Northern Zambales College, Inc.

Inhobol, Masinloc, Zambales

Basic Education Department

Recombinant DNA

The method of joining two or more DNA molecules to create a hybrid is known as Recombinant DNA
(rDNA). Restriction endonucleases and ligases, two forms of enzymes, activate the technology. A restriction
endonuclease recognizes a particular DNA sequence and cuts inside or near to it.
Genetic Engineering is a process of making changes on the genetic code of an organism. It goal is to
add one or more new traits that are not normally found in that organism. Through advanced studies in the
structure of DNA and its chemical properties, and make unlimited copies of DNA.

The Process of Genetic Engineering

Genetic engineering is a complex activity that requires sophisticated machinery and creative thinking. Since
the dawn of agriculture, humans have been selectively breeding different plants, resulting in more and better
food for all. This approach is genetic engineering, to put it in far simpler terms than we think about it today.

Now, when one thinks of genetic engineering, they are more likely to picture a complicated science which
involves altering the very building blocks of life. There are several steps in the process of genetic engineering.
Scientist follows a step by step process in order to alter the DNA of an organism. In describing the process, the
example of a soybean will be used as a guide to each step.

1. First, a gene is picked that will be altered, added or removed. This process generally requires the discovery
and isolation of the desired gene. If a new breed of soybean was given a pesticide, the pesticide's successful
bacteria will be isolated. In order to be placed in the soybean genome, the bacteria genes are needed.

2. The isolated gene is copied several times. The DNA from the bacteria is then copied several times. This is
done by splitting the DNA down the center of the double helix and pairing it with the appropriate chemical
(Goldbas).

3. The gene is transferred to the new organism. It is transferred into the tissue of the organism. Since it is
impossible to insert the DNA into each cell of an organism, the DNA is now injected into the tissue of the
soybean plant. The soybean plant is then grown to maturity in a greenhouse. A technique used today
involves shooting DNA from a .22 caliber charge into plant tissue (Goldsburg).

4. Create a new plant/animal/trait from the newly modified tissue. Now that the new DNA is present, the
soybeans will continue to grow, only now they possess a new trait.

5. Check the insertion produces desired results.

6. Check that the new gene can be found in the offspring (seeds) of the organism. This is most crucial step in
genetic engineering. If the offspring of the genetically modified organism does not possess the traits given
to the parent, the engineering has failed and must be done again.

Technologies and Tools Used in Recombinant DNA Technology

Molecular biologists have developed different technologies and tools that allow them to study and
manipulate DNA molecules. The process that will be discussed here are gel electrophoresis, DNA splicing, and
polymerase chain reaction.

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Gel Electrophoresis
Gel electrophoresis is a method used to separate DNA fragments based on the size. In this method, a
mixture of DNA fragments is placed at one end of a porous gel, and an electric voltage is applied to the gel. The
smaller the DNA fragments, fingerprinting, comparing the genomes of different organisms, and locating and
identifying one particular gene out of the millions of genes in an individual’s genome.

DNA Sequencing
DNA sequencing is a technique for identifying the appropriate sequence of bases (A, C, G, and T) in a
DNA molecule in a laboratory setting. The knowledge a cell requires to assemble protein and RNA molecules is
contained in the DNA base sequence. Scientists looking into the roles of genes need to know the DNA
sequence. The technology of DNA sequencing was made faster and less expensive as a part of the Human
Genome Project.

Polymerase Chain Reaction

The polymerase chain reaction (PCR) was originally developed in 1983 by the American biochemist
Kary Mullis. He was awarded the Nobel Prize in Chemistry in 1993 for his pioneering work. PCR is used in
molecular biology to make many copies of (amplify) small sections of DNA or a gene. Using PCR it is possible
to generate thousands to millions of copies of a particular section of DNA from a very small amount of DNA.
PCR is a common tool used in medical and biological research labs. It is used in the early stages of processing
DNA for sequencing, for detecting the presence or absence of a gene to help identify pathogens during
infection, and when generating forensic DNA profiles from tiny samples of DNA.

