Principle of cDNA cloning

 Complementary DNA (cDNA) cloning is termed for the gene cloning (cloning of
DNA fragments) obtained from cDNA.
 The principle of cDNA cloning is that it involves the copying of mRNA transcripts into
DNA, which are then inserted into bacterial plasmids and then placed into bacteria
by transformation.
 At this stage, it should be clear that mRNA used for cDNA preparation is a processed
transcript and not the original one transcribed from DNA.
 In-order to clone a DNA sequence that codes for a required gene product, the gene
should be removed from the organism and cloned it in the vector molecule.
 A gene library is a random collection of cloned fragments in an appropriate vector that
particularly consists of all the genetic information about that species.
 There are two methods for the formation of gene libraries. They are:
 Complementary DNA (cDNA)
 Genomic DNA libraries

Steps of cDNA cloning:

1. Isolation of mRNA
2. Synthesis of first strand of cDNA
3. Synthesis of second strand of cDNA
4. Cloning of cDNA
5. Introduction to host cells
6. Clone selection
Fig: steps of cDNA cloning
source: sciencedirect.com

1. Isolation of mRNA:

 A crude extract of the tissue with the gene of interest is prepared.

 The extract must be free from proteins, polysaccharides and all other contaminants.
 The technique of oligo-deoxythymine (oligo-dT) cellulose chromatography is used for
the further purification of many eukaryotic mRNAs from the total or polysomal
fraction.
 mRNAs consist of poly A (adenosine residues) tail at their 3′ end.
 Under favourable conditions, this tail will bind to a string of thymidine residues
immobilized on cellulose and then poly (A) + fraction can be eluted.
 Two or three passages of the poly (A)+ fraction through such a column produces a
fraction highly enriched for mRNA.
 This fraction includes different mRNA sequences, however certain techniques can be
employed for extracting a particular mRNA species.
 After the preparation of the fraction, it is essential to confirm if the extracted mRNA
consists of the sequence of interest.
 It is performed by translation of mRNA in vitro and identification of suitable
polypeptides in the products obtained.

2. Synthesis of first strand of cDNA:

  Reverse transcriptase is a RNA dependent DNA polymerase which is used to copy the
mRNA fraction into the first strand of DNA.
 This enzyme, like all other DNA polymerases, can only add residues at the 3′-OH
group of an existing primer, which is base paired with the template.
 The most commonly used primer is oligo-dT for cloning of cDNAs.
 Oligo-dT primer is 12-18 nucleotides in length, that binds to the poly (A) tract at the 3′
end of mRNA molecules.
 The RNA strand of the resulting RNA-DNA hybrid is destroyed prior to second strand
synthesis through alkaline hydrolysis.

3. Synthesis of second strand of cDNA:

 The second strand of cDNA can be synthesized by two techniques. They are:
 i. Self-priming cDNA:
 In Self-priming, the mRNA hybrid obtained is denaturated for the synthesis
of second strand on the single strand of cDNA by the klenow fragment of DNA
polymerase I.
 The transitory hairpin structure at the 3′ end of single-stranded DNA can be used
to prime the synthesis of second strand of cDNA by the klenow fragment
of Escherichia coli DNA polymerase I.
 Single-strand specific S1 nuclease digests the hairpin loop and any single-
stranded overhung at the other end.
 The ultimate product is a population of double-stranded, blunt-ended DNA
molecules complementray to the original mRNA fraction.
 ii. Replacement synthesis:
 In this method, the cDNA:mRNA hybrid works as a template for a nick
translation reaction.
 In the mRNA strand of the hybrid, RNase H produces nicks and gaps, creating a
series of RNA primers.
 These RNA primers are used by E. coli DNA polymerase I during the synthesis
of second strand of cDNA.
 The advantages of this technique are:
 – very efficient
 – can be performed directly using the products of the first strand reaction
 – eliminates the need to use nuclease S1 to cleave the single-stranded
hairpin loop in the double stranded cDNA.

4. Cloning of cDNA:

 The most frequently used technique for cloning cDNAs involves the addition of
complementary homopolymeric tracts to double stranded cDNA and to the plasmid
vector.
 To the cDNA, strings of cytosine residues are added using the enzyme terminal
transferase to form oligo-dC tails on the 3′ ends.
 Likewise, a plasmid is cut open at a unique restriction endonuclease site and tailed with
oligo-dG.
  Now, the vector and the double stranded cDNA are joined by hydrogen bonding
between the complementary homopolymers.
 It results in the formation of open circular hybrid molecules capable of transforming E.
coli.

