605T_Phamaceutical biotecnology_unit 2.pptx

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Assistant Professor (Pharmaceutics)
Pharmaceutical sciences
Rama University
GT Road Mandhana Kanpur
By Mr. Akhilesh Kumar

Study of cloning vectors (Gene cloning)
Introduction: A clone is an exact copy of an organism, organ, single cell, organelle or macromolecule.
• Cell lines for medical research are derived from single cells allowed to replicate millions of times, producing masses of identical clones.
• Gene cloning is the act of making copies of a single cell.
• Once a gene is identified, clones can be used in many biomedical and industrial research areas.
• Genetic engineering is the process of cloning genes into new organisms or altering a genetic sequence to change the protein product.
Principles of Gene Cloning
Principle of Gene cloning based on the given principles:
1. Polymerase chain reaction
2. Restriction enzymes
3. Visualizing DNA by agarose gel
4. Join two pieces of DNA
5. Selection of small self-replicating DNA
6. Method to move a vector into a Host cell
7. Methods to select transgenic organisms
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Mr. Akhilesh Kumar (Pharmaceutical Biotechnology_BP605T)

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Polymerase chain reaction: The discovery of thermostable DNA polymerases, such as Taq Polymerase. made it
possible to manipulate DNA replication in the laboratory and was essential to the development of PCR. Primers specific
to a particular region of DNA, on either side of the gene of interest, are used, and replication is stopped and started
repetitively, generating millions of copies of that gene. These copies can then be separated and purified using gel
electrophoresis.
Restriction Enzymes: The discovery of enzymes known as restriction endonucleases has been essential to protein
engineering. These enzymes cut DNA at specific locations based on the nucleotide sequence. Hundreds of different
restriction enzymes, capable of cutting DNA at a distinct site, have been isolated from many different strains of bacteria.
DNA cut with a restriction enzyme produces many smaller fragments, of varying sizes. These can be separated using gel
electrophoresis or chromatography.
Visualizing DNA by Agarose Gel: Purifying DNA from a cell culture, or cutting it using restriction enzymes wouldn't
be of much use if we couldn't visualize the DNA i.e., to find a way to view whether or not the extract contains anything,
or what size fragments have to be cut it into.
Join Two Pieces of DNA: In genetic research it is often necessary to link two or more individual strands of DNA, to
create a longer strand, or close a circular strand that has been cut with restriction enzymes. Enzymes called DNA ligases
can create covalent bonds between nucleotide chains. The enzymes DNA polymerase I and polynucleotide kinase are
also important in this process, for filling in gaps or phosphorylating the 5' ends, respectively.
Selection of Small Self-Replicating DNA: Small circular pieces of DNA that are not part of a bacterial genome, but are
capable of self-replication, are known as plasmids. Plasmids are often used as vectors to transport genes between
microorganisms. In biotechnology, once the gene of interest has been amplified and both the gene and plasmid are cut by
restriction enzymes, they are ligated together generating what is known as recombinant DNA. Viral (bacteriophage)
DNA can also be used as a vector, as can cosmids recombinant plasmids containing bacteriophage genes.
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Method to Move a Vector into a Host Cell: The process of transferring genetic material on (small circular pieces of
DNA) into new host cells is called transformation. This technique requires that the host cells are exposed to a heat-shock,
which makes them "competent" or permeable to the plasmid DNA of The larger the plasmid, the lower the efficiency with
which it is taken up by cells. Larger DNA segments are more easily cloned using bacteriophage, retrovirus or other viral
vectors or cosmids in a method called transduction. Phage or viral vectors are often used in regenerative medicine but
may cause insertion DNA in parts of our chromosomes where we don't want it, causing complications and even cancer.
Plasmid
Methods to Select Transgenic Organisms: Not all cells will take up DNA during transformation. It is essential that there
be a method of detecting the ones that do. Generally, plasmids carry genes for antibiotic resistance and transgenic cells
can be selected based on expression of those genes and their ability to grow on media containing that antibiotic.
Alternative methods of selection depend on the presence of other reporter proteins such as the x-gal/ lac Z system, or
green fluorescence protein, which allow selection based on colour and fluorescence, respectively.
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Features of Cloning Vectors
The cloning vectors possess the following features:
1) A cloning vector should possess an origin of replication so that it can self-replicate inside the host cell.
2) It should have a restriction site for the insertion of the target DNA.
3) It should have a selectable marker with an antibiotic resistance gene that facilitates screening of
the recombinant organism.
4) It should be small in size so that it can easily integrate into the host cell.
5) It should be capable of inserting a large segment of DNA.
6) It should possess multiple cloning sites.
7) It should be capable of working under the prokaryotic and eukaryotic systems.
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Types of Cloning Vectors
There are the following different types of cloning vectors:
• Plasmids
• Bacteriophage
• Phasmid
Plasmids
These are extrachromosomal double stander, circular, self-replicating DNA molecules. Almost all the bacteria
have plasmids containing two copy members (1-4 per cell) or a high copy number (10-100 per cell). The size of
the plasmid varies from 1 to 500 kb (kilobase)
• These were the first vectors used in gene cloning.
• These are found in bacteria, eukaryotes, and archaea.
• These are natural, extrachromosomal, self-replicating DNA molecules.
• They have a high copy number and possess antibiotic-resistant genes.
• They encode proteins which are necessary for their own replication.
• pBR322 (Plasmid Bolivar and Rodriguez) , pUC18, F-plasmid are some of the examples of plasmid vectors.
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Bacteriophage
The virus which can replicate within bacteria. Phase
vectors can accept short fragments of foreign DNA but
they can carry large segments than plasmids. Eg.
Bacteriophage λ – virus found in E. Coli, it can
carry 25kb.
• These are more efficient than plasmids for cloning large
DNA inserts.
• Phage λ and M13 phage are commonly used
bacteriophages in gene cloning.
• 53 kb DNA can be packaged in the bacteriophage.
• The screening of phage plaques is much easier than the
screening of recombinant bacterial colonies

