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
20. A Pipetman is:
(A) the new biomedical device made by tissue engineering
and now used to treat the damaged blood vessels of heart
attack victims
(B) a radical group of bioengineered superheroes in
the Hollywood movie GATTACCA
(C) a molecular biology tool used in the lab to measure
small volumes of liquid common in biotechnology
(D) a new type of bio-engineered crop plants that are
drought tolerant
(E) a new surgical tool used in to extract cancer cells
21. An ultracentrifuge consists of a rotor that spins
tubes containing materials and is:
(A) a component on a new type of microscope to allow cell
components to be easily visualized
(B) the laboratory tool developed by Robert Hooke in the
1660s that he used to discover cells
(C) a tool used by cell biologists for separating and
comparing cell components based on size and density
(D) a component on the recent Rover Mars mission used to
look for life on other planets
(E) used only to enrich uranium for nuclear warheads
22. Cloning a gene:
(A) is a method that has been banned in Europe but widely
used in the US
(B) is a method used to copy a specific segment of DNA
(C) is a method used to reproduce an entire new organism
from a donor cell
(D) is a science fiction idea that has not yet been
accomplished or proven
(E) is a good idea in theory, but has not yet been
accomplished
23. The atomic number of carbon is 6. Its nucleus
must contain:
(A)
(B)
(C)
(D)
(E)
6 neutrons and 6 protons
3 protons and 3 neutrons
6 neutrons and no electrons
6 protons and no electrons
6 protons and 6 electrons
24. A covalent bond is formed when:
(A) two non-polar molecules associate with each other in a
polar environment
(B) a positively charged particle is attracted to a negatively
charged particle
(C) one atom gives up electrons to another atom
(D) two atoms share electrons
(E) two polar molecules associate with each other in a nonpolar environment
25. Enzymes are usually:
(A)
(B)
(C)
(D)
(E)
proteins
carbohydrates
helpful bacteria
only available in health food stores
none of these answers are correct
26. Changing one amino acid within a protein could
change what about that protein?
(A) the primary structure of the protein
(B) the overall shape of the protein
(C) the function of the protein itself
(D) the sequence of amino acids specified in the DNA
sequence called a gene
(E) all of the above
27. A shortage of phosphorus would make it difficult
for an organism to manufacture:
(A)
(B)
(C)
(D)
(E)
DNA
proteins
cellulose
fats
none of these answers are correct
28. Which of the following is not a chemical
reaction?
(A) sugar and oxygen combine to form carbon dioxide and
water
(B) sodium metal and chlorine gas unite to form sodium
chloride
(C) hydrogen gas combines with oxygen to form water
(D) ice melts to form liquid water
(E) none of these answers are correct
29. The chemical units of information in DNA are:
(A)
(B)
(C)
(D)
(E)
ATGC
UAGC
DAMN
RNAI
XYZQ
30. In DNA replication an (A) adenine always pairs
with:
(A)
(B)
(C)
(D)
(E)
cytosine (C)
thymidine (T)
guanine (G)
tyrosine
monosodium glutamate
31. Observation of and wonder at the workings of
nature are what initiate “why” and “how” type
questions. Science is a system of:
(A) relying on one’s best intuition, inspiration and
perspiration to solve problems
(B) advertising as a devious enterprise conceived by
entrepreneurial western capitalists to make money from the
ideas of inventive and often eccentric minds
(C) making theories that fit certain beliefs about why and
how things happen
(D) hypothesis making about the mysteries of life
(E) hypothesis making and testing to discern and validate
observable facts
32. Water can absorb and store a large amount of
heat while increasing only a few degrees in
temperature. Why?
(A) the heat must first be used to break the hydrogen bonds
rather than raise the temperature
(B) the heat must first be used to break the ionic bonds rather
than raise the temperature
(C) the heat must first be used to break the covalent bonds
rather than raise the temperature
(D) an increase in temperature causes an increase in adhesion
of the water
(E) an increase in temperature causes an increase in cohesion
of the water
33. Water is split during photosynthesis is plants to
yield what to compounds?
(A)
(B)
(C)
(D)
(E)
methane gas and carbon dioxide
oxygen and water
hydrogen and oxygen
sugars and proteins
RNA and DNA