Genetic Engineering and Biotechnology Genetic Engineering and Biotechnology

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22 Οκτ 2013 (πριν από 3 χρόνια και 10 μήνες)

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Genetic Engineering and Biotechnology
Genetic Engineering and Biotechnology
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Genetic Engineering and Biotechnology
Genetic Engineering and Biotechnology
So, what is biotechnology??
The use of living organisms to carry out defined chemical
processes for industrial or commercial application.
The office of Technology Assessment of the U.S. Congress defines
biotechnology as "any technique that uses living organisms or their
products to make or modify a product, to improve plants or
animals, or to develop microorganisms for specific uses."
GENETIC ENGINEERING
However Today, Biotechnology often implies and organism which has been
manipulated for carrying out a process –
What type of manipulation -- isolation, manipulation and sequencing
of DNA, regulation of and control of gene products—
Use of in vitro techniques, especially molecular cloning, which result in genetically
modified organisms is often termed GENETIC ENGINEERING
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Genetic Engineering and Biotechnology
Genetic Engineering and Biotechnology
Molecular Aspect
Tools which allow --
isolation, identification, verification,
purification and manipulation
of the gene of interest.
Applied Aspect
Production of useful products
2 MAIN ASPECTS TO THIS DISCUSSION….
Useful products – insulin, growth hormone, frost free strawberries, bacteria
which eat oil, vaccines, industrial enzymes
Tools --
Vectors –
Plasmids, Cosmids, YAC, Bacmids, Transposons
Enzymes –
Reverse Transcriptase, Ligase, Restriction Endonuclease, DNA
polymerase
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Genetic Engineering and Biotechnology
Genetic Engineering and Biotechnology
Restriction Endonuclease – WHAT? WHY?
Three types
Type I, Type II, and Type III
Type I and III
ATP dependent
Don’t cut at recognition site
Type II
No ATP dependence
Cut within the recognition site
Molecular Scissors
WHY IN THE WORLD WOULD A BACTERIA HAVE SOMETHING THAT CUTS UP
DNA?
HOW DO THEY PROTECT THERE OWN DNA?---
METHYLASE MODIFICATION OF OWN DNA
HAVE METHYLASES FOR EACH ?? RESTRICTION
ENZYME YOU HAVE --
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Genetic Engineering and Biotechnology
Genetic Engineering and Biotechnology
Type II Restriction Enzymes
Cut within recognition sequence
symmetrical palindromes (mostly)
sticky end or blunt end
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Genetic Engineering and Biotechnology
Genetic Engineering and Biotechnology
Sticky Ends
Blunt ends
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Genetic Engineering and Biotechnology
Genetic Engineering and Biotechnology
Cloning Vectors
Early cloning vector
WHY GOOD CLONING VECTOR????
Small 4.3kb
Sequence known
High Copy Number
20-30 copies/cell
stably maintained
Accept up to 10kb inserts
Many single cleavage sites
2 Antibiotic Resistance Markers
Easily Transformed
Tet resistance is INSERTIONALLY INACTIVATED
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Genetic Engineering and Biotechnology
Genetic Engineering and Biotechnology
A cloning we will go –
Creating Recombinant DNA
SPECIFIC GENE CLONING (ALREADY KNOW WHAT YOU WANT) VS.
CREATION OF A DNA LIBRARY (FISHING FOR STUFF)
DON’T FORGET TO GO ALL THE WAY TO TRANSFORMATION == INCREASE
THE NUMBER OF COPIES BY REPLICATION WITHIN THE HOST BACTERIUM.
CAN ALSO INCREASE THE NUMBER OF COPIES BY ---- PCR
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Genetic Engineering and Biotechnology
Genetic Engineering and Biotechnology
PCR –
Polymerase Chain Reaction
Shared the noble prize in chemistry in 1993 for his invention
$10,000 bonus
$300,000,000 sale to Roche
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Genetic Engineering and Biotechnology
Genetic Engineering and Biotechnology
POINT OUT TEMPERATURES ---
DENATURING – 94-96oC
ANNEALING – 45-64oC DEPENDING ON PRIMERS
EXTENSTION – 68 TO 72oC DEPENDING ON TAQ USED
TAQ POLYMERASE THERMOPHILE THERMOSTABLE
POLYMERASE
Thermus aquaticus Hot Springs in Yellowstone – 1976 first reported
REPEAT
25-40 TIMES
LOGARRITHMIC AMPLIFICATION
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Genetic Engineering and Biotechnology
Genetic Engineering and Biotechnology
But how do you know if you got the right clone??
How to pick from a plate full of colonies
enzyme – detect activity -- assays
protein – detect with an antibody to the
protein
gene – will it hybridize with DNA probe?
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Genetic Engineering and Biotechnology
Genetic Engineering and Biotechnology
Practical Application of Genetic Engineering –
What we do with what we can do
Therapeutic products
blood proteins – TPA, Factors VII, VIII, IX
hormones – Insulin, Somatotropin, FSH
immune products – Interferons (a/b)
vaccines – hepatitis B
Transgenic Plants and Animals
Bt corn, flavor saver tomatoes, vaccines(?), vitamin A
Gene Therapy
augmentation or replacement(?) of a non or dys
functional gene with a functional one
Bioremediation
Mammalian proteins can be produced –
cheaper, more safely, and in larger amounts than isolating from tissue
TPA == Tissue plasminogen activator – dissolved clots
Factors == Clotting agents
Somatotropin == growth hormone
VACCINES – CLONE GENE FOR PROTECTIVE VIRAL COAT (THIS IS THEN THE ANTIGEN) OR ATTENUATED
VACCINES – THOSE CREATED BY RECOMBINANT DNA TECHNOLOGIES THAT DO NOT HAVE THE VIRULENCE
FACTORS.
GENETICALLY MODIFIED ORGANISMS (EUROPE DOESN’T ACCEPT MANY OF THESE) GMO’S OR GM PLANTS
NO DISIGNATION IF DONE IN VIVO, (BEEN GENETICALLY ALTERING PLANTS AND ANIMALS FOR
OVER 10,000YRS) BUT IN VITRO USUALLY ADDS GENES FROM OTHER ORGANISMS SO CALLED TRANSGENIC
ORGANISMS.
BT == BACILLUS THURINGIENSIS
ROUNDUP READY == RESISTANCE TO GLYPHOSATE – FROM AGROBACTERIUM
FLAVOR SAVOR TOMATOES == BLOCKS PROUCTION OF POLYGALACTMORASE – THIS BREAKS DOWN CELL
WALL AND LEADS TO MUSHY TOMATOES
Vaccine – GM petunia and honey parvo vaccine – Hepatitis B and bananans
GOLDEN RICE
GENE THERAPY -- NOT REALLY REPLACED
Introduce functional copy of a gene using retroviruses or adenovirus(DNA)
Problems –
mostly center on vectors being used
give stable integration, but site of insertion is unpredictable
amount of cloned DNA is limited
protein expression is often transient
vectors are rapidly limited infectivity and rapidly inactivated
adenovirus can also present adverse reactions to the host