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Phasmids
It passes both plasmid and bacteriophage λ, carry 40kb.
• These are artificial vectors.
• They are used in combination with M13 phage.
• They possess multiple cloning sites and an inducible lac gene promoter.
• They are identified by blue-white screening.
• Bacterial Artificial Chromosomes
• These are similar to E.coli plasmids vectors.
• It is obtained from naturally occurring F‘ plasmid.
• These are used to study genetic disorders.
• They can accommodate large DNA sequences without any risk.

Applications of Gene Cloning:
The cloned genes are utilized commercially in various fields such as pharmaceuticals, industry, agriculture, pollution
control, medical science, etc. Some of the examples have been discussed here
1. Production of Pharmaceuticals: The production of medically useful human peptides and proteins such as recombinant
human growth hormone (hGH), recombinant insulin, recombinant vaccines.
2. Diagnosis of Diseases
i) Use of DNA Probe in Diagnosis: This diagnosis system is very effective for viruses, bacteria andprotozoa.
Tuberculosis caused by Mycobacterium tuberculosis is diagnosed by this method.
ii) Use of PCR in Disease Diagnosis: The PCR can detect even a single organism that has infected the humans which
is present even in low number. From the suspected patients, sample of sera is taken and the region from DNA samples
is amplified. The PCR as diagnostic tool may be used in some diseases as given:
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3. Insect Pest Control: In controlling insects it can work in the following methods
i) Bacterial Biopesticides (Bioinsecticides): Microbial biopesticides have been produced by many companies by
using genetically engineered microbial cells of the preparation of B. thuringiensis.
ii) Production of Transgenic Plants: Transgenic plants are those plants in which a gene of foreign origin. i.e., other
organisms has been introduced. The leaves of transgenic plants, produced through cell culture, secrete lethal toxins.
When the bollworm eats upon cotton leaves, toxins are taken up by them. Toxin activates in their guts and results in the
death of bollworms. Toxin does not harm the spiders, humans, and other mammals.
iii) Viral Pesticides: A number of viruses has been discovered which belong to groups Baculoviruses and Cytoplasmic
Polypeptides Viruses (CPV). Use of viral preparations in disease control is done in the field of agriculture, horticulture
and forestry. Nuclear Polyhedrosis Viruses (NPV's) have been used for preparation of potential pesticides. Heliothis sp.
is a cosmopolitan insect that attacks 30 plant species. is controlled by application of NPV's of Baculovirus Heliothis.
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Restriction Endonucleases: The ability to join DNA
molecules together for cloning is dependent on the type of
enzymes occur in bacteria. These restriction enzymes are called
restriction endonucleases or molecular scissors. Restriction
endonucleases recognize specific sequences in the incoming
DNA and cleave the DNA into fragments. The existence of these
enzymes was first postulated by Werner Arber in the early 1960s
while studying bacterial viruses. He found that when virus DNA
entered in the bacteria, it was cut into small pieces. Arber also
proposed that restriction enzymes act at specific sites on the viral
DNA. Hamilton Smith and his colleagues (1970) isolated the
first restriction enzyme (Hind III) from Haemophiles influenza.
This enzyme recognizes a particular target sequence in a duplex
DNA molecule and breaks the polynucleotide chain.
The restriction enzymes name is designated by a three letter
abbreviation for the host organism (e.g. Escherichia coli - Eco).
A strain or type identified is written as subscript (e.g. Escherichia