5. Introduction to host cells:

 For the transforming of bacteria, the recombinant plasmids are used, usually the E.
coli K-12 strain.
 The uptake of plasmid molecules from the surrounding medium is performed by E. coli
cells treated with calcium chloride.
 Any gaps in the recombinant plasmid will be repaired by the host cells.
 The transformed bacteria can be isolated from non-transformed ones on the basis of
antibiotic resistance.
 Majority of cloning plasmids contain two antibiotic resistance genes, one of which is
destroyed during cloning.
 For instance, in the case of pBR322, cloning into unique PstI site destroys ampicillin
resistance but leaves tetracycline resistance intact.
 Bacteria transformed with a recombinant plasmid will be sensitive to ampicillin but
resistant to tetracycline.

6. Clone selection:

 The antibiotic resistance selection already performed has recognized which clones
carry a recombinant plasmid, however there will be thousands of various inserts.
 The cloning process generally commences with a whole population of mRNA
sequences.
 Selection of clones carrying the sequence of interest is the tough job.
 If the gene is expressed, then the simplest selection is to screen for the presence of the
protein.
 It can be screened either by bacterial phenotype it produces or by the protein detection
methods usually based on immunological or enzymological techniques.
 If the protein is not expressed, then other methods such as nucleic acid hybridization
are used.
 Identification of the gene is discussed after the genomic DNA cloning.

Genomic Library
A genome is an organism’s complete set of DNA, including all of its genes. Each genome
contains all of the information needed to build and maintain that organism. In humans, a copy
of the entire genome is more than 3 billion DNA base pairs which are present in all cell
nuclei. The nuclear genome contains protein-coding and non-coding genes, as well as other
junk DNA and functional regions of the genome.
Most eukaryotes are diploid, which means that each chromosome has two copies in the
nucleus, but the term genome refers to just one copy of each chromosome.
Genomic DNA Library
A genomic library or gene bank is a complete collection of cloned DNA fragments that
constitutes the entire genome of an organism. It represents all the genes – expressed, non-
expressed, intron, exons, etc. Genomic libraries can be kept for many years and the copies
can be used for research purposes.
Advantages of a Genomic Library

 Genomic libraries derived from eukaryotic organisms are critical for studying the
genome sequence of a particular gene of interest.
 It is useful for prokaryotes with small genomes to identify a clone encoding a specific
gene of interest.
 It helps researchers to explore more about an organism’s genomic structure and
function. It is also used to study genetic mutations.
 Pharmaceutically important genes can also be identified by this method.
Disadvantages of a Genomic Library

 It requires sophisticated software and a vast amount of computing power. Also, the
process is prone to errors.
 Eukaryotic genome libraries with very large genomes contain many DNA that do not
code for proteins, as well as non-coding DNA like repetitive DNA and regulatory
regions, making them less than ideal.

Construction of a Genomic DNA Library

The Genomic Library or gene bank is constructed by a shotgun experiment where the entire
genome of the cell is cloned in the form of random and unidentifiable clones. It uses the chain
termination method (Sanger’s sequencing) to sequence the DNA molecules. The DNA clones
are produced by the following steps:

 Isolating the DNA fragments that are to be cloned and joining them with suitable
vectors like the lambda phage.
 Now, it is introduced into the host cell at high efficiency to get a large number of
independent clones.
 Finally, the desired clones are selected and used for the construction of the genomic
library.
Steps to Construct a Genomic DNA Library

1. First, the purification of the desired eukaryotic cell nuclei is done and this is
accomplished through protease digestion and organic extraction.
2. The derived genomic DNA is too large to be incorporated into a vector and must be
fragmented into desired sizes. Both physical and enzymatic methods can be used to
fragment DNA.
3. There are several vectors available for cloning large DNA fragments. Phage, bacterial
artificial chromosome, yeast artificial chromosome, and other such vectors are
suitable for larger DNA. The λ replacement vectors are the most preferred ones for
the construction of a genomic DNA library.
 4. Usually, the T4 DNA ligase enzyme is used to ligate the chosen DNA sequence into
the vector.
5. The recombinant vectors and the insert combinations are grown in a bacterial host cell
(E. coli). They replicate their genome along with the vector genome contained within
them.
6. The collection of clones that contain all the sequences from the original source
(including the sequence of interest) forms the gene bank or genomic library.

