Recombinant DNA Technology

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Recombinant DNA Technology
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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)
Corn damaged by corn borer and fungi
bt-corn, with a bacterial gene
Human insulin, created in bacteria
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• Recombinant DNA Technology
• 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.

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• Recombinant DNA Technology
• Overview
• 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.

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• Recombinant DNA Technology
• Overview
• 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

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• Recombinant DNA Technology
• Overview
• 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.

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• Recombinant DNA Technology
• Overview
• 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

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• Recombinant DNA Technology
• Overview
• 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!!!)

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• Recombinant DNA Technology
• Overview
• 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

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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).
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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?
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or a virus, capable of infecting other cells
with their new gene.
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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!)
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• Recombinant DNA Technology
• Overview
• 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.

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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.
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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)
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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.)
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PCR
4. Repeat Cycle Heat up and denature, anneal,
polymerize
3 hrs, 25 cycles 225 copies
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PCR
Benefits - with correct primer, it is very
sensitive and amplifies very specific sequences
from very small samples - genetic testing -
forensics - molecular paleontology
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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)
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• Recombinant DNA Technology
• Overview
• 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.

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Isolate m-RNAs 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.
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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
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You can also use reverse transcription PCR to
amplify a particular c-RNA directly.
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• 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.
• 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.

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• Recombinant DNA Technology
• Overview
• Creating a DNA Library

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• Recombinant DNA Technology
• Overview
• 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?
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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.