coli strain K - Eco K). Roman numerals are used to indicate the
different restriction - modification systems in a strain, when
more than one enzyme is obtained from the same organism (e.g.
Haemophiles influenzae, serotype d, enzyme III - Hind III).
Some commonly used restriction enzymes are given in Table.
Arrows indicate in the table are the recognition sites.
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DNA ligase
DNA ligases seal the cut ends of two DNA molecules. Mertz
and Davis (1972) demonstrated that cohesive termini of
cleaved DNA molecules could be covalently sealed with
Escherichia coli DNA ligase and it produces recombinant DNA
molecules (see structure). These enzymes were originally
isolated from viruses. They also occur in E. coli and eukaryotic
cells. There are two types of DNA ligases: E. coli DNA ligase
and T, DNA ligase. DNA ligase catalyzes the formation of
phosphodiester bonds between 3'-OH and 5'-PO, group of a
nick and turns into an intact DNA. The T, DNA ligase enzyme
requires ATP as a co-factor while the E. coli DNA ligase
enzyme requires nicotinamide adenine dinucleotide (NAD) as
a co-factor for the joining reaction of the nick. The cofactor
splits and forms an enzyme adenosine monophosphate (AMP)
complex. The complex binds to the nick which must expose a
5’-PO4 and 3’-OH group and makes a covalent bond in the
phosphodiester chain. T4 DNA ligase enzyme has the ability to
join the blunt ends of DNA fragments while E. coli DNA
ligase joins the cohesive ends produced by restriction
enzymes. These enzymes actively participated in the cellular
DNA repair process.
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Recombinant DNA technology
Introduction
• The most important application of molecular genetics in
biotechnology is genetic engineering or recombinant (rDNA).
• Genetic engineering is the process of producing an organism that
contains a gene or genes not naturally present in that organism.
• This technique has tremendous potential for developing
microorganisms that can produce many useful products that are
difficult or impossible to produce by other methods.
• This is a useful tool for the production of vaccines and antigens.
Recombinant DNA technology has also been used for the
production of proteins of therapeutic interest such as insulin,
interferons, human growth, tissue plasminogen activator, tumor
necrosis factor, interleukin-2, fibroblast growth factor,
erythropoietin, and other biologicals.
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Application Recombinant DNA technology

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Applications of Genetic Engineering in medicine
Genetic engineering can be applied to:
• Manufacturing of drugs
• Creation of model animals that mimic human conditions and,
• Gene therapy
• Human growth hormones
• Follicle-stimulating hormones
• Human albumin
• Monoclonal antibodies
• Antihemophilic factors
• Vaccines

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Application in the pharmaceutical industry and medicine
• Through genetic engineering, a variety of medical products are available today. Among these products, insulin and
human growth hormone were t h e first commercially available products obtained from recombinant E. coli.
Recombinant insulin is the result of successful genetic engineering.
• The initial production of insulin involved the separate synthesis of the insulin A- and B-chains in two bacterial strains. Both
the insulin A and B chains genes were placed under the control of the lac promoter for inducible expression by lactose
inducer. After purification of the A- and B-chains from the bacteria, the chains were then linked chemically to produce the
final insulin. Recombinant insulin is now commercially available in several forms and is involved in diabetes therapy.

