Download ppt

Recombinant DNA Technology
Recombinant DNA Technology combines DNA from different sources
– usually different species
Utility:
this is done to study DNA sequences
to mass-produce proteins
to give recipient species new characteristics
as a therapy/curative for genetic disorders (‘gene therapy’)
Human insulin, created in bacteria
Corn damaged by
corn borer and fungi
“bt-corn”, with a
bacterial gene
Recombinant DNA Technology
A. Overview:
1. Purify DNA
2. Cut it with restriction enzymes – create a DNA ‘library’ of the fragments
3. Insert it into a vector, creating a recombinant DNA molecule
4. Insert the vector into a host cell
5. Create a population of cells (clone) that have this new DNA.
6. The DNA or protein product can be isolated and purified, or the new
organisms with this cell type can be used.
Recombinant DNA Technology
A. Overview:
B. Creating a DNA Library
- the more you know about the location of your gene of interest,
the better; the LESS DNA you will have to manipulate.
Recombinant DNA Technology
A. Overview:
B. Creating a DNA Library
- the more you know about the location of your gene of interest, the
better; the LESS DNA you will have to manipulate.
- Best case – know the location of the gene and can pinpoint it
Recombinant DNA Technology
A. Overview:
B. Creating a DNA Library
- the more you know about the location of your gene of interest, the
better; the LESS DNA you will have to manipulate.
- Best case – know the location of the gene and can pinpoint it
- know the chromosome it is on
- Worst case – have to screen the entire genome.
Recombinant DNA Technology
A. Overview:
B. Creating a DNA Library
- the more you know about the location of your gene of interest, the
better; the LESS DNA you will have to manipulate.
- Best case – know the location of the gene and can pinpoint it
- know the chromosome it is on
- Worst case – have to screen the entire genome.
Three Ways:
- cut up DNA with restriction enzymes and isolate gene
- make copies of gene with PCR
- use m-RNA to make copies of gene with reverse transcriptase
Recombinant DNA Technology
A. Overview:
B. Creating a DNA Library
- the more you know about the location of your gene of interest, the
better; the LESS DNA you will have to manipulate.
- Best case – know the location of the gene and can pinpoint it
- know the chromosome it is on
- Worst case – have to screen the entire genome.
1. - cut it with a restriction enzyme that cuts at specific sequences and
leaves specific “tails” (the fewer fragments the better!!!)
Recombinant DNA Technology
A. Overview:
B. Creating a DNA Library
- the more you know about the location of your gene of interest, the
better; the LESS DNA you will have to manipulate.
- Best case – know the location of the gene and can pinpoint it
- know the chromosome it is on
- Worst case – have to screen the entire genome.
1. - cut it with a restriction enzyme that cuts at specific sequences and
leaves specific “tails” (the fewer fragments the better!!!)
- cut a ‘vector’ with the same restriction enzyme and ligate them with
ligase…
The vector could be plasmid (for placement in bacteria)…
Absorbed by inducing a state of ‘competence’ with calcium
salts, and transforming the bacteria (uptake of exogenous
DNA).
…or a ‘yeast artificial chromosome’ – YAC - (“yak”)
for the manipulation and study of eukaryotic genes…
Bacterial plasmid that has
had yeast centromeres,
telomeres, and replication
origins inserted.
Eukaryotic gene is
inserted into the plasmid,
then the chromosome is
“linearized” with BamHI
and inserted into a yeast
colony.
Why insert a eukarytoic
gene into yeast, rather
than bacteria?
…or a virus, capable of infecting other cells with their new gene.
Identify cells that have absorbed a recombinant plasmid.
- grow them on an ampicillin plate – only cells that have accepted a plasmid can grow
- since the fragments insert into a functional gene for lactose metabolism, cells that have
accepted a recombinant plasmid will be white (no lactose metabolism).
- WHICH of THESE WHITE cells absorbed the gene of interest??? (small library is better!)
Recombinant DNA Technology
A. Overview:
B. Creating a DNA Library
- the more you know about the location of your gene of interest, the
better; the LESS DNA you will have to manipulate.
- Best case – know the location of the gene and can pinpoint it
- know the chromosome it is on
- Worst case – have to screen the entire genome.
1. - cut it with a restriction enzyme that cuts at specific sequences and
leaves specific “tails” (the fewer fragments the better!!!)
