Transcript Document

Recombinant DNA
and Other Topics in Biotechnology
Recombinant DNA Technology
Preparing Recombinant DNA
Amplification of DNA by the
Polymerase Chain Reaction
Applications of Recombinant DNA
Technology
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The potential
The area of recombinant DNA seeks to engineer
changes in an organism’s genome to perform
useful tasks.
New lab methods have made DNA easy to work
with. We can now cut well defined fragments
and splice it into the DNA of another species.
Promising potential
• Gene replacement therapy
• DNA finger printing
• Improved agricultural products
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Recombinant DNA technology
Gradual increase in DNA knowledge over the last
100 years.
1868
Discovery of DNA.
1944
Confirmed as the carrier of genetic
information.
1953
Determination of structure and the
methods of replication, transcription,
translation and regulation.
1960’s
Deciphering of the genetic code.
mid 1970’s Genetic engineering.
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Recombinant DNA
DNA recombination or molecular cloning
Covalent insertion of a DNA fragment
from one cell or organism into the
replicating DNA of another.
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Basic steps
• Select and isolate a DNA molecule to serve as
the carrier (vector) for the foreign DNA.
• Cleave the vector DNA strands with a restriction
endonuclease.
• Prepare and insert the foreign DNA, producing a
recombinant or hybrid DNA molecule.
• Introduce the hybrid into a host organism, often
a bacterial cell - transformation.
• Develop a means to screen and identify host
cells that have accepted the hybrid DNA.
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Major breakthroughs
• Isolation of a mutant strain of E. coli that
did not have restriction endonucleases could not degrade foreign DNA.
• Development of bacterial plasmids and
bacteriophage DNA as cloning vectors.
• Discovery of restriction endonucleases
that permit selective DNA cleavage.
• Discovery of DNA ligase that catalyzes
the formation of phosphodiester link for
final site closure.
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Cloning vectors
Bacterial Plasmids
• Found in most bacterial
cells.
• Self-replicating, extra
chromosomal DNA.
• Closed, circular, double-stranded.
• Smaller than chromosomal DNA with only
3,000-30,000 base pairs.
• Contain information for translation of
specialized and protective proteins.
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Plasmid replication
Two possible modes
Stringent - only a few copies are made
Relaxed - many, many copies are made.
Some relaxed plasmids may continue to
replicate forming 2,000 - 3,000 copies,
accounting for 30-40% of all cellular DNA.
A typical plasmid will accept foreign DNA
up to 15, 000 base pairs.
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Ideal plasmid cloning
vector properties
It should:
• replicate in a relaxed fashion so that many copies
are produced.
• be small so it is easy to separate from
chromosomal DNA and easier to handle without
damage.
• have only a few sites for attack by restriction
enzymes.
• have identifiable markers for screening.
• have a single cleavage site for a given restriction
enzyme - within a gene.
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Ideal plasmid cloning
vector properties
Examples:
E. coli E1
Bacterial strain that has been shown to be
useful for recombinant DNA work.
pBR322
A plasmid of E. coli E. It has 4363 base
pairs. It is cleaved at a single site by the
restriction endonuclease EcoRI
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Bacteriophage DNA
 phage is the most widely used.
• Double stranded DNA with ~50,000 base pairs.
• It can produce many DNA copies within a host
cell.
• It acts as a package to introduce DNA by
infecting host bacteria.
• It is easily screened for.
• DNA fragments up to 23,000 base pairs can be
added.
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Preparing recombinant DNA
Foreign double stranded DNA must be
prepared for insertion.
Methods include
Chemical synthesis
Use of restriction endonucleases
Reverse transcription of mRNA
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Preparing recombinant DNA
Fragments prepared by restriction endonuclease
have either “sticky” or blunt ends.
'5 C G T
OH
P
C
T
'3 G C A G A A T
T
A C A T
G
T
P
OH
G 3'
A C
5'
Sticky or cohesive ends
'5 C G T
OH
P
C
A T G 3'
'3 G C A G
P
T
A C 5'
OH
Blunt ends
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Preparing recombinant DNA
Unpaired cohesive ends, up to 5 nucleotide
bases, can be joined to a plasmid that was
cleaved by the same endonuclease.
T
T
T
C A C A
T
G C A G A
A
G T
A C
C G T
C
G T
G
T
It can lead to regions that may not be stable.
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Preparing recombinant DNA
Blunt ends can be attached using DNA ligase.
ATP,
OH
P
C
A
T G
G C A G
T
A C
C G T
P
DNA ligase
C G T
C
A
T G
G C A G
T
A C
OH
5’ end must have phosphate groups and the 3’
end must have a free hydroxyl group.
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Use of homopolymer tails
This is a more widely used approach.
• Segments of 50-100 poly-A or poly-G are added
to one fragment - typically the plasmid.
• Equal length segments of poly-T or poly-C are
added to the other fragment.
• Both are accomplished using deoxynucleotidyl
transferase.
• They are then allowed to incubate together to
allow hydrogen bonding to bring them together.