Screening of Clone
Each transformed bacterial host cell in a library will have only one vector with one DNA
insert. The entire library can be plated over media on a filter. The filter, as well as colonies,
are then hybridised, labelled with a probe and identified using detection methods such as
autoradiography. Other techniques like PCR and the blue-white selection method can also be
used to screen the clone.
Cloning of insulin gene

Cloning of Human insulin in a bacterial host

Gene segments corresponding to the mature A & B chains of insulin are engineered into
separate bacterial plasmids with an antibiotic resistance gene and a B-gal structural gene
as markers. The plasmids are separately transformed into E. coli. Culture of the E. coli in
the presence of antibiotics selects those cells that have been successfully
transformed. Induction of the B-gal gene expression (by addition of B-galactose sugar) also
causes transcription of the insulin genes, which are then translated by the bacterial cells. The
bacterial culture is done on an industrial scale to produce thousands of litres of cloned human
insulin.

Following chemical cleavage and purification away from the plasmid protein,
the A & B chains spontaneously assemble to form biologically-active insulin. As this
process in vitro is relatively inefficient, commercial human insulin is now made by cloning
the proinsulin mRNA (without the signal peptide of pre-proinsulin) in a single host, and
specific cleavage with the proteolytic enzymes.

Transgenic Animals
Trangenic animals are the animals with the modified genome. A foreign gene is inserted into
the genome of the animal to alter its DNA. This method is done to improve the genetic traits
of the target animal.
Initially, the improvement of genetic traits was done by selective breeding methods. In this,
the animals with desired genetic characteristics were mated to produce an individual with
improved genetic characteristics. Since this technique was time-consuming and expensive, it
was later replaced by recombinant DNA technology.
Transgenesis is the phenomenon in which a foreign gene with desired characteristics is
introduced into the genome of the target animal. The foreign gene that is introduced is known
as the transgene, and the animal whose genome is altered is known as transgenic. These
genes are passed on to the successive generations.
The transgenic animals are genetically engineered and are also known as genetically modified
organisms. The first genetically modified organism was engineered in the year 1980.

ethods for Creating Transgenic Animals

The transgenic animals are created by the following methods:

Physical Transfection
In this method, the gene of interest is directly injected into the pronucleus of a fertilized
ovum. It is the very first method that proved to be effective in mammals. This method was
applicable to a wide variety of species. Other methods of physical transfection include
particle bombardment, ultrasound and electroporation.

Chemical Transfection
One of the chemical methods of gene transfection includes transformation. In this method,
the target DNA is taken up in the presence of calcium phosphate. The DNA and calcium
phosphate co-precipitates, which facilitates DNA uptake. The mammalian cells possess the
ability to take up foreign DNA from the culture medium.
Retrovirus-Mediated Gene Transfer
To increase the chances of expression, the gene is transferred by means of a vector. Since
retroviruses have the ability to infect the host cell, they are used as vectors to transfect the
gene of interest into the target genome.

Viral Vectors
Viruses are used to transfect rDNA into the animal cell. The viruses possess the ability to
infect the host cell, express well and replicate efficiently.

Bactofection
It is the process by which the gene of interest is transferred into the target gene with the help
of bacteria

Examples of Transgenic Animals

Following are the examples of transgenic animals:

Dolly Sheep
Dolly the sheep was the first mammal to be cloned from an adult cell. In this, the udder cells
from a 6-year-old Finn Dorset white sheep were injected into an unfertilized egg from a
Scottish Blackface ewe, which had its nucleus removed. The cell was made to fuse by
electrical pulses. After the fusion of the nucleus of the cell with the egg, the resultant embryo
was cultured for six to seven days. It was then implanted into another Scottish Blackface ewe
which gave birth to the transgenic sheep, Dolly.

Transgenic Mice
Transgenic mice are developed by injecting DNA into the oocytes or 1-2 celled embryos
taken from female mice. After injecting the DNA, the embryo is implanted into the uterus of
receptive females.

Applications Of Transgenic Animals

The transgenic animals are created because of the benefits they provide to the man. Let us
discuss a few of them here.

Normal Physiology and Development

In transgenic animals, a foreign gene is introduced due to which the growth factor is altered.
Hence, these animals facilitate the study of gene regulation and their effect on the everyday
functions of the body.