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Recombinant DNA Technology
Recombinant DNA, is molecules of DNA from two different species that are inserted into a host organism to produce new
genetic combinations that are of value to science, medicine, agriculture, and industry. Since the focus of all genetics is the
gene, the fundamental goal of laboratory geneticists is to isolate, characterize, and manipulate genes. Although it is
relatively easy to isolate a sample of DNA from a collection of cells, finding a specific gene within this DNA sample can
be compared to finding a needle in a haystack. Consider the fact that each human cell contains approximately 2 meters (6
feet) of DNA. Therefore, a small tissue sample will contain many kilometers of DNA. However, recombinant DNA
technology has made it possible to isolate one gene or any other segment of DNA, enabling researchers to determine its
nucleotide sequence, study its transcripts, mutate it in highly specific ways, and reinsert the modified sequence into a
living organism.
Isolation of Genetic Material
The first step in rDNA technology is to isolate the desired DNA in its pure form i.e. free from other macromolecules.
Since DNA exists within the cell membrane along with other macromolecules such as RNA, polysaccharides, proteins,
and lipids, it must be separated and purified which involves enzymes such as lysozymes, cellulase, chitinase,
ribonuclease, proteases etc. Other macromolecules are removable with other enzymes or treatments. Ultimately, the
addition of ethanol causes the DNA to precipitate out as fine threads. This is then spooled out to give purified DNA.

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Restriction Enzyme Digestion
Restriction enzymes act as molecular scissors that cut DNA at specific locations. These reactions are called ‗restriction
enzyme digestions‘. They involve the incubation of the purified DNA with the selected restriction enzyme, at conditions
optimal for that specific enzyme. The technique of Agarose Gel Electrophoresis‘ reveals the progress of the restriction
enzyme digestion. This technique involves running out the DNA on an agarose gel. On the application of current, the
negatively charged DNA travels to the positive electrode and is separated out based on size. This allows for separating and
cutting out the digested DNA fragments. The vector DNA is also processed using the same procedure.
Amplification Using PCR
Polymerase Chain Reaction or PCR is a method of making multiple copies of a DNA sequence using the enzyme – DNA
polymerase in-vitro. It helps to amplify a single copy or a few copies of DNA into thousands to millions of copies.
Ligation of DNA Molecules
The purified DNA and the vector of interest are cut with the same restriction enzyme. This gives us the cut fragment of
DNA and the cut vector, that is now open. The process of joining these two pieces together using the enzyme DNA ligase‘ is
ligation‘. The resulting DNA molecule is a hybrid of two DNA molecules – the interested molecule and the vector. In the
terminology of genetics, this intermixing of different DNA strands is called recombination. Hence, this new hybrid DNA
molecule is also called a recombinant DNA molecule and the technology is referred to as recombinant DNA technology.

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Insertion of Recombinant DNA into Host
In this step, the recombinant DNA is introduced into a recipient host cell mostly, a bacterial cell. This process is
Transformation‘. Bacterial cells do not accept foreign DNA easily. Therefore, they are treated to make them competent
to accept new DNA. The processes used may be thermal shock, Ca++ ion treatment, electroporation, etc.
Isolation of Recombinant Cells
The transformation process generates a mixed population of transformed and non-transformed host Cells. The selection
process involves filtering the transformed host cells only. For the isolation of recombinant cell from non-recombinant
cell, marker gene of plasmid vector is employed. For examples, the PBR322 plasmid vector contains different marker
gene (Ampicillin resistant gene and Tetracycline resistant gene. When pst1 RE is used it knock out Ampicillin resistant
gene from the plasmid, so that the recombinant cell become sensitive to Ampicillin.

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Application of r DNA technology and genetic engineering in the production of:
i) Interferon
ii) Vaccines- hepatitis- B
iii) Hormones-Insulin.
Interferons
• In the year 1957 Alec Issacs and Jean Linderman discovered a wonder molecules names as Interferons with an intention
that the molecule will interfere with viral replication without endangering cellular metabolism as well as it may serve as a
potential antiviral agent.
• Interferon is the molecule which interferes with viral replication without disturbing the cellular metabolism and it may
serve as an antiviral agent.
• The large quantity of interferons produced by gene cloning (e.g. Introducing rDNA of interferon gene into E.coli.)
• Interferon is available in the market with trade names intro A, Roferon, Wellferon, and Shanferon.
(IFNs, Interferons) are a group of signaling proteins made and released by host cells in response to the presence of several
viruses. In a typical scenario, a virus-infected cell will release interferons causing nearby cells to heighten their anti-viral
defenses. IFNs belong to the large class of proteins known as cytokines, molecules used for communication between cells to
trigger the protective defenses of the immune system that help eradicate pathogens Interferons are named for their ability to
"interfere“ with viral replication by protecting cells from virus infections. However, virus-encoded genetic elements have the
ability to antagonize the IFN response contributing to viral pathogenesis and viral diseases. IFNs also have various other
functions: they activate immune cells, such as natural killer cells and macrophages; they increase host defenses by up-
regulating antigen presentation by virtue of increasing the expression of major histocompatibility complex (MHC) antigens.
Certain symptoms of infections, such as fever, muscle pain and "flu-like symptoms", are also caused by the production of
IFNs and other cytokines.