Application of Recombinant DNA

Genetic engineering has always been a topic of controversy as the balance it aims to reach between the
benefits accrued to humans and attendant ethical considerations is open to debate. In each of the diverse fields
of agriculture, medicine, bioremediation and biotechnology concerns vary in a discipline-specific manner.
However, the principal source of apprehension often involves the ecological impact, real or perceived, of the
use of recombinant DNA technology, in particular the release of genetically modified organisms into the
environment. 

The use of recombinant (r-) DNA technology to produce genetically engineered organisms started in the
early 1970s with the pioneering transfer of genes between bacteria of the same Escherichia
coli species. Following these successful pilot experiments, in 1978 Cohen and colleagues progressed to transfer
an insulin synthesis gene into a plasmid of E. coli, with that producing the first genetically modified organism
(GMO).

Agriculture

In agriculture development of genetically modified crops with a purpose to improve both yield and
resistance to plant pests or herbicides seems to have gained a degree of public acceptance and is already
practised in a commercial context in several countries. The genetically modified tomato CGN-89564-2 was the
first commercially grown, genetically engineered crop product to be granted a licence for human consumption.
This was developed in 1994 to express the trait of delayed softening of tomato flesh as a practical means to

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minimize post-harvest crop losses. Ironically given its brand name of ‘Flavr Savr’, this failed in the marketplace
due not to public apprehension over eating a genetically altered food per se but to an apparent lack of taste.
Nevertheless, the introduction of a genetically modified fruit paved the way for use of GMOs in food and today
genetic modification is widespread. In the US, 88% of corn and 93% of soybeans are genetically altered and
much of this finds its way unlabelled into processed foods.

The introduction of pest-resistant brinjal (also known as eggplant or aubergine) was met with criticism
in some countries, in contrast to the concurrent popularity of pest-resistant cotton. Both attempts at
implementation followed incorporation of the identical crystal protein gene (Cry1Ac) from the soil
bacterium Bacillus thuringiensis (Bt) into the genome of the host plant expression of which synthesizes so-
called Bt toxins that confer resistance to predation by lepidopteran insects. However, of the two uses as a food
and as clothing the one which caused anxiety among the general public involved human consumption. The
benefits to humans of using Bt toxin should be stressed in an attempt to overcome the initial unpopularity of
consuming Bt-brinjals in developing countries such as India, Bangladesh and Philippines.  This would reduce
public scepticism founded on the misperception that eating a plant product containing a ‘toxin’ is in fact toxic to
humans irrespective of its unrelated target and benign mechanism of action.

The following are some bacteria used in Recombinant DNA Technology:

1. Psuedomonas syriangae – The recombinant variant of this bacterium is called the ice-minus bacterium,
which lacks the gene responsible for ice formation. The ice-minus bacteria prevent frost crystals from
forming on plants.
2. Psuedomonas flourescens – This is a nonpathogenic bacterium that has the ability to produce proteins
rapidly. This characteristic is advantageous in developing biotherepeutics and vaccines.

3. Agrobacterium tumefaciens – In its natural state, this bacterium that has a tumor-inducing (Ti) plasmid
that causes crown gall diseases in plants. The said Ti plasmid in the bacterium can be removed and
replaced with a recombinant plasmid. This enables the now-modified bacterium to introduce beneficial
genes to plants.

Medicine

An important contribution of recombinant DNA technology in the field of medicine is the use of bacteria
to create substances that our body needs whether to maintain good health or to treat a disease. For example,
human insulin is created using recombinant DNA to help diabetics. Also, the modified human growth hormones
is wildly used to support the development of the people who have a malfunctioning pituitary gland. This is great
benefit because it avoids the practice of getting growth hormones from dead bodies, which can pose serious
health risks. Other important substances such as blood clotting factors (for people suffering from hemophilia)
and hepatitis B virus surface antigens (for people suffering from hepatitis B) are also products of recombinant
DNA technology. Certainly, this technology has given us many benefits in the field of medicine.

