, join many DNA fragments into many different vector molecules, and
then transform those molecules into E. coli. The different E. coli cells that contain
different DNA molecules are then pooled, resulting in a "library" of E. coli cells that
contain, collectively, all of the genes present in the original organism.
Another variation is to make a library of all expressed genes (genes that are used to make
proteins) from an organism or tissue. In this case, RNA is isolated. The isolated RNA is
converted to DNA using the enzyme called reverse transcriptase. The resulting DNA
copy, commonly abbreviated as cDNA, is then joined to vector molecules and put into E.
coli. This collection of recombinant cDNAs (a cDNA library) allows researchers to study
the expressed genes in an organism, independent from nonexpressed DNA.
Applications

Recombinant DNA technology has been used for many purposes. The Human Genome
Project has relied on recombinant DNA technology to generate libraries of genomic DNA
molecules. Proteins for the treatment or diagnosis of disease have been produced using
recombinant DNA techniques. In recent years, a number of crops have been modified
using these methods as well.
As of 2001, over eighty products that are currently used for treatment of disease or for
vaccination had been produced using recombinant DNA techniques. The first was human
insulin, which was produced in 1978. Other protein therapies that have been produced
using recombinant DNA technology include hepatitis B vaccine, human growth hormone,
clotting factors for treating hemophilia, and many other drugs. At least 350 additional
recombinant-based drugs are currently being tested for safety and efficacy. In addition, a
number of diagnostic tests for diseases, including tests for hepatitis and AIDS, have been
produced with recombinant DNA technology.
Gene therapy is another area of applied genetics that requires recombinant DNA
techniques. In this case, the recombinant DNA molecules themselves are used for
therapy. Gene therapy is being developed or attempted for a number of inherited human
diseases.
Recombinant DNA technology has also been used to produce genetically modified foods.
These include tomatoes that can be vine-ripened before shipping and rice with improved
nutritional qualities. Genetically modified foods have generated controversy, and there is
an ongoing debate in some communities about the benefits and risks of developing crops
using recombinant DNA technology.
Since the mid-1970s, recombinant DNA techniques have been widely applied in research
laboratories and in pharmaceutical and agricultural companies. It is likely that this

relatively new area of genetics will continue to play an increasingly important part in
biological research into the foreseeable future.

he Basics of Recombinant DNA


So What Is rDNA?
That's a very good question! rDNA stands for recombinant DNA. Before
we get to the "r" part, we need to understand DNA. Those of you with
a background in biology probably know about DNA, but a lot of ChemE's haven't
seen DNA since high school biology. DNA is the keeper of the all the information
needed to recreate an organism. All DNA is made up of a base consisting
of sugar, phosphate and one nitrogen base. There are four nitrogen bases,
adenine (A), thymine (T), guanine (G), and cytosine (C). The nitrogen
bases are found in pairs, with A & T and G & C paired together. The sequence
of the nitrogen bases can be arranged in an infinite ways, and their structure is known as
the famous "double helix" which is shown in the image below. The sugar used in
DNA is deoxyribose. The four nitrogen bases are the same for all organisms. The
sequence and number of bases is what creates diversity. DNA does not
actually make the organism, it only makes proteins. The DNA is transcribed
into mRNA and mRNA is translated into protein, and the protein then forms the
organism. By changing the DNA sequence, the way in which the protein is
formed changes. This leads to either a different protein, or an inactive protein.

Now that we know what DNA is, this is where the recombinant comes in.
Recombinant DNA is the general name for taking a piece of one DNA, and
and combining it with another strand of DNA. Thus, the name recombinant!
Recombinant DNA is also sometimes referred to as "chimera." By combining two or
more different strands of DNA, scientists are able to create a new strand of DNA.
The most common recombinant process involves combining the DNA of two
different organisms.

How is Recombinant DNA made?


