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BIOLOGY
TOPIC 12: BIOTECHNOLOGY

Content Covered
DNA technology
Gene therapy
Tissue culture
Cloning

12.1 DESCRIBE RECOMBINANT DNA TECHNOLOGY AND ITS APPLICATION (E.G. INSULIN PRODUCTION)

12.1.1 Recombinant DNA Technology

Also known as genetic engineering

Synthesizes recombinant DNA by taking DNA from 2 sources

Steps:

Gene of interest to be cloned

Molecular scissors to cut gene of interest
Molecular carrier or vector on which gene of interest is placed
An expression system
How to get gene?

3 ways

Isolate from chromosome: Cutting chromosome on flanking genes sites using special
enzymes called restriction endonucleases.

synthesize chemically in laboratory (if they are small)

Synthesize from mRNA using reverse transcription (cDNA)

Molecular scissors

Restriction Endonucleases:

Natural enzymes of bacteria

Restrict growth of virus in bacteria

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Cut down viral DNA without any harm to bacterial chromosome

First restriction enzyme was isolated in 1970 by Hamiltom O.Smith at Johns Hopkins University

Bacteria produce variety of such enzymes

Far more than 400 isolated

20 commonly used in Recombinant DNA technology

Palindromic Sequence:

Such enzymes are characterized by specific sequence of 4 to 6 nucleotides arranged symmetrically in

reverse order

EcoR1: (E-Coli Type – I) – endosomes

Commonly used restriction enzyme

Cut down double stranded DNA when base sequence is at cleavage
Single stranded but complementary ends of 2 DNA molecules – sticky ends – binding by complementary
base ends.

Molecular carrier – vector

Gene is place on vector

Plasmid Antibiotic resistant Gene for

pSC 101 Tetracycline

pBr 322 Ampicillin + Tetracycline

Preparations for Recombinant DNA

Isolation

Plasmid is cut by some enzyme used for isolating gene of interest

Ligation:
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Gene of interest is joined with sticky ends produced after cutting plasmid – by help of DNA ligase

Formation

Sealed – 2 different pieces of DNA joined – chimeric DNA/ Recombinant DNA

Expression system

a. Bacterium
b. Viruses

IInsulin Production:

12.2 DESCRIBE THE PRINCIPLE AND STEPS OF POLYMERASE CHAIN REACTION

Developed by Kary B. Mullis in 1983

PCR takes name from DNA polymerase enzyme used for DNA replication in this process

Produce specific and millions of copies (but less than recombinant DNA technology)

Use 1/million part of sample DNA for producing clones

Primers:

Sequence of about 20 bases that are complementary to bases on either side of target DNA

DNA polymerase:

Also known as Taq polymerase

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Temperature insensitive (thermo stable)

Extracted from bacterium (which live in hot springs) – Thermus Aquaticus

Used in DNA replication

12.2.1 Components required in PCR:

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Template DNA
Taq polymerase
dNTPs
Primers
NA ligase
MgCl2
Water
12.2.2 Process
1. Heating:
Heating of DNA for 1 minute at 90-95oC for denaturing 2 strands of DNA
2. Cooling:
Cool it for 2 minutes at 50oC
3. Addition of primers (Annealing):
Primers are added to start the replication process (cannot be done by DNA polymerase) +
dNTPs (5 minutes) – 72oC
4. Addition of DNA polymerase:
Continues the replication process initiated by primers (wait for 1.5 minutes)
5. Repetition of cycle:
Same cycle is repeated until desired number of copies of genes is produced
12.2.3 Terms
Genome:

Full set of individual genes

Genomic library:

Collection of bacteria/bacteriophage clones, each containing DNA from source cell

Probe:

Single stranded nucleotide sequence that is used to search genetic library by hybridizing with certain pieces
of DNA – Radioactive or Fluorescent

12.3 UNDERSTAND THE FOLLOWING TERMS:- DNA FINGER PRINTING, (DNA ANALYSIS)

12.3.1 DNA finger printing

Cutting of 3 DNA samples by using restriction endonucleases and adding into test tubes(RFLPs –
Restriction Fragment Length Polymorphism)
Gel electrophoresis of DNA samples is performed using agarose gel (to separate fragments according to
their length, weight, or size)