Study of Diseases
Transgenic animals are specially designed to study the role of genes in the development of
certain diseases. Moreover, in order to devise a cure for these diseases, the transgenic animals
are used as model organisms. These transgenic models are used in research for the
development of medicines. For example, we have transgenic models for diseases such as
Alzheimer’s and cancer.

Biological Products
A number of biological products such as medicines and nutritional supplements are obtained
from transgenic animals. Research for the manufacture of medicines to treat diseases such as
phenylketonuria (PKU) and hereditary emphysema is going on. The first transgenic cow,
Rosie (1997), produced milk containing human protein (2.4 grams per litre). This milk
contains the human gene alpha-lactalbumin and could be given to babies as an alternative to
natural cow milk.

Vaccine Safety
Transgenic animals are used as model organisms for testing the safety of vaccines before they
are injected into humans. This was conventionally done on monkeys
DNA FINGERPRINTING

DNA (deoxyribonucleic acid) represents the blueprint of the human genetic makeup. It exists
in virtually every cell of the human body and differs in its sequence of nucleotides. The
human genome is made up of 3 billion nucleotides, which are 99.9% identical from one
person to the next. The 0.1% variation, therefore, can be used to distinguish one individual
from another. It is this difference that can be used by forensic scientists to match specimens
of blood, tissue, or hair follicles to an individual with a high level of certainty. DNA carries
exons and introns in the form of specific nucleotide sequences. The introns exist in between
the exons. The sequences of exons code for specific proteins during the protein synthesis
process. The nucleotide sequences of introns do not code for any protein or RNA. DNA
fingerprinting usually involves introns because exons are much more conserved and
therefore, have less variability in their sequence. These non-coding sequences have stretches
of repetitive nucleotide sequences inside the introns. They constitute the major portion of
humans DNA. These sequences show high level of polymorphism in all kind of tissues, as a
result of which they prove to be very useful in forensic studies. They are the basis of DNA
fingerprinting too.

Complete DNA of each individual is unique, with the exception of identical twins. A DNA
fingerprint, therefore, is a DNA pattern that has a unique sequence such that it can be
distinguished from the DNA patterns of other individuals. DNA fingerprinting is also called
DNA typing or DNA profile

DNA can be extracted from two different sources within the cell. DNA found in the nucleus
of the cell, also called nuclear DNA (nDNA) is larger and contains all the information that
makes us who we are. It is tightly wound into structures called chromosomes. DNA can also
be found in an organelle within the cell called the mitochondria, which functions to produce
energy that drives all the cellular processes necessary for life. Mitochondrial DNA (mtDNA)
is much smaller, contains only 16,569 nucleotide bases (compared with nDNA, which
contains 3.9 billion nucleotide bases) and it is not wound up into chromosomes. Instead, it is
circular and there are many copies of it.
DNA fingerprinting procedure

The original DNA fingerprinting procedure uses Variable Number Tandem Repeats (VNTR)
/ Tandem Repeats, which are repetitive DNA sequences that are spread throughout the
genome in non-coding regions (introns). The pattern, length and number of these repeats are
unique for each individual. The Tandem Repeats may have a repeat-size composed of
hundreds of nucleotides, which can be repeated a hundred times.

Differences in the length of Tandem Repeats could be as a result of unequal crossing over
during meiosis. DNA fingerprinting technique is based on the DNA polymorphism (i.e.
differences in the length of the DNA sequences between individuals of same species such as
humans).

DNA samples from the crime scene and the suspects DNA can be collected followed by
amplifying the DNA using PCR technique. The PCR technique may create multiple copies of
the DNA of interest. After creating multiple copies of DNA, it can be subjected to DNA
digestion using the desired restriction enzymes.

Due to the dissimilarity of DNA repeats among individual, each individual will generate
different sizes of the fragments after the restriction digestion.

Six steps of DNA fingerprinting

1. Extracting the DNA from cells 4. Transferring the DNA onto paper

2. Cutting up the DNA using an enzyme 5. Adding the radioactive probe

3. Separating the DNA fragments on a gel 6. Setting up the X-ray film

 Biopharming
It involves using transgenic plants or animals to produce (or ‘farm’) pharmaceutical products
for therapeutic use
 This involves the insertion of target genes into hosts (crops or animals) that would not
normally express those genes
 The desired compound can potentially be expressed in a form that is routinely harvested (e.g.
milk, eggs, fruits, etc.)