There are three different methods by which Recombinant DNA is made. They are
Transformation, Phage Introduction, and Non-Bacterial Transformation. Each
are described separately below.
Transformation
The first step in transformation is to select a piece of DNA to be inserted
into a vector. The second step is to cut that piece of DNA with a restriction
enzyme and then ligate the DNA insert into the vector with DNA Ligase. The insert
contains a selectable
marker which allows for identification of recombinant molecules. An antibiotic
marker is often used so a host cell without a vector dies when exposed to a certain
antibiotic, and the host with the vector will live because it is resistant.

The vector is inserted into a host cell, in a process called transformation. One
example of a possible host cell is E. Coli. The host cells must be specially
prepared to take up the foreign DNA.
Selectable markers can be for antibiotic resistance, color changes, or any other
characteristic which can distinguish transformed hosts from untransformed hosts.
Different vectors have different properties to make them suitable to different
applications. Some properties can include symmetrical cloning sites, size, and
high copy number.
Non-Bacterial Transformation
This is a process very similar to Transformation, which was described above. The
only difference between the two is non-bacterial does not use bacteria such as E. Coli
for the host.
In microinjection, the DNA is injected directly into the nucleus of the cell being
transformed. In biolistics, the host cells are bombarded with high velocity
microprojectiles, such as particles of gold or tungsten that have been coated
with DNA.
Phage Introduction
Phage introduction is the process of transfection, which is equivalent to transformation,
except a phage is used instead of bacteria. In vitro packagings of a vector is used.
This uses lambda or MI3 phages to produce phage plaques which contain recombinants.
The recombinants that are created can be identified by differences in the
recombinants and non-recombinants using various selection methods.

How does rDNA work?


Recombinant DNA works when the host cell expresses protein from the recombinant
genes.
A significant amount of recombinant protein will not be produced by the host unless
expression
factors are added. Protein expression depends upon the gene being surrounded by
a collection of signals which provide instructions for the transcription and translation
of the gene by the cell. These signals include the promoter, the ribosome binding
site, and the terminator. Expression vectors, in which the foreign DNA is inserted,
contain these signals. Signals are species specific. In the case of E. Coli, these
signals must be E. Coli signals as E. Coli is unlikely to understand the signals of
human promoters and terminators.
Problems are encountered if the gene contains introns or contains signals which act
as terminators to a bacterial host. This results in premature termination, and the

recombinant
protein may not be processed correctly, be folded correctly, or may even be degraded.
Production of recombinant proteins in eukaryotic systems generally takes place in
yeast and filamentous fungi. The use of animal cells is difficult due to the fact
that many need a solid support surface, unlike bacteria, and have complex growth
needs. However, some proteins are too complex to be produced in bacterium,
so eukaryotic cells must be used.

Why is rDNA important?


Recombinant DNA has been gaining in importance over the last few years, and
recombinant DNA will only become more important in the 21st century as genetic
diseases become more prevelant and agricultural area is reduced. Below are
some of the areas where Recombinant DNA will have an impact.

Better Crops (drought & heat resistance)


Recombinant Vaccines (ie. Hepatitis B)
Prevention and cure of sickle cell anemia
Prevention and cure of cystic fibrosis
Production of clotting factors
Production of insulin
Production of recombinant pharmaceuticals
Plants that produce their own insecticides
Germ line and somatic gene therapy

What does the future hold?


Now that we've figured out the basics behind what Recombinant DNA are, it's
time to look at how Recombinant DNA will impact the future. Which industries
and fields will be shaped by rDNA? How will rDNA effect the health and
lifestyles of RPI students in the next generation? Click over to our
rDNA Impact Statement to find out the answer!

Pop Quiz Time!


To help you determine how well you know Recombinant DNA, we
have generously decided to provide you with a basic quiz that even a
senior ChemE should be able to do. Be sure and look over the additional
information provided below, because these questions could be tricky! All
the information needed to answer the questions can be found on this page,
or the associated pages. When you're ready, click below.

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