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Transferring of gel pattern on membrane (DNA denatured)
Hybridization of denatured DNA by using radioactive probes
Autoradiogram obtained due to complementary arrangement of bases in probe
(black bands appeared) – recorded on X-ray film
PCR Amplification & analysis:
Used to:
Diagnose viral infections, genetic disorders, cancer
In forensic labs to identify criminals
Determine evolutionary history of human population
12.3.2 Gene sequencing

Main principle:

Generating of different sized DNA pieces(same starts, different ends)

Separation by gel electrophoresis(by agarose gel)
Reducing of sequence

Generation of different sized DNA pieces

Two methods:
a. Sanger’s method:
Use of dideoxyribonucleoside triphosphates for terminating DNA synthesis at different sites
b. Maxam –Gilbert method:
Cutting of DNA threads chemically

Gel electrophoresis

Using agarose gel

3. Reading:

Sequencing has been made automated (robotic devices)

Genomes sequenced yet:

Plants chloroplasts
Animals mitochondria
Many bacteria, yeasts
Nematode worm
Model plant – Arabidopsis
Mouse

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Human various human pathogens

12.3.3 Human Genome

Two primary goals:

To construct genetic map of genome

To construct base sequence map

Human chromosome No. 22= sequence completed in 1999

Human genome = completed in 2001

= 25 times larger than other genomes

= 3 billion base pairs

= Encyclopedia of 200 volumes, each of 1000 pages

Entire genome sequenced = J.Craig Venter (Celeria)

12.4 EXPLAIN GENE THERAPY WITH REFERENCE TO HOW GENETIC DISEASE (I.E. CYSTIC FIBROSIS, SEVERE
COMBINEDIMMUNO-DEFICIENCY SYNDROME, ANDHYPERCHOLESTEROLEMIA) CAN BE TREATED WITH GENE
THERAPY

12.4.1 Gene Therapy

Insertion of genetic material into human cells for the treatment of a disorder
12.4.2 Methods
a. Ex-vivo gene therapy: (SCID, familial hypercholesterolemia)
Genes - cultured cells – patient bone marrow cells
b. In-vivo gene therapy: (hemophilia, diabetes, AIDS, Parkinson’s disease)
Gene – patient (particular tissue)

Severe Combined Immunodeficiency Syndrome (SCID):

Inherited immune disorder associated to T-lymphocytes (less extent) and B- lymphocytes dysfunction
Lack enzyme Adenosine Deaminase (ADA) required for B and T lymphocyte maturation

Hypercholesterolemia:

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Liver cells lack low density lipoprotein (LDL) receptors for removing cholesterol from blood
Can cause heart attack at young age

Cystic Fibrosis:
Lack gene that encodes for trans-membrane carrier of chloride ions
Patients die due to respiratory tract infection
Genetic disease
Lipoprotein + solution – liposomes+microscopic vesicles – coated with gene – used as nose spray
Method not yet efficient due to limited gene transfer

Cancer:
Chemotherapy: (old method)
Healthy cells – making patients tolerant
Tumor cells – making vulnerable for patients
Gene therapy:
TNF (Tissue Necrosis Factor)
Suicide therapy
Two gene therapies
Gene replacement therapy

Coronary Artery Angioplasty:

Old methods: Use of balloon catheter to open, close artery and Artery closed again
New method: Coating of balloon with gene for vascular endothelial growth factor

Hemophilia:
Regular dose of cells with normal clotting factor genes
Placing such cells in organoids (artificial organs of abdominal cavity)

Parkinson’s Disease:
Drafting of dopamine producing cells directly into brain
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12.5 DESCRIBE THE DETAIL OF TRANSGENIC ORGANISMS (BACTERIA, PLANTS AND ANIMALS), TISSUE CULTURE,
CLONING AND ITS APPLICATIONS

TRANSGENIC BACTERIA:

Biotechnology products:

Bioreactors= bacterial reproduced in large vats

Insulin

Human growth hormone

Tissue plasminogen activator

Hemophilia factor VIII

Hepatitis B Vaccine

Health of plants:

Promote health of plants by

Encouraging formation of ice crystals from frost – plus to minus

Bacteria in corn roots is endowed with insect toxin

Clean beaches:

Used to clean beaches after oil spills

Bio filters:

Prevent airborne chemical pollutants from being air ventilated

Remove sulfur from coal before combustion

Clean up toxic waste dumps

Produce suicide genes (for self-destruction after job completion)

Organic Chemicals:

Produce phenylalanine (organic chemical to make aspartame)

Aspartame is a dipeptide sweetener – NutraSweet

Mining of metals:

Extract Cu, U, and Au, from low grade sources

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Improve biochemical capabilities

TRANSGENIC PLANTS:

Introduction of foreign genes into immature plants embryos or into cell wall removed plants (protoplasts)

Insect toxin:

Cotton, corn and potato strains with foreign genes become pests resistant because they produce
toxins

Soybean – resistant to common herbicide

Some corn and cotton – Both pest + herbicide resistant

Green evolution:

Development of special high hybrid plants

Stomata:

Altered to boast up CO2 intake + cut down of water loss

Rubisco enzyme:

Efficiently increased to capture more CO2 in plants

C4 cycle:

Efforts by Japanese to introduce C4 cycle in plants i.e. rice

Such plants avoid inefficiency of carboxylase by using different ways for CO2 capture

Mouse – Eared Cress Weed:

Produce biodegrable plastic – polyhydroxy butyrate (ion granules cells)

Human hormones; clotting factors; Antibodies:

Antibody by corn – Deliver radioisotopes to tumor cells

Antibody by soybean – treatment of genital herpes

TRANSGENIC ANIMALS:

Done by

Microinjection – Gene insertion into: eggs by hand

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Vortex Method – Eggs – Agitator + DNA + Silicon – Carbide needles – tiny holes – tiny holes by needles
for DNA to enter – Fertilization – transgenic animals

Large yields:

Fishes, cows, pigs, rabbits, sheep

Bovine growth hormone:

Taken up by many animals

GE Fishes:

Kept in pond offering no escape to wild to destroy ecosystem

Gene Pharming:

Use of transgenic farm animals to produce pharmaceuticals

Drugs for treatment of

cystic fibrosis

cancer

blood diseases etc.

Antithrombin III:

Used for preventing blood clot during surgery

Produce by goats

In vitro fertilization

Mice:

US Department of agriculture produce GE mice to produce human growth hormone in the urine
instead of their milk

Urine is preferable vehicle for biotechnology products instead of milk:

Both male and female urinate - only female give milk

Urination starts at birth – lactation start after puberty

Easy protein extraction – Difficult protein extraction

CLONING:

To achieve eugenic aims for welfare of humans

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Gene Improvement

First Cloning:

By Campbell and Tan Wilmit in 1997 (Scotland)

Mammal cloning (somatic cell)
Sheep was named Dolly
Asexual reproduction method

Have genes from one animal only = unfertilized egg

Involvement of 2 individuals = fertilized egg

100% identical to parent cell except mutation occurs

Cloning of 1 cell makes 4 clones

Clone: An individual + all asexually reproduced copies

Cloning of mice: 2n nuclei was taken from cumulus cells (that are cells which cling to egg after ovulation)

TISSUE CULTURE:

Growth of Tissue in an artificial liquid culture medium

GOTTLIEB HABERLANT: In 1902, said plant cells are totipotent (each cells has full genetic potential of
organism to become complete plant)
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F.C STEWARD:
Tiny piece of phloem of carrot  Complete carrot plant
Micropropagation:
Commercial method to produce millions of identical seedlings in limited space
Meristem culture (tips of roots/ shoots of growing cells)
Single shoot tip – addition of auxins + cytokinins – new shoots in liquid medium
Advantages:
All plants have same traits
Virus free
Somatic Embryos
They are encapsulated in protective hydrated gel shipped everywhere
Artificial seeds
Leaf – mesophyll tissue – cell wall digesting enzyme – protoplasts – new cell wall regenerate –
cell division – somatic embryos – mature plant
Advantages:
At once, millions of copies produced in bioreactors
Done for certain vegetables
Tomatoes
Asparagus
Chery
Also done for ornamental plants
Lilies
Begonians
African violets
Soma clonal variations in new plants due to mutations and give plants with desired traits
Anther Culture
Matured anthers are cultured in medium containing
Vitamins
Growth regulators
Pollen grains – Haploid tube cells – cell division – proembryoes (20-40 cells) – rupturing of
pollen grains – Haploid embryos – Haploid plant or chemical agent added – diploid embryos –
mature plants
Advantages:
Resulting plants are diploid and homozygous
Direct way to produce plants expressing recessive alleles
Cell Suspension Culture
Rapidly growing cultures – cutting – small pieces – adding to – liquid nutrient medium– shaking
– suspension – chemicals (as entire plant)
Advantages:
Suspension culture Production

Cinchona ledgeriana Quinine

1. Digitalis lanta Digitoxin

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