There are several established examples of biopharming, including:

 Transgenic sheep that produce human α-1-antitrypsin in their milk (individuals deficient in
this enzyme develop emphysema)
 Crops that express attenuated antigenic fragments for specific pathogenic diseases (i.e. edible
vaccines)

Antithrombin is a blood protein which inactivates certain enzymes in the coagulation system
to prevent excessive clotting
 Individuals with a genetic or acquired antithrombin deficiency are at an increased risk of
developing damaging blood clots

Genetically engineered antithrombin has been biopharmed from transgenic goats to produce
stocks for commercial use
 A human gene encoding for antithrombin is introduced into the fertilised egg of a goat
 The modified eggs are implanted into the uterus of surrogates, which give birth to transgenic
offspring
 The transgenic offspring will produce human antithrombin in its milk, which can then be
extracted and purified
 Each year, the transgenic goat can produce a quantity of antithrombin equivalent to 90,000
human blood donations

Biopharming of Antithrombin

Nuclear Transplantation
Nuclear transplantation is a method in which the nucleus of a donor cell is relocated
to a target cell that has had its nucleus removed (enucleated). Nuclear transplantation
has allowed experimental embryologists to manipulate the development of an
organism and to study the potential of the nucleus to direct development. Nuclear
transplantation, as it was first called, was later referred to as somatic nuclear transfer
or cloning.

nuclear transfer, the introduction of the nucleus from a cell into an enucleated egg cell (an
egg cell that has had its own nucleus removed). This can be accomplished through fusion of
the cell to the egg or through the direct removal of the nucleus from the cell and the
subsequent transplantation of that nucleus into the enucleated egg cell. The donor nucleus
used for nuclear transfer may come from an undifferentiated embryonic cell or from
a differentiated adult cell (somatic cell); in the latter case, the technique is called somatic cell
nuclear transfer (SCNT).
Xenotransplantation
Xenotransplantation is any procedure that involves the transplantation, implantation or
infusion into a human recipient of either (a) live cells, tissues, or organs from a nonhuman
animal source, or (b) human body fluids, cells, tissues or organs that have had ex vivo contact
with live nonhuman animal cells, tissues or organs.

 One of the biggest obstacles to transplantation is organ rejection. In the above case,
scientists have addressed the problem by genetically altering pigs’ organs. The donor
pig underwent 10 genetic modifications, by which the genes responsible for the rapid
rejection of foreign organs by the human body were inactivated or knocked out.

History of Xenotransplantation
Xenotransplantation was first tried in humans in the 1980s. American Baby Fae who was
born with a congenital heart defect had received a baboon heart in 1984. The surgery was
successful, but the baby died within a month of the transplant after it was rejected by her
body’s immune system.

 Notably, pig heart valves have been used for replacing damaged valves in humans for
over 50 years now.
 Also some patients with diabetes have received porcine pancreas cells.
Why pigs are most used for xenotransplantation?

 Pigs are increasingly becoming popular candidates for organ transplantation.

  Pigs offer advantages over primates for organ procurements, because they are easier
to raise and achieve adult human size in six months. Also pigs have large litters.
Hence pigs could provide an unlimited supply of organs, tissues, and cells.
 Pig organs have similarities to human organs in respect of anatomy and physiology.
For instance, physiologically, cardiac output and stroke volume, which are major
indicators of cardiac function, have been reported to be comparable in pigs and
humans.
 Also porcine components are more tuned for genetic engineering. From a scientific
viewpoint, pigs are genetically modifiable to reduce the chances of rejection by the
human body.
 Pigs are produced for food, so using them for organs raises fewer ethical concerns.

Xenotransplantation Significance
According to the World Health Organization, more than 114,000 organ transplants are carried
out annually in the world, but they fulfil only less than 10% of global needs.
Xenotransplantation, if found compatible in the long run, could help provide an alternative
supply of organs to those with life-threatening diseases. This development would help solve
the global organ shortage.

 The ample availability of organs will help overcome issues of organ trafficking and
ethical issues concerning the practice of commercial transactions between a potential
recipient and a paid living donor.
 Recent evidence has suggested that transplantation of cells and tissues may be
therapeutic for certain diseases such as neurodegenerative disorders and diabetes,
where, again human materials are not usually available. Certain procedures, some of
which are being investigated in early clinical trials, aim to use cells or tissues from
other species to treat life-threatening and debilitating illnesses such as cancer,
diabetes, liver failure and Parkinson’s disease.

