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This presentation is all about biotechnology. It is about the basic aspects of Biotechnology and covers a lot of topics under biotechnology, recombinant DNA technology. This is specifically for the HSC students of Mumbai. I hope that it helps.
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Biotechnology: Process and Application
Definition
• Biotechnology is the integration of natural science and organisms, cells, parts thereof and molecular analogues for products and services.
Oldest form of biotechnology
Making breads and curds with the help of micro organisms.
Application of fermentation in production of
wine and other alcoholic
beverages is also a
biotechnological technique
But with time biotechnology gradually became more
sophisticated.
Biotechnology
DNA manipulation
Tissue culture
Protoplast fusionCell catalysis
Immobilized enzymes
Protein engineering
Biotechnology led to production of many products and provides many services for human welfare.
Dragon Fly
There were 180,000 people employed by U.S. biotechnology companies in 2006.
There are more than 400 biotech drug products and vaccines currently in clinical trials targeting more than 200 diseases, including various cancers, Alzheimer’s disease, heart disease, diabetes, multiple sclerosis, AIDS and arthritis.
The biotechnology industry has mushroomed since 1992, with U.S. health care biotech revenues from publicly traded companies rising from $8 billion in 1992 to $58.8 billion in 2006.
Some of the most famous biotechnology products
In-Vitro Fertilization
Also called as Test tube baby
Plant tissue culture
DNA vaccines
The recombinant DNA technique was first proposed by
Peter Lobann A. Dale Kaiser
The present day rDNA technology flourished after the work of
Salmonella typhimurium
E. coli
They successfully linked a gene coding for antibiotic resistance with a native plasmid of Salmonella typhimurium with the vector plasmid and then cloning it in E.coli.
Plasmid
Gene coding for antibiotic resistance
Vector
Cloning
• Technique of manipulating the genome of a cell or organism so as to change the phenotype desirably.
What is recombinant DNA ?
Seedless guava Calorie free sugar
Basic steps involved in process
Isolating genomic DNA
Basic steps involved in process
Isolating genomic
DNA
1.
Isolating genomic DNA from the donor.
Fragmenting this DNA2
.
Fragmenting this DNA using molecular scissors.
Basic steps involved in process
Insertion of DNA in a vector
3.
Screening the
fragments
4.
Screening the fragments for a “desired gene”.
Inserting the fragments with the desired gene in a ‘cloning vector’.
Basic steps involved in process
Introducing in Host
Culturing the cells
Transformation of host
cell
Introducing the recombinant vector into a competent host cell
Culturing these cells to obtain multiple copies or clones of desired DNA fragments
Using these copies to transform suitable host cells so as to express the desired gene.
5.
6.
7.
Example: Production of Insulin
Tools used in recombinant DNA technology
• Enzymes • Vectors
Tools used in recombinant DNA technology
• Enzymes Act as biological scissors. Most commonly used are:
Restriction endonuclease DNA ligase DNA polymerase Alkaline phosphatases
Tools used in recombinant DNA technology
• Vectors Low molecular weight DNA molecules. Transfer genetic material into another
cell. Capable of multiplying independently.
Vector
Vector
Insertion of vector in target cell is called
• Bacterial cells – Transformation• Eukaryotic cells – Transfection• Viruses - Transduction
Insertion of vector in target cell
Vectors used:• Bacteria- plasmids, cosmid,
lambda phage• Insects- baculoviruses• Plants- Ti plasmid• Yeast cells- YAC (yeast artificial
chromosome)
Process HOST DONOR
DNA
Fragmented by Restriction Endonuclease
DNA strands with sticky ends
Sticky ends base pair with complementary sticky ends
DNA ligase links them to form rDNA Cloned
In vivoIn vitro
Prokaryotic or eukaryotic cell, mammalian tissue culture cell
DNA
Polymerase chain reaction (PCR)
Some examples of therapeutic products made by recombinant DNA techniques
¶ Blood Proteins: Erythropoietin, Factors VII, VIII, IX; Tissue plasminogen activator; Urokinase.
¶ Human Hormones: Epidermal growth factor; Follicle stimulating hormone, Insulin.
¶ Immune Modulators: α Interferon, β Interferon; Colony stimulating hormone; Lysozyme; Tumor Necrosis factor.
¶ Vaccines: Cytomegalovirus; Hepatitis B; Measles; Rabies
Transposons • Transposons are sequences of
DNA that can move or transpose themselves to new positions within the genome of a single cell.
• Also called ‘Jumping genes’.