Food Industry

Recombinant technology has also improved the food industry. Some of the crops that we eat are now
resistant to pests, diseases, and environmental stress. As a result, crop yields have increased and production cost
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have been kept lowered. Recombinant crops are also beneficial because of their improved nutritional quality
and longer shelf life. Recombinant technology can also be used for processing high-quality fermented foods

History of Life on Earth

Alexander Ivan Oparin (1894-1980) – Russian chemist, proposed the widely accepted idea of how
life on Earth formed. He proposed that the atmosphere of early Earth was very reactive, with numerous
incidents of lightning and high levels of ultraviolet radiation.

The Origin of Life

Geological pieces of evidence show that Earth was not formed from a single event. Our planet was
formed by different pieces of cosmic debris that were probably attracted to one another over a course of about
100 million years. As it was forming, it was struck by one or more meteor like objects, possibly one as large as
Mars. The collision most probably produced enough heat to melt the entire globe. When Earth melted, the
different elements contained in it rearranged themselves according to density. The densest elements formed the
core of Earth. From there, radioactive decay generated enough heat to convert the interior of Earth into molten
rock. Moderate dense elements floated to the surface, forming Earth’s solid crust. The least dense elements,
which include hydrogen and nitrogen, formed the atmosphere. The infant planet was so different from modern
Earth because the sky was most probably pinkish orange, and its early atmosphere contained hydrogen
cyanide, carbon dioxide, carbon monoxide, nitrogen, hydrogen sulfide, and water. No life-form would have
been able to survive that kind of harsh environment.

First Organic Molecules

Stanley Miller and Harold Urey – American chemists conducted an experimented in the 1950’s , which
involved simulating the early conditions of Earth inside the laboratory. In their experiment, they filled a flask
with hydrogen , methane, ammonia, and water to represent the early atmosphere of Earth. Then, they passed
electric sparks through the mixture to simulate lightning. They discovered that several amino acids – the
building blocks of proteins – began to accumulate in their setup. Their experiment was still significant because
it suggested that organic compounds necessary for life might have arisen from simple inorganic materials
present on primitive Earth. This experiment has not only encouraged many other experiments to find out the
origin of life: it also helped in contributing knowledge to science.

How Life Begins

Scientist believe that microscopic cell-like structures similar to modern bacteria appeared 200-300
million years after Earth cooled enough to carry water. These cell-like structures were actually large organic
molecules known as protienoid microspheres. Like cells, they had a selectively permeable membrane that
allowed certain molecules to pass through them. They also means of storing and releasing energy in and out of

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their systems. Several hypotheses suggest that structures similar to protenoid microspheres might have
acquired more cellular characteristics.

Paleontology – it is the scientific study of the existence of life, including the origin and eventual
destruction or extinction of different groups of organisms. It is a science that incorporates different disciplines
such as biology, geology, ecology, archaeology, and even computer science to study the evolution of
organisms and how they interact with the environment.
Paleontologist – specialize in studying the ecologies of the past and the evolution of organisms that thrived
in these ecologies through careful observation and documentation of fossils. They work to identify the forms
of life that existed millions of years ago.
Geology – it is the study of life on Earth based on the evidence found in rocks.
Geologist – are scientist who carefully study the different materials that make up Earth. They work to
understand the history of Earth by focusing on the changes of Earth over time in relation to changes in climate
and land formation.
Geological Timescale – it is used to represent evolutionary time. Geological timescale shows significant
events in history of Earth and of the evolution of living things. The information obtained from these pieces of
evidence made it possible for them to identify the relative age of Earth.

