Biotechnology

Introduction

Biotechnology is essentially the use of living organisms and their

products for health, social or economic purposes.

Biotechnology is widely considered to be the growth technology of the 21st century which will lead to huge growth in the Biotechnology industry and exciting opportunities for graduates

Application of Biotechnology

Its use and application ranges from fields like agriculture to industry (food, pharmaceutical, chemical, bioproducts, textiles etc.), medicine, nutrition, environmental conservation, animal sciences etc. making it one of the fastest growing fields.

The work is generally carried out in the laboratories, as it is a scientific research oriented field.

Application of Biotechnology

Applications of biotechnology are widespread, including the following:

diagnosis and treatment of human diseases. improved production of therapeutic agents. development of improved crop plant species. Development of improved farm animalsdevelopment of improved pest/pathogen

control processes

Application of Biotechnology

development of biosensors for environmental pollutants.

development of improved waste treatment processes and methods for remediation contaminated sites.

production of transgenic organisms for production of new drugs, improved transplantation success and improved animal and plant.

Selective BreedingHumans use selective

breeding, which takes advantage of naturally occurring genetic variation in plants, animals, and other organisms, to pass desired traits on to the next generation of organisms

Selective Breeding

Breed only those plants or animals with desirable traits

People have been using selective breeding for 1000’s of years with farm crops and domesticated animals.

Selective BreedingNearly all domestic

animals -- including horses, cats, and farm animals – and most crop plants have been produced by selective breeding No freaking way!

HybridizationLouis Burbank was the

greatest selective breeder of all time. He developed the disease-resistant potato and more than 800 varieties of plants.

Louis Burbank used the technique of hybridization and bred dissimilar individuals to combine the best traits of both parents.

The hybrids produced by these crosses were hardier than their parents

Inbreeding

To maintain the desired characteristics of a line of organisms, breeders often use the technique of inbreeding.

Inbreeding is the continued breeding of individuals with similar characteristics

Increasing VariationIn order for selective breeding to be

successful, there must be a lot of genetic variation in the population

Breeders increase the genetic variation in a population by inducing mutations, which are the ultimate source of genetic variability

Increasing VariationBreeders increase the mutation rate by

using radiation and chemicals

Molecular Biology

Molecular Biology

Molecular biology refers to the field of study regarding the investigation and manipulation of biological structures, processes, and phenomena at the molecular level.

Involves several classical basic techniques such as restriction enzymes, gel electrophoresis, and PCR, as well as more complex methods such as DNA fingerprinting, DNA sequencing, and genetic engineering

Outline

Restriction-enzyme analysisGel electrophoresisThe polymerase chain reaction (PCR)DNA FingerprintingDNA sequencingBlotting techniquesRecombinant DNA

Restriction Enzyme Analysis

aka restriction endonucleasesRecognizes specific base sequences and

cleave the nucleic acidPALINDROMES

Two-fold rotational symmetry

Generates fragments of DNA

Restriction Enzyme and Gel Electrophoresis

DNA fragments produced by restriction enzymes can be separated by gel electrophoresisAgarose (>20 kb)PAGE (1 kb)

VisualizationAutoradiographyEthidium bromide

Gel Electrophoresis

A technique for separating DNA (or protein) molecules on the basis of size.

DNA plus restriction enzyme

Mixture of DNA fragments

Gel

Power source

Longer fragments

Shorter fragments

Gel Electrophoresis

Utilizes agarose or polyacrylamide gels for the separation of DNA

DNA plus restriction enzyme

Mixture of DNA fragments

Gel

Power source

Longer fragments

Shorter fragments

Electrophoresis

Electrophoresis

• Electrophoresis is used to map the structure of a DNA fragment

Electrophoresis Stained gel

result

Visualization may be achieved through UV dyes or radioactive agents

Polymerase Chain Reaction

A rapid and versatile in vitro method to amplify defined target DNA within a heterogeneous collection of DNA sequences (genomic DNA or cDNA)

PCR Requirements

Template (genomic DNA or cDNA population)Oligonucleotide primersDNA polymerase (Taq polymerase)dNTPThermal cycler

Cycles (25 – 30)

Denaturation95o CSeparate strands

DNA Synthesis70 – 75 oC (ideal temp for Taq polymerase)Thermus aquaticus (Taq)

Annealing50 – 70 oC (~5o C lower than Tm)

Utility of PCR in Medical Diagnostics

Detection of bacteria and viruses by specific primersHIV virus in people who have not mounted an

immune responseMycobacterium tuberculosis bacilli

Detection of certain cancer cellsras genes and leukemias caused by chromosomal

rearrangementMonitoring cancer chemotherapy

Utility of PCR in Forensics

DNA fingerprinting Restriction fragment length polymorphisms PCR-Based analysis

Can be used to determine biological parentage Can be used to settle assault and rape cases

DNA Fingerprinting: A tool for forensics and paternity cases DNA analysis can be used for catching criminals,

establishing parentage, finding how closely organisms are related and many other applications.