Xenotransplantation Issues

 Xenotransplantation raises several medical challenges.

 Animal to human transplantation brings with it huge risks for the patient in the form
of immunological rejection. This can result in the immediate death of the recipient in
some cases. Even well-matched human donor organs can be rejected after they are
transplanted – and with animal organs, the danger is likely to be higher.
 Pigs have a shorter lifespan than humans, meaning that their tissues age at a quicker
rate. Hence, there is a question of whether the organ will function in the long term or
not.
 Disease transmission (zoonosis) remains a major concern in terms of public health.
The use of xenotransplantation raises concerns regarding the potential infection of
recipients with both recognized and unrecognized infectious agents and the possible
subsequent transmission to their close contacts and into the general human population.
 Apart from the medical challenge, xenotransplantation raises many ethical issues as
well.
  Similar to objections to animal testing, animal rights activists have also objected to
xenotransplantation on ethical grounds. Animal rights groups strongly oppose killing
animals to harvest their organs for human use.
 Permanent alteration to the genetic code of animals are also causes for concern.
Activists say it is wrong to modify the genes of animals to make them more like
humans.

Mapping and Sequencing the Human Genome

A primary goal of the Human Genome Project is to make a series of descriptive diagrams
mapsof each human chromosome at increasingly finer resolutions. Mapping involves (1)
dividing the chromosomes into smaller fragments that can be propagated and characterized
and (2) ordering (mapping) them to correspond to their respective locations on the
chromosomes. After mapping is completed, the next step is to determine the base sequence of
each of the ordered DNA fragments. The ultimate goal of genome research is to find all the
genes in the DNA sequence and to develop tools for using this information in the study of
human biology and medicine. Improving the instrumentation and techniques required for
mapping and sequencinga major focus of the genome projectwill increase efficiency and cost-
effectiveness. Goals include automating methods and optimizing techniques to extract the
maximum useful information from maps and sequences.

A genome map describes the order of genes or other markers and the spacing between them
on each chromosome. Human genome maps are constructed on several different scales or
levels of resolution. At the coarsest resolution are genetic linkage maps, which depict the
relative chromosomal locations of DNA markers (genes and other identifiable DNA
sequences) by their patterns of inheritance. Physical maps describe the chemical
characteristics of the DNA molecule itself.

Geneticists have already charted the approximate positions of over 2300 genes, and a start
has been made in establishing high-resolution maps of the genome

Mapping Strategies

Genetic Linkage Maps

A genetic linkage map shows the relative locations of specific DNA markers along the
chromosome. Any inherited physical or molecular characteristic that differs among
individuals and is easily detectable in the laboratory is a potential genetic marker. Markers
can be expressed DNA regions (genes) or DNA segments that have no known coding
function but whose inheritance pattern can be followed. DNA sequence differences are
especially useful markers because they are plentiful and easy to characterize

On the genetic map, distances between markers are measured in terms of centimorgans (cM),
named after the American geneticist Thomas Hunt Morgan. Two markers are said to be 1_cM
apart if they are separated by recombination 1% of the time. A genetic distance of 1_cM is
roughly equal to a physical distance of 1 million bp (1 Mb). The current resolution of most
human genetic map regions is about 10 Mb.
 Physical Maps

Different types of physical maps vary in their degree of resolution. The lowest-resolution
physical map is the chromosomal (sometimes called cytogenetic) map, which is based on the
distinctive banding patterns observed by light microscopy of stained chromosomes. A cDNA
map shows the locations of expressed DNA regions (exons) on the chromosomal map. The
more detailed cosmid contig map depicts the order of overlapping DNA fragments spanning
the genome. A macrorestriction map describes the order and distance between enzyme
cutting (cleavage) sites. The highest- resolution physical map is the complete elucidation of
the DNA base-pair sequence of each chromosome in the human genome. Physical maps are
described in greater detail below.

Ethical, Legal, and Social Issues in Biotech (ELSI)

Ethical issues in biotechnology are specific issues that are morally wrong. Such issues can
affect the fundamental moral principle that creates a massive conflict with the normal
working of the society. We have divided the ethical issue related to biotechnology into
specific subcategories. These groups are-

 Socio-economic issues
 Cultural issues
 Legal issues
 Environmental issues
 Religious issues

There are several principles relating to ethics, medical ethics, scientific and technological
ethics, justice and human dignity and its related ethics, and more of such ethical issues in
biotechnology.