- cut a ‘vector’ with the same restriction enzyme and ligate them with
ligase.
OR
2. - use the Polymerase Chain Reaction (‘PCR’) to clone DNA.
PCR (Mullis – 1993 Nobel in Chemistry)
- in preparation, you have purified the
DNA of interest… it may be in very
small amounts – even one cell or a
DNA fragment
Step 1: Denaturation of DNA:
Heating the sample to 90-95oC breaks
the H-bonds, denaturing the DNA
(separating the helices). ~ 1 min.
PCR
- in preparation, you have purified the
DNA of interest… it may be in very
small amounts – even one cell or a
DNA fragment
Step 1: Denaturation of DNA:
Heating the sample to 90-95oC breaks
the H-bonds, denaturing the DNA
(separating the helices). ~ 1 min.
Step 2: Anneal Primers:
The key to PCR is that you must know
something about the DNA sequence…
or be able to guess. You create
ss-DNA primers that anneal (h-bond)
to complementary sequences in DNA
when it is cooled down (50-70oC)
PCR
- in preparation, you have purified the
DNA of interest… it may be in very
small amounts – even one cell or a
DNA fragment
Step 1: Denaturation of DNA:
Heating the sample to 90-95oC breaks
the H-bonds, denaturing the DNA
(separating the helices). ~ 1 min.
Step 2: Anneal Primers:
The key to PCR is that you must know
something about the DNA sequence…
or be able to guess. You create
ss-DNA primers that anneal (h-bond)
to complementary sequences in DNA
when it is cooled down (50-70oC)
Step 3: Extension (Polymerization):
Use a thermally stable polymerase
(Taq) to polymerize DNA.
One
Cycle
(2-5 min.)
PCR
4. Repeat Cycle:
Heat up and denature, anneal, polymerize
3 hrs, ~ 25 cycles = 225 copies
PCR
Benefits:
- with correct primer, it is very
sensitive and amplifies very specific
sequences from very small samples
- genetic testing
- forensics
- molecular paleontology
PCR
Benefits:
- with correct primer, it is very
sensitive and amplifies very specific
sequences from very small samples
- genetic testing
- forensics
- molecular paleontology
- primers can be used as PROBES
that recognize sequences differing by
single bases (alleles)
Recombinant DNA Technology
A. Overview:
B. Creating a DNA Library
- the more you know about the location of your gene of interest, the
better; the LESS DNA you will have to manipulate.
- Best case – know the location of the gene and can pinpoint it
- know the chromosome it is on
- Worst case – have to screen the entire genome.
1. - cut it with a restriction enzyme that cuts at specific sequences and
leaves specific “tails” (the fewer fragments the better!!!)
- cut a ‘vector’ with the same restriction enzyme and ligate them with
ligase.
OR
2. - use the Polymerase Chain Reaction (‘PCR’) to clone DNA.
OR
3. - create c-DNA (‘complementary DNA’) from the isolated m-RNA
transcript, using reverse transcriptase.
Isolate m-RNA’s in eukaryotic
cell.
Use a “poly-T primer” to
recognize the Poly-A tails of the
m-RNA.
Use reverse transcriptase to
polymerize the DNA.
Partially digest with Rnase.
Add DNA Polmerase I (repair),
which cuts out rest of RNA and
polymerizes DNA across template.
Add ligase to complete ds-DNA
You can also use “reverse
transcription PCR” to amplify a
particular c-RNA directly.
Benefits of a c-DNA Library:
1. The absence of introns means that vectors and bacteria can
handle the size and structure of the eukaryotic c-DNA gene.
2. If you can localize the cell that is producing the protein of
interest, then the library will only contain DNA of active
(translated) genes – not ALL genes like in a whole genome
library.
3. If made from m-RNA, you can amplify genes that are very low
in productivity, and can amplify genes at different times of
development and get a picture of gene activation.
Recombinant DNA Technology
A. Overview:
B. Creating a DNA Library
Recombinant DNA Technology
A. Overview:
B. Creating a DNA Library
C. Recover the clone of interest
The white cells have
absorbed recombinant
plasmids… but which one
has absorbed the fragment
we want – the one with our
gene of interest?
Probes are short sequences of
DNA that will bind to the gene
of interest. They are either
radioactive or colorimetric.
Often the probe is a stretch of
c-DNA.