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Gene Cloning: Definition
• Cloning is making an exact replica of
something.
• Gene cloning: making many, many copies of
identical pieces of DNA.
• Why do we need to clone genes, and how do
we do this?
Basic Science
To explore a gene’s function, or understand the differences between a mutant
and normal gene, researchers need to have a large supply of the gene at hand.
Medicine (Applied Science)
We lack the technology to make proteins from scratch, so we need to use
bacteria to make them for us.
For bacteria to make a protein, they need to have copies of the genetic information from a
gene.
Gene Therapy: Eventually we will be able to replace damaged genes with healthy
ones. We can do this already in some cases. To do these kinds of medical
miracles, large quantities of the DNA must be made.
Health & the Economy
Transgenic organisms: Organisms that have beneficial DNA transferred to them.
We can modify certain organisms in a beneficial way, or in a way that
enhances their value to humans.
How to We Synthesize Cloned DNA?
• There are two basic strategies to clone DNA.
Which one we use depends on the source of
our original DNA, and what we plan to do with
the final results.
• These two strategies are genetic engineering
(using recombinant DNA technology) and
PCR, or polymerase chain reaction.
– We will go through each one of these processes.
Recombinant DNA Technology
Genomic DNA
Find a source of DNA
If we are interested in studying a gene’s DNA
sequence, and not the protein product, we can
use genomic DNA as our source DNA.
If we wish to ultimately look at proteins, we need
DNA without introns (right?) This kind of DNA
(which we make from the mRNA) is called
complimentary DNA, or cDNA (more later)
mRNA
cDNA
Reverse transcriptase
Put in something you can move it around in – a
“Vector”
To actually manipulate the DNA and put it into the
host cell, we need a vector. Vectors are simply a
“carrier” that allow us to manipulate the DNA
sequence that is important to us.
Vectors can be either plasmids or viruses.
Note: DNA from two or more different sources
(bacteria or virus DNA, and another organism’s
DNA such as humans) is called Recombinant
DNA.
Plasmid
virus
Recombinant DNA Technology
Plasmid
Enzymes as Tools
To actually put a foreign piece of DNA into a
plasmid or vector, we literally cut and paste DNA.
We can cut it VERY specifically, by taking
advantage of the primitive bacterial immune
system.
This consists of a variety of enzymes that cut
DNA at specific sequences – we call these
restriction enzymes.
Normally, bacteria use these to destroy foreign
DNA (such as that coming in from
bacteriaphages).
Humans have purified hundreds of these
enzymes, creating a library of precise tools that
can cut DNA at almost any sequence!
Once we cut the DNA, we can paste in our cDNA
(or genomic DNA). To do this, another enzyme,
DNA ligase, is used.
Restriction enzyme
cDNA insert
DNA Ligase
Recombinant DNA Technology
The final result is a recombinant plasmid with
useful DNA in it.
Host
once we have our vector, we need a way to
actually use it.
We stick it special strains of bacteria such as e.
coli
Transformation
You should already be familiar with this
concept.
PCR: Polymerase Chain Reaction
• Another way to clone DNA – in a test tube – is
to allow it to undergo many rounds of
replication.
• The basic idea behind PCR is simple: DNA is
replicated in a test tube over and over again.
Double-stranded DNA
DNA is “melted” (the hydrogen
bonds between base pairs
break because of heat)
DNA is cooled – a little bit.
In order to begin replication in a
test tube, DNA polymerase
needs a “landing pad” that
consists of double-stranded
DNA to extend.
We use small pieces of DNA,
called “primers”, to set the
starting points of replication.
Once primers bind (called
hybridization), DNA polymerase
extends each strand.
5’
3’
3’
5’
Increased heat
“melts” the DNA
double helix.
5’
DNA
polymerase
AGGTCCTG
elongation
3’
3’
elongation
CGTATCGA
DNA
polymerase
5’
The cycle is repeated.
Each new cycle produced twice
the amount of DNA from the
previous cycle. There is a
geometric expansion.
Within a few hours, a single
piece of DNA can be cloned into
over a million pieces.
