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MBG 202: Molecular Biology - Biotechnology

Canik Başarı University - Credits:Biology (Campbell) 9th edition, copyright Pearson 2011, & The Internet. Provided under the terms of a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. Adapted from David Knuffke.
by

Ferhat Ozturk

on 19 March 2015

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Transcript of MBG 202: Molecular Biology - Biotechnology

A way to produce many copies of a DNA molecule without the use of cells.

Nobel Prize: 1993
Copying a genetic sequence. A single gene can be cloned. That's not really what people mean when they talk about "cloning".

They mean creating new organisms without sexual reproductive events.

Normal in most domains of life.

Controversial when used for organisms that do not naturally reproduce asexually.

Has been achieved in all domains of life.
Biotechnology
Applications
Tools & Techniques
Restriction Enzymes
Electrophoresis
PCR
DNA Sequencing
Genetic Engineering
Genetic Testing
Vectors
Microarrays
Labeling
Gene Libraries
Cloning
Cloning produces specific cells to be used for medical purposes.

Requires collection of "
pleuripotent stem cells
" from an early embryo, or from an adult.
What they Are:
What they Are:
What it is:
What they are:
What it is:
What they are:
What it is:
What it is:
How they work:
How they work:
How it works:
How it works:
How it works:
How they work:
How it works:
How it works:
Officially: "
Restriction Endonucleases
"
Only cut DNA at specific sequences (hence "restriction")
Cutting locations: "
Restriction sites
" Usually 4-8 bp
Essentially enzymatic DNA scissors.
Hundreds have been isolated.

Probably serve as a bacterial immune system against phages

Nobel Prize: 1972
EcoRI: Cuts at GAATTC

Restriction Enzymes are named for the bacteria they are isolated from
There are many different types of restriction enzymes.

Generally speaking:
They recognize "palindromic DNA sequences"
They either cut in the middle of the sequence ("
blunt cuts
"), or produce a 5' overhang of a few bases ("
sticky ends
").
Different restriction enzymes recognize different restriction sites.
DNA from different sources can be recombined by treatment with the same, sticky end making, restriction enzyme.

Ligase can be used to seal the break between strands.
DNA molecules that can be modified to store and replicate other DNA sequences.

Examples: Bacterial plasmids, phages, viruses, artificial chromosomes.
We'll focus on
plasmids
To be useful, plasmids must minimally have:
An
origin of replication
A region containing many restriction sites (a "
multiple cloning site
")
A gene/genes that enable screening of cells that have successfully taken up the plasmid (usually
ampicillin resistance
)
There is a size limit on plasmids.

To get more genes in to cells,
artificial chromosomes
can be used.

In 2010, the J. Craig Venter Institute created the first cell with a completely synthetic chromosome.
A way to separate fragments of DNA based on their size.
The DNA is placed in to a matrix gel made of
agarose
.

The gel is exposed to an electric field.

DNA migrates to the positive electrode.

Different sized fragments move through the gel at different rates (smaller = faster)

"
DNA Fingerprint
": The unique banding pattern of a particular restriction enzyme digest of a particular DNA sequence.
A way to identify sequences of interest.
DNA fragments are isolated and denatured (strands separated).

The fragments are exposed to a complementary
oligonucleotide
probe, tagged with radioactivity (old) or flourescence (new).

The oligonucleotide bonds to the complementary sequence among the fragments.
The "Polymerase Chain Reaction"
A "target" DNA sequence is analyzed.

Oligonucleotide
primers
that bracket the sequence are created.

The DNA, primers, free nucleotides, coenzymes, and special
Taq polymerase
are put in a thermalcycler.

The
thermalcycler
is programmed and it runs.

One sequence can be copied 2 times overnight.
1.
Denaturation
:
94-98 degrees Celsius
DNA strands separate
2.
Annealing
:
50-65 degrees Celsius
primers bond to strands
3.
Extension
:
75-80 degrees Celsius
Taq polymerase attaches to primers and replicates target sequence
Taq
Polymerase
For PCR to work, a polymerase that is not destroyed by high temperatures is required.

Taq
polymerase
: isolated from
Thermus aquaticus
, a bacterium first found in hot springs in Yellowstone National Park
30
collections of DNA sequences, stored in vectors.
large sequences are cut into smaller pieces.

the pieces are placed in to vectors.

the vectors are introduced into cells.
A way to determine the sequence of bases in a piece of DNA
Nobel Prize: 1980
Kary Mullis
Fred Sanger
(his second Nobel Prize)
DNA is replicated in the presence of
dideoxynucleotides
(
ddNT's
).

When a ddNT is incorporated into a strand of DNA, it terminates replication.

