Definition

• Biotechnology is the

integration of natural

science and organisms,

cells, parts thereof and

molecular analogues for

products and services.

Oldest form of biotechnology

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 fusion

Cell 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.

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

Isolating genomic

DNA

Fragmenting this DNA Screening

the fragments

Insertion of DNA in a vector

Introducing in Host

Culturing the cells

Transformation of host cell

Isolating genomic

DNA

Isolating

genomic DNA

from the donor.

Fragmenting this DNA

Fragmenting

this DNA using

molecular

scissors.

Insertion of DNA in a

vector

Screening the

fragments

Screening the

fragments for a

“desired gene”.

Inserting the

fragments with the

desired gene in a

„cloning vector‟.

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.

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

Bacteriophage DNA

Artificial DNA

Cosmid

Plasmid

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)

HOST DONORDNA

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:

Retrotransposons

• Follows method of “Copy and Paste”.

• Copy in two stages.

DNA DNARNA

Reverse 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.

Genetic material can be

ssRNA, dsRNA, ssDNA,

dsDNA.

For Single genes- Plasmids are used

For Large pieces of DNA- Bacteriophages

48.5 kb in length.

Cos sites of 12 bp at the ends.

Cohesive ends allow circularizing DNA in host.

(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

EndonucleaseExonuclease

They remove

nucleotides

from the ends

of the DNA

They make cuts

at specific

positions within

the DNA

TYPES OF REN

REN

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.

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

dNTPsThermostable

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

deoxyribonucleotid

es 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 Agriculture

Ethical issues

Impact on human health

and environment

Genetically

modified

organisms

Genetic modification of organisms

can lead to Contamination 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.