2
4
8
16
32
64
128
256
512
1024
2048
4096
8192
16384
32768
65536
131072
262144
524288
1048576
Double-stranded DNA
Sources of DNA
• Primers can be easily synthesized in a lab.
• So if we know at least a little bit of a gene’s
sequence, we can clone it.
• There are thousands of unexplored genes in
the human genome, waiting to be
investigated!
Quick example
• A researcher finds a correlation between a kind
of leukemia and a strange looking karyotype.
• “8 out of 10 patients with this form of leukemia
also have a translocation between chromosome
15 and 17.”
• We look at the genes in this region. We find a
few that look “interesting” (maybe there’s an
unknown gene that has sequence similarity with
a yeast gene known to be involved in cell growth)
Quick example
• The researcher makes a primers that flank the
gene of interest.
• They do PCR on genomic DNA from people
that don’t have leukemia and people that do.
– They isolate a “wild type” and “mutant” gene.
• They put the gene into a plasmid, and clone it
in bacteria
• They sequence both genes, and find that
there is a mutation.
Quick example
• They go back to the patient and “wild type”
person, and purify the mRNA from their cells
• They use reverse transcriptase to make cDNA
• They use PCR to make a LOT of this cDNA
• They use genetic engineering to put this piece of
cDNA into a plasmid.
• They transform bacteria
• The bacteria produce wild-type or normal
protein.
Quick example
• The researcher can purify the 5two kinds of
protein, and perform various experiments on
it.
• THE END
• PCR can also be used to determine whether
individuals have certain disease genes.
• Genetic screening can show if people are
predisposed to certain diseases, and can
therefore allow them to alter their lifestyle to
encourage an improved quality and longevity.
DNA Fingerprinting
• Take someone’s DNA and cut it with restriction enzymes.
• Because there are SO many sequences, there will be – due
to chance alone – many regions of this person’s genome
that is cut by the enzyme.
• This DNA is run on a gel, and a unique set of bands can be
detected. This is called a person’s “DNA fingerprint” or
“genomic fingerprint”
• We use this for many reasons:
–
–
–
–
Identifying the father
Criminal cases when “DNA evidence” is used
Identifying distinct populations of organisms
Many other uses – limited only by human imagination.
Uses of cloning
• Biotechnology is a huge business.
– Genentech has a $72 B market cap
– Amgen has a 56.7 B market cap
– Compare: GE has a 378 B cap; Apple is 170 B
Uses of cloning
• Transgenic Organisms
– Organisms that contain DNA that was not part of the
original genome.
• Transgenic bacteria
– Produce important proteins and compounds such as
insulin (diabetes), and phenylalanine (for nutrasweet)
– They can use oil as a source of food.
– Produce complex organic compounds we can’t easily
synthesize (phenylalane for nutrasweet)
• Transgenic plants & animals
– Interesting list here
In the News
• The end of the stem cell debate?
– The science news article
• Once the kinks are worked out, "the whole field is going to
completely change," says stem cell researcher Jose Cibelli of
Michigan State University in East Lansing. "People working on ethics
will have to find something new to worry about.“
• Fox News Article (by… Father John)
– Shows that the good scientists really don’t have an ego.
• Professor Wilmut will not take advantage of his licence from the United
Kingdom to attempt human embryonic cloning because he is now convinced
that two independent research teams in Japan and the United States have
discovered a process that is much more efficient than therapeutic cloning (his
own discovery) and offers a more realistic and timely hope for therapies of
serious illnesses, such as stroke, heart disease and Parkinson’s disease.
•
•
•
•
•
•
•
1. genetically engineered microorganisms (GEMS)
2. transgenic plants
3. transgenic animals
D. Gene therapy
1. Discuss the concept and importance.
2. List technical approach(es) such as viral vectors.
3. Provide examples of the therapy such as cystic
fibrosis, adenosine deaminase deficiency.
• 4. Define somatic cell and germ cell gene therapy.
• 5. List positive and negative aspects with a discussion
of eugenics.
Genomics
• Genome Sequences
• Proteomics
• Bioinformatics