• 1st transposons were discovered by
Barbara McClintock
in Zea mays (maize)
Types of transposons
• According to their mechanism they are classified as:
Transposons
Retrotransposons DNA transposons
Retrotransposons
• Follows method of “Copy and Paste”.
• Copy in two stages.
DNA DNARNAReverse Transcription
Transcription
DNA transposons
• Follows the method of “Cut and Paste”.
• Do not involve RNA intermediate.
Enzyme Transposase
Cuts out transposon
Ligates in new position
Plasmid
• Plasmids are small, extra chromosomal, double stranded, circular forms of DNA that replicate autonomously.
• The term was introduced by in 1952.
Joshua Lederberg
Plasmid • Found in bacterial, yeast and
occasionally in plants and animal cells. • Transferable genetic elements or
‘Replicons’.• Size- 1 to 1000 kilo bp.• Related to metabolic activity.• Allows bacteria to reproduce under
unfavorable conditions.
Nomenclature Lower case P (p)First letters of researchers name or place
where it was discovered.Numerical numbers given by workers.
Plasmid
Plasmid
Eg. Plasmid pBR 322
BR is for Bolivar and Rodriguez, who designated it as 322
Plasmid Eg. Plasmid pUC 19
UC stands for University of California
Plasmid- Cosmids
• Cosmids are plasmids with cos sequence.• They are able to accommodate long DNA
fragments that plasmids can’t.
A bacteriophage is a virus that infects bacteria. Virulent portion is deleted.
Bacteriphages
Genetic material can be ssRNA, dsRNA, ssDNA, dsDNA.
For Single genes- Plasmids are used
For Large pieces of DNA- Bacteriophages
Vectors used
48.5 kb in length. Cos sites of 12 bp at the ends. Cohesive ends allow circularizing DNA in host.
Phage Lambda () as vector
Lytic Cycle (replication of bacteriophges)(1) Phage attaches to a specific host
bacterium.(2) Injects its DNA, (3) Disrupting the bacterial genome
and killing the bacterium, and (4) Taking over the bacterial DNA and
protein synthesis machinery to make phage parts.
(5) The process culminates with the assembly of new phage, and
(6) The lysis of the bacterial cell wall to release a hundred new copies of the input phage into the environment.
RESTRICTION FRAGMENTS
A restriction fragment is a DNA fragment resulting from the cutting of a DNA strand by the restriction enzyme.
Process is called restriction.
RESTRICTION FRAGMENTS
Steward Linn along with Werner Arber in 1963 isolated two enzymes.
One of them is Restriction Endonuclease. Restriction Endonuclease can cut DNA. Restriction Endonuclease are basic
requirement for gene cloning or rDNA technology.
RESTRICTION FRAGMENTS
Nucleases
Endonuclease
Exonuclease
They remove nucleotides from the ends of the DNA
They make cuts at specific positions within the DNA
TYPES OF RENREN
Type I Type II Type III
Mostly used in rDNA technology.More than 350 types of type II endonucleases with recognition sites are known.Can be used to identify and cleave within specific DNA.
NOMENCLATURE OF REN
First letter- genus name of bacteria (in italics).
Next- first two letters of the species name (in italics).
Next- strain of the organism. Roman number- order of discovery.
Eg. - EcoR I
E- Escherichia, co- coli, R-strain Ry 13,
I- first endonuclease to be discovered. Eg.- Hind III
H- Haemophilus, in- influenzae, d- strain Rd,
III- third endonuclease to be discovered.
NOMENCLATURE OF REN
RECOGNITION SEQUENCE (RESTRICTION SITES) It is the site/ sequence where REN cuts the
DNA. Sequence of 4-8 nucleotides. Most restriction sites are Palindromes.
In DNA, palindrome is a sequence of base pairs that reads the same on the two strands when orientation of reading is kept same.
CLEAVAGE PATTERNS OF REN
REN recognizes the restriction site.
Cleave the DNA by hydrolyzing Phosphodiester bonds.
Isolate a particular gene.
Single stranded ends called sticky ends.
These sticky ends can form hydrogen bonded base pairs with complementary sticky ends or any other cleaved DNA.
CLEAVAGE PATTERNS OF REN
Restriction fragments yield a band pattern characteristic of the original DNA molecule & restriction enzyme used.
RESTRICTION FRAGMENTS CAN BE ANALYZED BY Gel
electrophoresis
Bands
PREPARING AND CLONING A DNA LIBRARY Collection of DNA fragments from a particular
species that is stored and propagated in a population of micro organisms through molecular cloning.
GENOMIC LIBRARY Collection of all clones of DNA fragments of
complete genome of an organism. All DNA fragments are cloned and stored as
location of desired gene is not known. Screening of DNA fragments can be done by
Complementation Or by using Probes.