The results of the process are read, either manually (old school) or automatically (modern way).
A way to visualize simultaneous gene expression for multiple genes
The microarray contains a complementary sequence for different mRNAs on thousands of microscopic spots.
mRNA is isolated from a cell and exposed to the microarray.
Each well that has a sequence complementary to an mRNA fluoresces.
The fluorescence pattern is analyzed.
"
Expression analysis
"
What it is:
Combining genetic information from two or more organisms to create a "
transgenic
" organism that expresses new traits.

Involves restriction enzymes and vectors.

Has been accomplished in all domains of life.

Used in medicine, agriculture, industry, research
Details:
cDNA:
Recombinant
Screening
The frequency of successful engineering events is quite low.

How do you know if an organism is expressing the engineered trait?

Reporter genes
: Genes in vectors that enable detection of successful engineering.

Examples: Ampicillin resistance, GFP, luciferase
"
Complementary DNA
"

DNA copies of RNA molecules (produced using reverse transcriptase).

Increases stability, removes introns for prokaryotic expression of eukaryotic genes
Eukaryotic
Engineering
More difficult than prokaryotic engineering due to nuclear membrane.

Requires different strategies.
Gene Therapy
Engineering cells in a multi-cellular organism.

Tricky and mixed results to this point in time.
Complex
Engineering
Engineering multi-gene traits is becoming more common.
Ethics
What it is:
Using biotechnological tools to look for the presence/absence of particular genetic sequences.

Restriction Enzymes for DNA digestion. PCR for amplification, Gel Electrophoresis for analysis.
Health:
Identity
Research
Look for genetic markers associated with various traits.
Typically analyze "
Restriction Fragment Length Polymorphisms
" (aka "
RFLP's
"), though modern approaches also compare expression patterns.
In this example, the RFLP is due to a
single nucleotide polymorphism
(
SNP
) leading to loss of a
DdeI
restriction site, which can be visualized on a gel.
Look at areas of the genome that have many repeating sequences ("
Short Tandem Repeats
":
STR's
).

By comparing enough of these areas, identity can be confirmed or ruled out.
Soon, whole-genome sequencing will be cheap enough to be covered by most insurance plans.
The only limitation for genetic testing in research is experimental creativity.
What it is:
Reproductive Cloning
Cloning produces a new organism.
Somatic Cell Nuclear Transfer
Therapeutic Cloning
Consent
Ownership
Conceptions of "Life"
How much do we have a right to know?
How much do other folks have a right to know about us?
Can a gene be owned?
Can an organism be owned?
Should there be boundaries on what we are allowed to do to other living things?
Where does "life" begin?
The Thermalcycler
Essentially, a very fancy oven that can move between specific temperatures very quickly.
GFP Expression in these bacteria identifies successful transformants.
Ampicillin resistance identifies all bacteria who took in the plasmid.
The development of GFP as a reporter gene has spawned a variety of applications outside of genetic engineering.

Shown here: various flourescent proteins showing gene expression during
Drosophila
development.

Nobel Prize in 2008.
Agrobacterium is a bacterium that naturally inserts a plasmid (the "
Ti plasmid
") into plant cells during it's life cycle.

The Agrobacterium plasmid induces the formation of a tumor in the plant, where the bacterium lives.

By engineering the Ti plasmid, scientists can introduce novel genes into plants.
This is a "
gene gun
", which can be used to shoot micro-pellets coated with DNA into a eukaryotic cell's nucleus.
This goat produces a human blood clotting protein in its milk, an example of what is called a "
pharm animal
".
Golden Rice
is an engineered variety of rice that expresses beta carotene (a precursor of vitamin A) in its grain.

1-2 million people die of vitamin A deficiency every year
The gene "
cassette
" that was constructed to create Golden Rice.
xkcd.com
CC, the first cloned cat
The Asilomar conference (1975), where the regulations for genetically engineering organisms were first established
AM Chakrabarty, the plaintiff in the major precedent case
The BRCA-1 protein
Monsanto Terminator Seeds
Genetically engineered salmon (background)
A fast-aging knockout mouse
A cloned rhesus monkey
cell removal from an early embryo
"23 & Me": Consumer Genetic Testing
How to take a DNA sample.
Genetically Modified (GM) foods.
Big Questions:
Make Sure You Can:
What is the relationship between science and technology?

How have developments in genetics lead to the developments of new technologies?

What are the implications of genetic technologies for society?

What ethical and moral responsibilities do scientists have?
Tools & Techniques:
Explain all tools and techniques discussed. Inputs, process, outputs, and uses
The Banding Pattern is visualized with DNA stains (ethidium bromide shown)
Steps of PCR:
Old School:
Applications:
Explain all applications discussed. Inputs, process, outputs, and multiple real-world and realistic hypothetical examples
Ethics:
Provide valid ethical justifications for and against all applications of biotechnological tools discussed in this presentation using evidence.
Making Recombinant DNA Molecules
PCR
PCR!
PCR!?!
Sanger Sequencing
Automation!
DNA Microarrays
Full transcript