 Socio-economic issues

The scientific community and society promise significant advancement in biotechnology but
never yours the harmlessness regarding socio-economic life. Such arguments and interactions
are some of the most problematic ones to be considered out there. Social-economic issues
interfere with the natural way or the initial god’s process of learning the environment. Search
issues could be related to the cultural backgrounds, and also it could be on the level of
different public awareness. The development of technology destroys the environment, which
is a tricky thing.

 Cultural issues
 Different cultures have different thoughts and values. These values are the reflection of their
long-lasting concept. Sometimes biotechnology hinders these cultural concepts, which is not
something to look out for. Biotechnology has given us everything to improve the quality of
life. Still, if one interferes with the cultural aspect of the human, there can be ethical issues
arising from such matters.

 Environmental issues

Environmental issues are also severe to look out for regarding ethical issues in biotechnology.
Ecological issues might arise when one destroys the living flora or the fauna. Gene
manipulation of different crops to produce hybrids is one of the most problematic issues.
Even for most biotech experiments, other organisms are made to sacrifice. Due to such harsh
sacrifices, numerous plant and animal species have been mutated regularly. Crops such as
sugarcane have gone through genetic mutation and recombination, which has continuously
destroyed the original constituent of the crop. Due to the prolonged mutation, these crops get
mutated for a generation. The genes pass from one progeny to another, degrading the quality
of the organism.

 Religious issues

One of the most faced issues with biotechnology is that it hurts people’s sentiments on a
religious basis. The cow is considered to be very sacred by Hindus. People pray for the
animals in countries such as India. Many plants are considered very sacred. Religious issues
can be exceptionally sentimental and personal, and people get free attached to such matters.
Some holy plants and animals are blessed and very sacred to lots of communities. These
communities protect the organisms at any cause. Killing these organisms is a matter of deep
concern.

 Other legal issues

Special laws protect many plants and animals. These laws protect these animals and plants
from being disturbed to overcome. Many sacred plants and animals are manipulated for other
experimental uses from their natural home. Sometimes due to the limitation of the organism
reacting to biotechnology, the choice becomes very harsh. In such a case, these organisms are
used, but this can create a severe law issue if the sacrifice for the mutation goes wrong.
Different governments have different restrictions and rules regarding these species and their
uses. Hence, a scientist must be aware that it must be legal to operate according to the
country’s laws before using specific species for Biotechnology.
Intellectual Property Rights

 Intellectual property rights (IPR) are the rights given to persons over the creations of
their minds: inventions, literary and artistic works, and symbols, names and images used
in commerce. They usually give the creator an exclusive right over the use of his/her
creation for a certain period of time.
 These rights are outlined in Article 27 of the Universal Declaration of Human
Rights, which provides for the right to benefit from the protection of moral and material
interests resulting from authorship of scientific, literary or artistic productions.
 The importance of intellectual property was first recognized in the Paris Convention
for the Protection of Industrial Property (1883) and the Berne Convention for the
Protection of Literary and Artistic Works (1886). Both treaties are administered by
the World Intellectual Property Organization (WIPO).
Intellectual property rights are customarily divided into two main areas:

(i) Copyright and rights related to copyright:

 The rights of authors of literary and artistic works (such as books and other writings,
musical compositions, paintings, sculpture, computer programs and films) are protected
by copyright, for a minimum period of 50 years after the death of the author.
(ii) Industrial property: Industrial property can be divided into two main areas:

 Protection of distinctive signs, in particular trademarks and geographical

indications.

o Trademarks distinguish the goods or services of one undertaking from those of

other undertakings.
o Geographical Indications (GIs) identify a good as originating in a place where a
given characteristic of the good is essentially attributable to its geographical
origin.
o The protection of such distinctive signs aims to stimulate and ensure fair
competition and to protect consumers, by enabling them to make informed
choices between various goods and services.
o The protection may last indefinitely, provided the sign in question continues to be
distinctive.
 Industrial designs and trade secrets: Other types of industrial property are protected
primarily to stimulate innovation, design and the creation of technology. In this
category fall inventions (protected by patents), industrial designs and trade secrets.
What is the need of IPR?