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More than twenty distinct IFN genes and proteins have been identified in animals, including humans. They are typically
divided into three classes: Type I IFN, Type II IFN, and Type III IFN. IFNs belonging to all three classes are important for
fighting viral infections and for the regulation of the immune system.
Interferon type I: All type I IFNs bind to a specific cell surface receptor complex known as the IFN- α/β receptor (IFNAR)
that consists of IFNAR1 (Interferon alpha and beta receptor subunit 1) and IFNAR2 chains. The type I interferons present in
humans is IFN-α, IFN-β, IFN-ε, IFN-κ (inf- Kalium), and IFN-ω (interferon omega). In general, type I interferons are
produced when the body recognizes a virus that has invaded it. They are produced by fibroblasts and monocytes.
However, the production of type I IFN-α is inhibited by another cytokine known as Interleukin-10. Once released, type I
interferons bind to specific receptors on target cells, which leads to the expression of proteins that will prevent the virus from
producing and replicating its RNA and DNA. Overall, IFN-α can be used to treat hepatitis B and C infections, while IFN-β can
be used to treat multiple sclerosis.
Interferon type II (IFN-γ in humans): This is also known as immune interferon and is activated by Interleukin-12.Type II
interferons are also released by cytotoxic T cells and type-1 T helper cells. However, they block the proliferation of type-2 T
helper cells. The previous results in an inhibition of Th2 immune response and a further induction of Th1 immune response.
IFN type II binds to IFNGR (Interferon gamma receptor), which consists of IFNGR1 and IFNGR2 chains.
Interferon type III: Signal through a receptor complex consisting of IL10R2 (also called CRF2-4) and IFNLR1 (also called
CRF2-12). Although discovered more recently than type I and type II IFNs, recent information demonstrates the importance of
Type III IFNs in some types of virus or fungal infections.

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Hepatitis B
It is a vaccine-preventable liver infection caused by the hepatitis B virus (HBV). Hepatitis B is spread when blood, semen, or
other body fluids from a person infected with the virus enters the body of someone who is not infected.
Signs and symptoms of hepatitis B range from mild to severe. They usually appear about one to four months after you've been
infected, although you could see them as early as two weeks post-infection. Some people, usually young children, may not
have any symptoms.
Hepatitis B signs and symptoms may include:
1) Abdominal pain
2) Dark urine
3) Fever
4) Joint pain
5) Loss of appetite
6) Nausea and vomiting
7) Weakness and fatigue
8) Yellowing of your skin and the whites of your eyes (jaundice).

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Production of Recombivax HB
 HB antigen-producing gene is isolated from
the HB virus.
 A plasmid DNA is extracted from a bacterium
and is cut with restriction enzyme forming the
plasmid vector.
 The isolated HB antigen-producing gene is
inserted into the bacterial plasmid vector on
forming the recombinant DNA.
 This recombinant DNA, containing the target
gene, is introduced into a yeast cell forming
the recombinant yeast cell.
 The recombinant yeast cell multiplies in the
fermentation tank and produces the HB
antigens.
 The HB antigens are extracted, purified, and
bottled. It is ready for vaccination in humans.

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Causes
Hepatitis B infection is caused by the hepatitis B virus (HBV). The virus is passed from a person to person through blood,
semen or other body fluids. It does not spread by sneezing or coughing.
Common ways that HBV can spread are:
• Sexual contact: You may get hepatitis B if you have unprotected sex with someone who is infected. The virus can pass to
you if the person's blood, saliva, semen or vaginal secretions enter your body.
• Sharing of needles: HBV easily spreads through needles and syringes contaminated with infected blood. Sharing IV
drug paraphernalia puts you at high risk of hepatitis B.
• Accidental needle sticks: Hepatitis B is a concern for health care workers and anyone else who comes in contact with
human blood.
• Mother to child: Pregnant women infected with HBV can pass the virus to their babies during childbirth. However, the
newborn can be vaccinated to avoid getting infected in almost all cases. Talk to your doctor about being tested for
hepatitis B if you are pregnant or want to become pregnant.

