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.