The progress and well-being of humanity rest on its capacity to create and invent new works
in the areas of technology and culture.
 Encourages innovation: The legal protection of new creations encourages the
commitment of additional resources for further innovation.
 Economic growth: The promotion and protection of intellectual property spurs
economic growth, creates new jobs and industries, and enhances the quality and
enjoyment of life.
 Safeguard the rights of creators: IPR is required to safeguard creators and other
producers of their intellectual commodity, goods and services by granting them certain
time-limited rights to control the use made of the manufactured goods.
 It promotes innovation and creativity and ensures ease of doing business.
 It facilitates the transfer of technology in the form of foreign direct investment, joint
ventures and licensing.
India and IPR

 India is a member of the World Trade Organisation and committed to the Agreement
on Trade Related Aspects of Intellectual Property (TRIPS Agreement).
 India is also a member of World Intellectual Property Organization, a body responsible
for the promotion of the protection of intellectual property rights throughout the world.
 India is also a member of the following important WIPO-administered International
Treaties and Conventions relating to IPRs.

patent
A patent is an exclusive right granted for an invention, which is a product or a process that
provides, in general, a new way of doing something, or offers a new technical solution to a
problem. To get a patent, technical information about the invention must be disclosed to the
public in a patent application.

 The history of Patent law in India starts from 1911 when the Indian Patents and
Designs Act, 1911 was enacted.
 The Patents Act, 1970 is the legislation that till date governs patents in India. It first
came into force in 1972.
 The Office of the Controller General of Patents, Designs and Trade Marks or
CGPDTM is the body responsible for the Indian Patent Act.
 The Patent Office has its headquarters in Calcutta and has branches in New Delhi,
Chennai and Mumbai. The office of the CGPDTM is based in Mumbai. Nagpur hosts
the office of the Patent Information System and also the National Institute for
Intellectual Property Management.
 The Controller General supervises the Act’s administration and also offers advice to
the government on related matters.
 The Patents Act has been repeatedly amended in 1999, 2002, 2005, 2006 respectively.
These amendments were required to make the Patents Act TRIPS compliant. TRIPS
stands for Trade-Related Aspects of Intellectual Property Rights.
  The major amendment in the Patent Act was in 2005, when product patents were
extended to all fields of technology like food, drugs, chemicals and microorganisms.
The Rules under Patent Act were also amended in 2012, 2013, 2014.

Patent Law Amendment Act 2005

Salient features of the Patents (Amendment) Act 2005 related to product patents:

1. Extension of product patent protection to products in sectors of drugs, foods and

chemical.
2. Term for protection of product patent shall be for 20 years.
3. Introduction of a provision for enabling grant of compulsory license for export of
medicines to countries which have insufficient or no manufacturing capacity;
provided such importing country has either granted a compulsory license for import or
by notification or otherwise allowed importation of the patented pharmaceutical
products from India (in accordance with the Doha Declaration on TRIPS and Public
Health)
4. Section 3 (d) regarding patentability.

Effects of Patent Amendment Act 2005

1. Due to the new patent regime, increased prices of products was considered to be a
major hindrance during the time. However, the government has taken proactive
measures to ensure low prices for essential drugs, and has used compulsory licensing
as a tool to keep exorbitant prices under check.
2. The amendment intended to make Indian drug and pharmaceutical industries
competitive at par with multinational companies.
3. Despite initial reservations, Indian pharmaceutical companies manufacturing generic
drugs have flourished in the last decade.
4. Also, MNCs have opened Research and Development Centres in India.

Pharmaceutical & Biotech Patents

Pharmaceutical and Biotech patents are registered in India after undergoing a stringent
examination process.
In Section 3, which specifies inventions that are not patentable, under clause (d) where new
use of the existing substance, process, the machine results in a new product or at best one
new outcome, can be patented.
Also, the provisions of the patent law allow patenting of products in chemicals,
biotechnology, food processing, drugs and pharmaceuticals, not just the process.
Rights granted by a Patent

1. If the patent is for a process, then the patentee has the right to prevent others from
using the process, using the product directly obtained by the process, offering for sale,
selling or importing the product in India directly obtained by the process.
2. If the grant of the patent is for a product, then the patentee has a right to prevent
others from making, using, offering for sale, selling or importing the patented product
in India.
Term of Patent
The term of every patent in India is 20 years from the date of filing the patent application,
irrespective of whether it is filed with provisional or complete specification.
However, in case of applications filed under the Patent Cooperative Treaty (PCT), the term of
20 years begins from the international filing date.