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Insulin
Humulin is the branded product of the famous pharmaceutical manufacturer, Eli-Lilly (Pharmaceutical Company
marketed in 1923), containing human insulin and its host of variants, being produced by it in different countries
across the globe. Insulin was for the first time applied to a diabetic nine year old boy in 1922.
Humulin is synthesized in a special non-disease-producing laboratory strain of Escherichia coli (E. coli) bacteria
that has been genetically altered to produce human insulin. Humulin N (Human insulin (rDNA origin) isophane
suspension) is a crystalline suspension of human insulin with protamine and zinc providing an intermediate-acting
insulin with a slower onset of action and a longer duration of activity (up to 24 hours) than that of Regular human
insulin. The time course of action of any insulin may vary considerably in different individuals or at different times
in the same individual. As with all insulin preparations, the duration of action of Humulin N is dependent on dose,
site of injection, blood supply, temperature, and physical activity. Humulin N is a sterile suspension and is for
subcutaneous injection only. It should not be used intravenously or intramuscularly The concentration of Humulin
N is 100 units/mL (U-100).
Description of Insulin
Insulin is a pancreatic hormone essentially involved in the regulation of blood glucose concentrations and also
having a specific role in the protein and lipid metabolism.

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Human-Insulin Variants
Insulin is-a hormone produced by the B-cells of the islets of Langerhans of the pancreas and essentially comprises of two
separate chains of amino acids, the A and B chains, joined together by two disulfide bridges.
Insulin produced has an amino-acid sequence very much similar to that of the human insulin. In actual usage there are several
human-insulin variants, such as:
1. Human Insulin (emp): Produced by the enzymatic modification of insulin obtained from the porcine pancreas. It is also
known as semisynthetic human insulin.
2. Human Insulin (crb): Produced by the chemical combination of A and B chains that have been duly obtained from
bacteria genetically modified by recombinant DNA technology.
3. Human Insulin (prb): Produced by proinsulin obtained from bacteria genetically modified by recombinant
4. Human Insulin (pyr): Produced from a precursor obtained from yeast genetically modified by recombinant DNA
technology.
Types of Insulin
Porcine Insulin
Human Insulin
Bovine insulin

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Production of Insulin
In one of the approaches, the production of insulin involves the chemical synthesis of genes, i.e., chains A and B.
Both the chains were cloned separately and attached the genes for B-galactosidase. This results in the synthesis of
a fusion polypeptide, that is relatively stable in E.coli. Two bacterial strains were constructed, each producing a
fused protein with A and B chains. The genes synthesized were devoid of promoters. To express the cloned genes
into a functional protein, it is essential to clone a gene into a plasmid vector close to bacterial promoter. A
synthetic gene lacks a ribosomal binding site, and therefore, it is necessary to insert the gene downstream from a
promoter and ribosomal binding site of the vector.
During the construction of synthetic genes, a signal sequences are to be cloned with an extra 15-30 amino acids at
the N-terminus. These sequences will have a central core of hydrophobic amino acids flanked by polar or
hydrophilic residues. During passage through the membrane, the signal sequence is cleaved off. Synthetic genes
do not contain any methionine residue (initiation codon). So the construction of synthetic genes is carried with
incorporation of methionine residue at the junction of fusion peptide. Authentic insulin can be obtained by
cleaving the methionine residue by treatment with cyanogen bromide, purified, and the two chains were linked
chemically in vitro to produce insulin.
In further developments, gene for precursor molecule is constructed by the synthetic approach. The proinsulin
gene is cloned in such a way as to create a B-galactosidase proinsulin hybrid protein. The proinsulin is chemically
cleaved from the B-galactosidase with cyanogen bromide. Proteolytic digestion with trypsin in vitro. cleaves out
amino acids (c-chain) in the middle of the molecule to generate insulin.

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Production of Human insulin by rDNA technology