The pattern of bands in a gel electrophoresis is known as a DNA fingerprint or a ‘genetic fingerprint’ or ‘genetic profile’

If a DNA fingerprint found in a sample of blood or other tissue at the scene of a crime matches the genetic fingerprint of a suspect, this can be used as evidence

A DNA sample can be obtained from the suspect using blood, cheek epithelial cells taken from the mouth lining or even the cells clinging to the root of a hair

Steps:1. Get DNA

sample2. Amplify with

PCR3. Cut with

restriction enzyme

4. Run resulting fragments on gel electrophoresis

5. Analyze result

A sample with the shorter DNA fragments travels through the gel faster than a sample with the larger fragments

V S S1 S2 S3

V Victim

S Sample from crime scene

S1 Suspect 1

S2 Suspect 2

S3 Suspect 3

More than 20 fragmentsfrom Suspect 1 match thosetaken from the crime scene

DNA profiles

Starting position of sample

1 2 3 4

Genetic fingerprint of …

1 mother

2 child

3 possible father A

4 possible father B

There is a match between one ofthe child’s restriction fragmentsand one of the mother’s.

There is also a match between the child’s other fragment and one from possible father A.Neither of the child’s restriction

fragments match those of possiblefather B

Famous cases

In 2002 Elizabeth Hurley used DNA profiling to prove that Steve Bing was the father

of her child Damien

Famous Cases

Colin Pitchfork was the first criminal caught based on DNA fingerprinting evidence.

He was arrested in 1986 for the rape and murder of two girls and was sentenced in 1988.

Famous Cases

O.J. Simpson was cleared of a double murder charge in 1994 which relied heavily on DNA evidence.

This case highlighted lab difficulties.

Sequencing by Sanger Dideoxy Method

Controlled termination of replicationUses 2’,3’ dideoxy

analog of nucleotide

Sequencing by Sanger Dideoxy Method

Electrophoresis

Fluorescence Detection

Automated DNA Sequencing

Southern blotting

Identification of restriction fragment

Southern blotting

Identification of restriction fragment

Research

Molecular biology

Genetic Engineering

Outline

Changing the Living WorldSelective BreedingIncreasing Variation

Manipulating DNAThe Tools of Molecular BiologyUsing the DNA Sequence

Outline

Cell TransformationTransforming BacteriaTransforming Plant CellsTransforming Animal Cells

Applications of Genetic EngineeringTransgenic OrganismsCloning

Introduction

Through genetic engineering scientists can combine DNA from different sources and this process is called “Recombinant DNA technology”

The secrets of DNA structure and functions have led to gene cloning and genetic engineering, manipulating the DNA of an organism

Genetic Engineering

A set of techniques used to manipulate DNA in order to elicit a desired characteristic in the target organism

Recombinant DNA technology, or the creation of recombinant DNA, is a necessary component of genetic engineering

Cutting DNA & Making Recombinant DNA

How Restriction enzymes work:The Enzymes recognize specific sequences on

Human and Bacterial PlasmidsThe Enzymes cut the strands.The cut produces DNA fragments with short strands

on each end that are complementary to each other“Sticky Ends”

Both the human DNA and the Plasmid “Open Up” with the same sticky ends remainingThey Bind Together

Restriction enzyme cleaving

Recognition sequences

DNA sequence

Recognition sequences

DNA sequence

Restriction enzyme EcoRI cuts the DNA into fragments.

Sticky end

Vectors = carriers of DNA fragments

PlasmidsNaturally occurring circular, double-stranded

DNA that act as accessory chromosomes in bacteria; DNA fragments (15, 000 bp)

λ phageBacteriophage (virus) DNA; for larger

DNA fragments (23, 000 bp) Yeast and bacterial artificial chromosome

Laboratory-designed carriers for larger DNA fragments

Confirmation of a Cloned Gene

Southern Blot can be used to identify a specific gene:

1. Cut DNA from bacteria with restriction enzymes.

2. DNA fragments are separated by a gel soaked in a chemical solution.

Confirmation of a Cloned Gene

3. The DNA separated is then transferred to a membrane (blotted) and a probe solution is added.

Probes: radioactive RNA or single-stranded DNA pieces that are complementary to the gene of interest

4. Only DNA fragments complementary to the probe will form and bind bands

Producing Recombinant Bacteria1. Remove bacterial DNA

(plasmid).