These pieces of evidence were used to mark the start and the end of a segment of geologic time. Other
techniques such as radioactive and relative dating were also used to confirm the specific ages of rock layers in
recent years. Scientist found out that these divisions did not turn out to be of standard length, such as 100
million years. Instead, geologic divisions vary in duration by several millions of years.
The geologic timescale has several levels of division. Each row in the geologic timescale will be
discussed thoroughly from the oldest to the latest geologic timeline.

Eons
Eons are the largest divisions in the geologic timeline. They composed of hundreds of millions of years
in duration.
Phanerozoic eon began more than 500 million years ago. This eon is characterized by the existence of
organisms that had skeletons or hard shells.
Archaeozoic eon (from 3.9 to 2.5 billion years ago), where the first life-forms represented bu single celled
organisms appeared.
Hadean eon (from 4.6 to 3.9 billion years ago), it is characterized by Earth’s formation of gases.

Eras
Eons are divided in smaller time intervals known as eras. The Phanerozoic eon is divided into three eras
namely, Paleozoic, Mesozoic, and Cenozoic eras. The boundaries of the era have been determined based on
the different organisms that were present during a certain period of time in the geologic timescale.

Paleozoic era
The Paleozoic era began about 540 million years ago and lasted for about 300 million years. Many kinds
of organisms lived during this era this is when fossil records became rich with evidence of many marine
organisms.
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Mesozoic era
The Mesozoic era began about 200 million years ago and lasted for about 180 million years. This era is
called the Age of Dinosaurs and, sometimes, the Age of Reptiles because this when many kinds of dinosaurs
existed. The Mesozoic era is also marked by the appearance of flowering plants.

Cenozoic era
The Cenozoic era is considered to be the latest era in the geologic timeline, which began from 65 million
years ago up to the present. It is composed of the Quaternary and Tertiary periods, which are characterized by
the presence of primitive mammals and modern human civilization. During the Cenozoic era, mammals have
evolved and adapted to live in various environments, including land, water, and even air. This is why the
Cenozoic era is sometimes called the Age of Mammals.

Periods
Periods are further subdivision of eras. The Proterozoic eon is composed of the Vendian/Ediacaran
period. Prokaryotic organisms began to appear during this period, first with the anaerobic life-forms, and then
followed by the photosynthetic organisms, which added oxygen to the atmosphere. However, life in this
period only thrived underwater, and only a few fossils existed because the animals were all soft-bodied.

The Mesozoic era was divided into the Cretaceous, Jurassic, and Triassic periods. These periods
represent the age when many forms of dinosaurs are believed to have existed on Earth. Their extinction could
have been brought about by an asteroid impact or volcanism.

Cretaceous period – this time when reptiles were dominant vertebrates on Earth. During this period, several
kinds of birds began to exist alongside Pterosaurs (flying reptiles). It was also the period when new kinds of
dinosaurs evolved. Iguanodon and Triceratops species emerged as common herbivores, and Tyrannosaurus
rex and Spinosaurus were some of the dominant carnivores in this period. Other aquatic organisms such as
rays, sharks, corals, and sea stars, as well as marine reptiles such as mosasaurs and plesiosaurs dominated
different bodies of water. The angiosperms, the flowering plants, also arose during this period.
Jurassic period – dinosaurs became the dominant animals on land for about 150 million years. However,
various types of land animals lived at different time frames. Dicraeosaurus was one of the largest dinosaurs in
this period. Archeopteryx, one of the earliest known birds, also appeared in this period. Because of the
presence of feathers, their similar body structure, and the presence of scales usually on their feet.
Archeopteryx made paleontologist believe that birds are close relatives of dinosaurs.
Triassic period – many organisms in this periods survived the mass extinction during the Permian period,
including fishes, insect, reptiles, and cone-bearing plants. About 225 million years ago, the first dinosaurs
appeared. One of the earliest dinosaurs species belonged to the genus Coelophysis, which were meat-eater that
had light, hollow bones to help them run swiftly on their hind leg.

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