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POLYMERASE CHAIN REACTION (PCR)
Polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete copies or
partial copies) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of
it) to a large enough amount to study in detail. PCR was invented in 1983 by the American biochemist Kary Mullis at
Cetus Corporation. It is fundamental to many of the procedures used in genetic testing and research, including the analysis
of ancient samples of DNA and the identification of infectious agents. Using PCR, copies of very small amounts of DNA
sequences are exponentially amplified in a series of cycles of temperature changes. PCR is now a common and often
indispensable technique used in medical laboratory research for a broad variety of applications including biomedical
research and criminal forensics. The majority of PCR methods rely on thermal cycling. Thermal cycling exposes reactants to
repeated cycles of heating and cooling to permit different temperature-dependent reactions-specifically, DNA melting and
enzyme-driven DNA replication.
PCR employs two main reagents-primers (which are short single-strand DNA fragments known as oligonucleotides that are
a complementary sequence to the target DNA region) and a DNA polymerase. In the first step of PCR, the two strands of the
DNA double helix are physically separated at a high temperature in a process called nucleic acid denaturation. In the second
step, the temperature is lowered and the primers bind to the complementary sequences of DNA. The two DNA strands then
become templates for DNA polymerase to enzymatically assemble a new DNA strand from free nucleotides, the building
blocks of DNA. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain
reaction in which the original DNA template is exponentially amplified.
PCR amplifies a specific region of a DNA strand (the DNA target). Most PCR methods amplify DNA fragments of between
0.1 and 10 kilobase pairs (kbp) in length, although some techniques allow for the amplification of fragments up to 40 kbp.
The amount of amplified product is determined by the available substrates in the reaction, which becomes limiting as the
reaction progresses. A basic PCR setup requires several components and reagents, including:

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Introduction: The development of the polymerase chain reaction (PCR) or gene amplification method in 1983 was a major
breakthrough in molecular biology. This technique was developed by Kary Mullis at Cetus Corporation (a biotech
company) in Emery Ville, California. The details of PCR techniques are described by Erlich (1989) in his edited book 'PCR
Technology'. It is an in-vitro method for producing large amounts of specific DNA fragments of defined length and
sequence from a small amount of complex templates. PCR is now considered as a basic tool for the molecular biologist.
PCR Technique
PCR is an in-vitro method for producing large amounts of specific DNA fragments. In this technique, microgram quantities
of DNA from picogram produce amounts of starting material. Target DNA, primers, polymerase and nucleotides are
combined in a test tube for the multiplication of genetic material. DNA is amplified by a polymerase chain reaction in an
enzymatic reaction which undergoes multiple incubations at three different temperatures. Each PCR contains four important
components.
• DNA Template: Any source that contains one or more target DNA molecules to be making a DNA amplified can be
taken as a template. RNA can also be used for PCR by first copy using the enzyme reverse transcriptase.
• Primers: Each PCR requires a pair of oligonucleotide primers. These are short single-stranded DNA molecules obtained
by chemical synthesis. These primers are designed to anneal on opposite strands of the target sequence so that they will
be extended towards each other by the addition of nucleotides.
POLYMERASE CHAIN REACTION (PCR)

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DNA polymerase: The most commonly used enzyme in PCR is Taq DNA polymerase isolated from a thermostable bacterium
called Thermus aquaticus. It survives at 95°C for 1 to 2 minutes and has a half life for more than 2 hours at the same
temperature. The DNA polymerase binds to a single-stranded DNA and synthesizes a new strand complementary to the origin
strand. The role of this enzyme in PCR is to copy DNA molecules.
Deoxynucleotide triphosphates: PCR requires four deoxynucleotide triphosphates, dNTPs (dATP, dGTP, dTTP, dCTP)
which are used by the DNA polymerase as building blocks to synthesize new DNA. Polymerase chain reaction (PCR)
involves three stages which are as follows :
• 1. Melting of DNA (95°C) to convert double-stranded DNA to single-stranded DNA (denaturation).
• 2. Promoting the primers (at 50 - 65°C) to attach themselves to either end of the target strip (annealing of primers).
• 3. Extension of the primers by DNA polymerase to form new double-stranded DNA across the segment by sequential
addition of deoxynucleotides (primer extension). When the temperature is again raised the new strands separate and the
process begins again. Temperature profile of typical PCR cycle is shown in The oligonucleotide primers are designed to
hybridize the region of DNA flanking a desired target gene sequence. The primers are then extended across the target
sequence using DNA polymerase derived from Thermus aquatics (Taq) in the presence of free deoxynucleotide
triphosphate. These three steps constitute one cycle of the reaction. These steps are repeated by manipulating the
temperature, by using the PCR machine. A cycle takes about 3 to 5 minutes and after 30 cycles (about 3 hours), a single
copy of DNA can be multiplied into 1,000,000 copies.