2. Cut the Bacterial DNA with “restriction enzymes”.

3. Cut the DNA from another organism with “restriction enzymes”.

4. Combine the cut pieces of DNA together with another enzyme and insert them into bacteria.

5. Reproduce the recombinant bacteria.

6. The foreign genes will be expressed in the bacteria.

When a bacteria or other cell takes in a foreign piece of DNA such as a plasmid, the process is called transformation

If transformation is successful, the recombinant DNA is integrated into one of the chromosomes of the cell.

Creating HGH

Human Cell

Gene for human growth hormone

Recombinant DNA

Gene for human growth hormone

Sticky ends

DNA recombination

DNA insertionBacterial Cell

Plasmid

Bacterial chromosome

Bacterial cell for containing gene for human growth hormone

Some Benefits of Recombinant Bacteria

1. Bacteria can make human insulin or human growth hormone.

2. Bacteria can be engineered to “eat” oil spills.

Genetically Engineered Drugs and Vaccines

Today, many pharmaceutical companies around the world produce important proteins using genetic engineering.

Vaccine: a solution containing all or part of a harmless version of a pathogen; used to prevent viral diseases (don’t respond to drugs)

Many vaccines are made using genetic engineering

The DNA of plants and animals can also be altered.

PLANTS

1. disease-resistant and insect-resistant crops

2. Hardier fruit

3. 70-75% of food in supermarket is genetically modified.

Improving Crops

Genetic engineers can add favorable characteristics to a plant

Plants become resistant to insects (no longer need pesticides); resistant to weed killer (so crops won’t die, but weeds will); improved nutrition – rice + corn

Pest Resistance: Bt Corn

Plant Transformation

Recombinant plasmid

Gene to be transferred

Agrobacterium tumefaciens

Cellular DNA

Transformed bacteria introduce plasmids into plant cells

Plant cell colonies

Complete plant is generated from transformed cell

Inside plant cell, Agrobacterium inserts part of its DNA into host cell chromosome

How to Create a Genetically Modified Plant

1.Create recombinant bacteria with desired gene.

2. Allow the bacteria to “infect" the plant cells.

3. Desired gene is inserted into plant chromosomes.

Genetically modified animals are called transgenic animals.

TRANSGENIC ANIMALS

1. Mice – used to study human immune system

2. Chickens – more resistant to infections

3. Cows – increase milk supply and leaner meat

4. Goats, sheep and pigs –

produce human proteins in their milk

Human DNA in a Goat Cell

This goat contains a human gene that codes for a blood clotting agent. The blood clotting agent can be harvested in the goat’s milk.

.

Transgenic Goat

Transgenic Cows

Growth hormones are given to cows to produce more milk

Human genes are added to farm animals in order to have human proteins in their milkThe human proteins are extracted from

milk and sold to pharmacy companies.

Useful for complex proteins that can’t be made in bacteria

Transgenic animals

How it works: an intact nucleus from an embryonic cell (whose DNA has recombined with a human gene) is placed into an egg whose nucleus has been removed.

The “new” egg is then placed into the uterus of an animal.

How to Create a Transgenic Animal

Animal Cloning

A clone is a member of a population of genetically identical cells produced from a single cell

The goal of cloning is to mass produce a certain individual with desired characteristics

No need for breeding, and the desired characteristics can be preserved.

How it works: an intact nucleus from a cell is removed

Cloning Animals

The nucleus is fused with a egg cell (whose nucleus has been removed) taken from another adult

The fused cell begins to divide and the embryo is placed in the uterus of a foster mother.

The “new” egg is then develops normally.

CloningA donor cell is taken from a sheep’s udder.

Donor Nucleus

These two cells are fused using an electric shock.

Fused Cell

The fused cell begins dividing normally.

Embryo

The embryo is placed in the uterus of a foster mother.

Foster MotherThe embryo develops normally into a lamb—Dolly

Cloned Lamb

Egg Cell

An egg cell is taken from an adult female sheep.

The nucleus of the egg cell is removed.

PERPETUATE: A pet cloning company

A company founded in 1998 by Dr. Heather Bessoff and Ron Gillespie

“PERPETUATE's packaged service provides the pet owner with the following three biotechnologies performed on their pet's cell biopsy”Cell culturing,DNA preservation,Genotyping.