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Use of homopolymer tails
P
OH
P
T
C G
C G
T
G C
A A
OH
n-dTTP
HO Tn G C
T
P
P
P
Tn OH
A A
P
OH
T
An OH
HO
P
P
HO An
n-dATP
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Use of homopolymer tails
HO P
Foreign DNA with
poly T tails
An C G
T T
Tn G C
A A An
OH
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OH
P
P
Tn
P O
H
Linearized plasmid
with poly A tails
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Getting the hybrid into the host
Transformation
• Introduction of the hybrid DNA into host cell.
• Current methods are inefficient with only 1
molecule in 10,000 being successfully
replicated.
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Transformation of E. coli cells
One can promote the introduction of hybrid
plasmid using CaCl2
• Wash cells with CaCl2 solution.
• Incubate with a solution of hybrid DNA.
• If DNA is incorporated, it is replicated.
• Selective markers can be tested for.
Cloning of bacteriophage DNA is done by
infecting the host bacteria.
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Screening and separation
pBR322 example.
• This plasmid has resistance for two
antibiotics - ampicillin and tetracycline.
• The foreign sequence is added within the
gene that imparts tetracycline resistance.
• The resistance is destroyed.
• Sequential testing using both antibiotics
can identify hybrid plasmids.
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Screening and separation
Yellow = Ampicillin treated
Green = Tetracycline treated
identical transfer
Circled colonies are
ampicillin immune
and tetracycline
sensitive. They
must be colonies
of cells containing
the hybrid plasmid.
incubate
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Restriction enzyme maps
EcoRI
XmnI
ScaI
Mapping is
valuable in the
selection of
plasmids for
cloning and
characterization
of DNA
fragments.
EcoRV
BamHI
EagI
NruI
BspMI
PstI
Amp'
Tet'
PpaI
pBR322
PflMI
HgiEII
AflIII
AccI
BapMII
PvuII
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Isolation and cloning
of a single gene
A human gene can contain 40,000 1,000,000 base pairs.
• That represents only about 0.03% of the
entire genome.
• We have the goal of identifying all human
genes - Human Genome Project.
• The first step is to construct a genomic
library - brute force, hit or miss approach.
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Constructing a genomic library
• Cut DNA into thousands of fragments using
restriction enzymes yielding random, overlapping
pieces.
• Separate fragments based on molecular size using
methods like gel electrophoresis.
• Add homopolymer tails and insert into vectors and
then into host cells.
• Clone the cells. This results in large population of
cells, each containing a different DNA fragment.
• Hope that all original genomic DNA is represented.
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Constructing a genomic library
Now one must screen to find which cells
contain a gene of interest.
• Grow each cell on an agar plate.
• Each colony has a different
recombinant DNA.
• Sample cells by blotting to identify
which colonies have the sequence of
interest.
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Blotting
• Apply nitrocellulose paper to the plate to
produce an imprint.
• Treat the paper with dilute NaOH to lyse
the cells, releasing the DNA which stays
on the paper.
• Add a radiolabeled hybridization probe complementary DNA or RNA molecule.
• Labeled spots on the paper identify which
colonies have the sequence of interest.
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Amplification of DNA
Polymerase chain reaction (PCR)
• Method is used to make multiple copies of
DNA without cloning.
• It requires that at least part of the sequence
for a DNA fragment be known.
• The advantage is that the process can be
repeated many times by altering
temperature.
• The amount of DNA increases exponentially.
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Amplification of DNA
Requirements for PCR
• Two synthetic oligonucleotide primers of
approximately 20 base pairs. They must
be complementary to the ‘flanking
sequences.”
• Heat stable DNA polymerase.
• All four deoxyribonucleotides as
triphosphates.
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Polymerase chain reaction
Target
sequence
I
II
III
IV
V
Separate strands by
heating to 95oC.
I
II
III
IV
V
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Polymerase chain reaction
I
II
III
IV
V
Hybridize primers
cooling to 54oC.
I
II
III
IV
V
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Polymerase chain reaction
I
II
III
IV
V
Synthesize DNA by
extending primers at 72oC.
I
II
III
IV
V
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Application of recombinant
DNA technology
Recombinant protein products
Bacteria can serve as factories for
various proteins like human insulin.
Bacteria can’t be used for many
eukaryotic genes since they are unable to
remove introns.
Genetically altered organisms
Alter DNA to remove defects or improve
quality/quantity of normal products.
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Examples
Bacteria
Modified Pseudomonas are being constructed to
degrade hazardous waste.
Plants
Flavr Savr tomato developed by Calgene. It was
altered to inhibit rotting and allow it to ripen longer
on the vine.
Animals
The rat growth hormone, somatotropin, was added to
a plasmid and added to mouse eggs. Resulted in
mice twice normal size. This was transgenic since
it was transferred to subsequent generations.
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Examples
Human gene therapy
There are approximately 4,000 known genetic
diseases.
There are several clinical trials ongoing or planned.
They involve:
• Removal of somatic cells from a patient.
• Insertion of a normal gene
• Reintroduction of the cells
Bone marrow, skin or liver cells are typically used.
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