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Issues in Biotechnology:
The Way We Work With Life
Dr. Albert P. Kausch
life edu.us
The Mechanics of DNA
Lecture 4
Some Techniques in Biotechnology
© life_edu
Issues in Biotechnology:
The Way We Work With Life
Dr. Albert P. Kausch
Kimberly Nelson
OnCampus Live
BCH 190, MIC 190, AFS 190, NRS 190, PLS 190
OnLine BCH 190
A Sweeping General Survey on Life and Biotechnology
A Public Access College Course
The University of Rhode Island
Issues in Biotechnology:
Biotechnology, Our Society and Our Future
life
edu.us
Issues in Biotechnology:
The Way We Work With Life
Dr. Albert P. Kausch
life edu.us
BCH 190
Section I.
The Mechanics of Life and
General Biotechnology
A Sweeping General Survey on Life and Biotechnology
© life_edu
The University
of Rhode Island
Issues in Biotechnology:
The Way We Work With Life
Dr. Albert P. Kausch
life edu.us
The Mechanics of DNA
3. Atoms, Cells and the Flow of Life
4. Some Techniques in Biotechnology
A Sweeping General Survey on Life and Biotechnology
A Public Access College Course
© life_edu
The University
of Rhode Island
Issues in Biotechnology:
The Way We Work With Life
Dr. Albert P. Kausch
life edu.us
The Mechanics of DNA
Lecture 4
Some Techniques in Biotechnology
© life_edu
Tools of the Trade
The eppendorf tube
and the pipetman
are the standard stock
and trade in the daily
work of a molecular
biologist
The eppendorf tube
and the pipetman
are the standard stock
and trade in the daily
work of a molecular
biologist
Tools of the Trade
“On the body of the traditional P-Series pipet it says, in
relief, “Gilson.” Warren Gilson, who earned his MD in
1940 at the Univ. of Wisconsin, invented and patented
the mechanical basis for the popular adjustable pipet
(US Patent No. 3,827,305, 1974), Nearly 40 yrs. After
the patent, the Pipetman continues to be manufactured
in France in a factory started by Gilson’s colleague,
Eugene Marteau D’Autry. Shortly before Gilson’s
patent issued Gilson sold the marketing and sales rights
to Ken Rainin President of Rainin Instrument, because,
Gilson says, ‘He was a good salesman.’”
Innovative technologies
become biotech
products
“Eppendorf tubes
and Pipetman
For the Gold Rush”
Separation Techniques: The need to separate the
components of Life
Precipitation/Dissolution
Filters
Centrifugation
Affinity
Blots
Magnetics
Electrophoresis
Etc.
The ultracentrifuge
is a centrifuge optimized for
spinning a rotor at very high speeds, capable of generating
acceleration as high as 2,000,000 G (approx 19 600 km/s²).
Ultracentrifuges find important uses in molecular biology,
biochemistry and polymer science, including separation of cellular
structures and molecules.
Gel Electrophoresis:
the separation of molecules,
DNA, RNA and proteins
by charge and size
Electro refers to the energy of
electricity. Phoresis, from the
Greek verb phoros, means
“to carry across.” Thus, gel
electrophoresis refers to the
technique in which molecules
are forced across a span of gel,
motivated by an electrical
current.
What is a Gel?
Agarose is a long chain of sugar molecules,
a polymer, derived from algae
used in electrophoresis to separate molecules
Two types of gel:
• Agarose (horizontal type)
• Polyacrylamide (vertical type)
How are Gels Loaded and Run?
Applications of Gel Electrophoresis
• DNA Fingerprinting
• DNA Recombinant Technology
• Forensics
• The Human Genome Project
DNA carries a net negative charge; it
is negatively charged because the
phosphates (red circles) that form the
sugar-phosphate backbone of a DNA
molecule have a negative charge.
The gel matrix acts as a sieve for DNA molecules. Large
molecules have difficulty getting through the holes in
the matrix. Small molecules move easily through the
holes. Because of this, large fragments will lag behind
small fragments as DNA migrates through the gel.
As the separation process continues, the
separation between the larger and smaller
fragments increases.
• Molecular weight markers are often electrophoresed
with DNA.
• Molecular weight markers are usually a mixture of
DNAs with known molecular weights.
• Molecular weight markers are used to estimate the
sizes of DNA fragments in a DNA sample.
Issues in Biotechnology
Gel electrophoresis is an important tool in molecular biology
and biotechnology. Electro refers to the energy of electricity.
Phoresis, from the Greek verb phoros, means “to carry
across.” Thus, gel electrophoresis refers to the technique in
which molecules are forced across a span of gel, motivated by
an electrical current. Gel electrophoresis allows for:
(A) the separation of biological molecules, including DNA, RNA
and
proteins by their charge and size
(B) all of the answers are correct
(C) the identification of DNA markers now commonly used in
forensics to implicate or exonerate persons accused of various crimes
(D) the rapid visualization of the products of PCR
(E) the acceleration of DNA into cells for genetic engineering
purposes
The Techniques of
Molecular Biotechnology
Technology has created new Fields
DNA detection
DNA synthesis
DNA sequencing
DNA cloning
Genomics
Bioinformatics
Pharmacogenomics
Transgenics
Expression cassette
construction
Computational
Biology
RNA detection
Population Genetics
Protein detection
Proteomics
The Techniques of
Molecular Biotechnology
Technology has created new Fields
DNA detection
DNA synthesis
DNA sequencing
DNA cloning
Genomics
Bioinformatics
Pharmacogenomics
Transgenics
Expression cassette
construction
Computational
Biology
RNA detection
Population Genetics
Protein detection
Proteomics
Proteins Are Used to Copy DNA
DNA does not replicate spontaneously, but is
facilitated by a group of proteins
Interestingly, each of these proteins is coded
for in DNA they also replicate
Enzymes were discovered that cut DNA
at specific sequences
And subsequently,
enzymes were discovered
that paste DNA together
The ability to cut and
paste DNA allowed
gene cloning
Plasmids are circular pieces
of DNA found in some bacteria
Many copies per cell
Antibiotic resistance gene
Plasmids can be cut and
pasted back together
Foreign genes can be
inserted
How is a gene cloned?
Foreign DNA (gene)
is inserted into a plasmid
that has a gene for
antibiotic resistance
The plasmid is introduced
into a bacterial cell and
grown on the antibiotic
Only bacteria with the
plasmid grow…the inserted
gene is copied many times
Gene Construction
Promoter
Coding Sequence
Protein coding sequence
Cell specificity
Developmental specificity
Start transcription
Terminator
Stop transcription
Message stability
Gene constructs can be moved into plants and the gene is expressed
driven by the promoter sequence
It is now possible to clone
any gene from any
organism and move it
into any other organism
Gene transfer from one organism to another is not new
Image of two species of
bacteria transferring viral
phage particles
Bacteria transfer genes
to other bacteria and plants
Now in nature there
is another organism
capable of
transferring DNA:
we call that organism
a human being
Tools and Techniques
used in Biotechnology
No Walls
The Clear bead at the center changes everything
There are no edges to my being now
I have heard it said that there is a window
That opens from one mind to another
But where there are no walls
There is no need for a window, or fitting a latch.
Rumi 1279 AD
For those who are interested in taking this
course for college credit through the
University of Rhode Island;
For more information please contact:
[email protected]
Credits
Lectures by:
Edited by:
Video Produced by:
Dr. Albert Kausch
Dr. Albert Kausch
and Kimberly Nelson
Thaddeus Weaver
Thank You to The University of Rhode Island
and all of the students of Issues in
Biotechnology over the years