8/2/2023 35

8/2/2023 36
TYPES OF PCR
RNA polymerase chain reaction is a modification of PCR technique that allows amplification beginning from an RNA template.
Recent research in the field of polymerase chain reaction has led to the development of the techniques contributing to the
effectiveness of the method. PCR is a highly versatile technique. Important variations of PCR are as follows:
• Inverse PCR: PCR can be used to the amplification of those DNA sequences which are away from the primer and not of
those which are flanked by the primer. The sequences to be amplified may be cloned in a vector and border sequence of the
vector may be used as a primer in such a way that the polymerization proceeds in the reverse direction.
• Anchored PCR: In this method, only one primer is used. One strand is copied first and then poly G tail is attached at the end
of the newly synthesized strand. This allows the use of complementary homopolymer, poly-C, to be used as primer for
copying the DNA single strands generated by PCR. It gives rise to the complete DNA duplex that can be amplified normally.
• RT-PCR: Reverse transcription-mediated PCR includes a single application combining the process of cDNA synthesis (by
reverse transcription) and PCR amplification. It can also be applied to double-stranded cDNA also which is synthesized from
mRNA using the enzyme reverse transcriptase. Thermostable enzyme rTth uses RNA templates from cDNA synthesis and
thus allows single enzyme RT-PCR viral reverse transcriptase from avian murine virus (AMV RTase).
• Asymmetric PCR: It is used to generate single-strand copies of a DNA sequence which can be directly used for DNA
sequencing. The two primers (100:1 ratio) are such adjusted in the reaction mixture that one of them is exhausted about 10 or
more cycles. After these cycles, only a single strand of DNA segment is copied and these copies are the ideal starting
materials for DNA sequencing. This variation is known as asymmetric PCR.
• AP-PCR: Arbitrary primed PCR (AP-PCR) is a type of random amplified polymorphic DNA(RAPD) where single
primers of 10 to 50 bases are used to amplify genomic DNA in PCR. Welsh and McClelland (1990) developed the arbitrarily
primed - PCR and carried out fingerprinting of genomes with arbitrary primers

8/2/2023 37
APPLICATIONS OF PCR
Polymerase chain reaction is useful in diverse areas of molecular biology, medicines and biotechnology.
 Diagnosis of pathogens: PCR is commonly used for the diagnosis of infections caused by viruses (e.g. HIV-1, HIV-2,
Herpes simplex virus, Hepatitis B virus etc.), bacteria (Mycobacterium tuberculosis, Helicobacter pylori, Mycoplasma
pneumonia etc), fungi (Candida albicans) and protozoa (Toxoplasma gondii, Trypanosoma cruzi etc).
 Diagnosis of plant pathogens: Various plant pathogens are detected by using PCR such as viruses (plum pox virus,
cauliflower mosaic virus), fungi (Verticillium spp., Laccaria spp., Phytophthora spp. etc), mycoplasms bacteria
(Agrobacterium tumefacient, Rhizobiumleguminosarum, Xanthomonas compestris etc.) and nematodes (Meloidogyne
incognita) etc.
 Inherited diseases: Inherited disorders are caused by gene mutations passed on from parents to their children e.g.
hemophilia, cystic fibrosis etc. PCR is used to amplify gene sequences which can then be screened for disease causing
mutations.
 Research: PCR is used extensively as a research tool for identification of new species. Many bioactive microbial species
are isolated from various extreme environment such as soil, water, air, sediments etc. DNA fingerprinting of new
microorganisms is carried out to confirm their identity by comparing with the DNA sequences of known microorganisms.
Cancer research: Polymerase chain reaction has been widely used in studies for the role of genes in cancer. Tumour-
suppressor genes and mutations in oncogenes have been identified in DNA from tumors using PCR-based strategies.
Biotechnology: PCR has played major role in the production of recombinant proteins. Insulin and growth hormones are
recombinant proteins, widely used as drugs and recombinant vaccines are developed for hepatitis B virus. It is an
important tool in the biotechnology industries of research institutes.

8/2/2023 38
 Forensic science: PCR is most applicable in forensic science where it is being used in search of criminals through DNA
fingerprinting technology. PCR allows amplification of DNA from individual hairs, stains of blood or seminal fluid having
partially degraded DNA. Analysis of variable sequences is also used in tissue typing to match organ donors with recipients.
 DNA polymorphism: PCR is used to study DNA polymorphism in the genome using known sequences as primers. PCR
can be used to study RFLPs (restriction fragment length polymorphisms) as well as RAPDs (random amplified
polymorphic DNA).
 Gene therapy: PCR proves to be an immense help in monitoring a gene in gene therapy experiments. This PCR
technology provides shortcuts for many cloning and sequencing applications.

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