Entire genome isolated
Cut into fragments by REN
Fragments inserted in Vector
Recombinant vectors are transferred into suitable organism
Transferred organisms are cultured and stored
Construction of Genomic Library.
CDNA LIBRARY cDNA is Complementary DNA. Produced using Teminism i.e. Reverse
Transcriptase. Constructed for eukaryotes.
cDNA is made from mRNAMature mRNA
StartAAAAAAA
Stop
TTTTTTT Add polyT primer, nucleotides, and Reverse Transcriptase
TTTTTTT AAAAAAA
TTTTTTT
DNA/RNA
RNA removed (by NaOH) and second strand synthesized
Complementary DNA cDNA
Gene Amplification (PCR)
It is obtaining multiple copies of a known DNA sequences that contain a gene.
Done artificially by using PCR (Polymerase Chain Reaction)
PCR (Polymerase Chain Reaction) Developed by in 1983.
In Vitro technique. Scientific technique to generate billions of
copies of a particular DNA sequence in a short time.
Kary Mullis
PCR Machine
Requirements for PCR technique
DNA segment
Primers
dNTPs
Thermostable DNA polymerase
PCR
A DNA segment 100-35,000 bp in length to be amplified.
Primers-forward and reverse, are synthetic oligonucleotides and complementary to the desired DNA segmentFour types of
deoxyribonucleotides i.e. dCTP, dGTP, dTTP, dATP
Enzyme that can withstand upto 94° C.
Steps of PCR technique
The double strand melts open to single stranded DNA, all enzymatic reactions stop (for example : the extension from a previous cycle).
Ionic bonds are constantly formed and broken between the single stranded primer and the single stranded template. Once there are a few bases built in, the ionic bond is so strong between the template and the primer, that it does not break anymore.
The bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template)
The exponential amplification of the gene in PCR.
Application of biotechnology in agriculture- Bt crops
Bacillus thuringiensis•Soil bacterium.•Produces a protein that
has insecticidal properties.
•Traditionally used as spray.
Mechanism of Bt
• Bt produces Bt toxins which are inactive protoxins.
• When an insect ingests it, inactive protoxin gets converted into active form due to alkaline pH of the insect’s gut.
• This led to swelling of gut and ultimately death of insect
Bt (in inactive form) sprayed on Crops
Eaten by insect
Toxin gets activated by alkaline pH of insect’s gut
Swelling of gut of insect
Death of insect
•Cry gene in Bt produces inactive protoxins.
Crop plants are now engineered to express Bt toxin.
Bt crops are now commercially available.For Eg.
Bt Rice Bt Cotton
Bt Tomato
Bt Brinjal Bt Soybean
Bt Potato
Bt Corn
Agrobacterium tumefaciens• Soil bacterium.• Causes crown gall tumors in
dicotyledonous plants.• T DNA (gall producing gene)
occurs in Ti plasmid.• Ti plasmid is used as vector for
higher plants.• Many genetically modified plants
are produced using A. tumifaciens.
Tumor
Mechanism
Ti Plasmid
•Desirable genes such as Cry gene an Nif gene is cloned inside A. tumifaciens and then transferred into another plant.
Nif Gene isolated from Rhizobium
Examples
1.Flavr savr tomato
Longer shelf life. Antisense DNA is introduced
that retards ripening
2.Golden Rice
Greater pro vitamin A content.
Genetically engineered.
Bio-Safety Issues
Biosafety issues
Impact on Agricultur
e
Ethical issues
Impact on human health
and environm
ent Genetically modified
organisms
Genetic modification of organisms can lead toContamination of gene pools.Consumption may lead to allergies.Hazardous microbes may escape
laboratory
Therefore manipulation of organisms needed regulation
Genetic Engineering Approval Committee
In India, GEAC takes decision regarding validity of GM research and introduction of GM products.
Biopiracy The patenting of plants, genes, and other
biological products that are indigenous to another country
Developed countries patent the knowledge and resources of underdeveloped countries and enjoy immense profits.
Biopatent
A patent is granted by the government to the inventor for biological entities, processes and products.
Case Study Texmati was derived by crossing Indian Basmati rice with a semi dwarf variety.A Texas based company got patent on rights of basmati.
Indian Basmati rice
Texmati rice
Some other Examples
Turmeric Neem Margosa
What can be done?
Genetic Literacy Movement in Schools and Colleges on rapid developments in Molecular Genetics
What will it do? Better understanding of opportunities
and risks of rDNA technology. Promote safe and responsible use of
tools of genetic engineering.