Download Explain what genetic recombination is, why it is important and ho it

Objective # 7
Module 4B – Biotechnology
Explain what genetic
recombination is, why it is
important and ho
important,
how it occ
occurs
rs
naturally.
In this module, we will examine
some of the techniques scientists
have developed to study and
manipulate the DNA of living
organisms.
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Objective 7
recombination involves
combining DNA from 2 different
sources into a single molecule.
Individual genes are not altered,
they
h are simply
i l jjoined
i d together
h iin
new combinations.
 Genetic recombination is
important because it produces new
genetic types.
Objective 7
 New
genetic types are the raw material
for evolution. As new genetic types are
generated, they may gradually replace
existing genetic types by the process of
natural
t r l selection
l ti orr by
b other
th r evolutionary
l ti r
mechanisms.
 Thus, the rate of evolution depends
directly on the rate at which new genetic
types are generated.
 Genetic
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Objective 7
Objective 7
 In
nature, combining DNA from 2
different individuals into a single
molecule involves 2 steps:
 first, DNA from 2 individuals is
combined in a single cell
 then DNA from both individuals is
joined to form a single molecule
 In
prokaryotes, several natural
mechanisms can combine DNA from
2 different individuals into a single cell:
 Transformation – a cell absorbs pieces
of foreign DNA from its environment.
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1
Genetic Recombination by transformation:
Recipient Cell
1
DNA
Foreign
DNA
2
Recombinant DNA
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Objective 7
 Plasmid
uptake – a cell absorbs
plasmids from the environment.
 Transduction – a virus acts as a vector
to transfer pieces of foreign DNA
from one cell to another.
another
 Conjugation – a temporary
cytoplasmic bridge connects 2 cells so
that DNA can be passed from one cell
to the other:
Objective 7
 Once
pieces of foreign DNA have
entered a recipient cell, they often
combine with the recipient cell’s
genome
m tto form
f rm recombinant
r mbi t DNA
DNA.
 Plasmids, for example, can be
integrated into, and excised from,
specific locations on the main bacterial
genome:
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Objective 7
prokaryotes, genetic recombination
generally occurs by transferring pieces
of foreign DNA into a recipient cell and
then combining it with the recipient
cell’s genome.
 In most eukaryotes, recombination has
become a regular part of the lifecycle.
It occurs through fertilization followed
by crossing over during meiosis:
Genetic Recombination in eukaryotes:
 In
Fertilization
Crossing Over
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Objective 7
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Objective # 8
 In
order to recombine DNA from 2
individuals through fertilization and
crossing over, the 2 individuals must
b able
be
bl tto m
mate
t with
ith each
h other.
th r
Therefore they must belong to the
same species.
Discuss the roles of restriction
enzymes and DNA ligase in
constructing artificially recombined
DNA.
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Objective 8
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Objective 8
 While
various natural mechanisms can
combine DNA from 2 individuals of the
same species, scientists have developed
q
to combine DNA from anyy 2
techniques
individuals.
 These techniques result in the
production of artificially recombined
DNA..
DNA
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Two key enzymes are used to make
artificially recombined DNA.
1) Restriction enzymes (also called
restriction endonucleases):
 cut DNA into fragments – so called
“molecular scissors”
 each one recognizes and cuts DNA
only where a specific sequence of
base pairs occurs.
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3
Objective 8
many do not cut straight through
both strands, but make a jagged cut
leaving unpaired bases at both ends.
Because these unpaired bases can
pair with complimentary bases, they
are called “sticky ends”.
2) DNA ligase is used to join DNA
fragments together. This is the
“molecular glue”.

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Objective 8
Objective 8
 Summary of procedure for making
 Mix
the DNA fragments together.
Because they were cut with the same
restriction enzyme, fragments from
different sources will have the same
“sticky ends” and can pair up.
artificially recombined DNA:
 Isolate
DNA from 2 different sources.
 Cut
the DNA from both sources into
fragments using the same restriction
enzyme.
 Use
the enzyme DNA ligase to join
the paired fragments together:
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Objective 8
 Recombinant
DNA technology can be
used to create recombinant plasmids (or
other recombinant agents such as
viruses) which are useful for inserting
foreign genes into recipient cells.
 Plasmids or other recombinant agents
that are used to insert foreign DNA
into recipient cells are called vectors:
vectors:
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Objective # 9
Objective 9
Describe how the following can be
used to produce multiple copies of a
DNA fragment:
g
cloning)
g
a) molecular cloningg (gene
b) polymerase chain reaction (PCR)
 Why
would scientists want to produce
multiple copies of a DNA fragment?
 to study its structure and function
 to co
compare
pa e the
t e fragment
ag e t with
w t DNA
DN
from other sources
 if it codes for a useful protein, to
produce large quantities of the protein
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Objective 9
Objective 9a
There are 2 basic strategies for producing
multiple copies of a gene:
a) With gene cloning, a vector is used to
insert the gene we wish to clone into
a host cell. The host cell then
replicates the foreign gene using the
same cellular machinery that it uses to
replicate its own DNA.
a) molecular cloning (gene cloning)
b) polymerase
l
chain
h i reaction
i (PCR)
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Objective 9a
 During
gene cloning, plasmids are
often used as vectors to insert foreign
genes into bacterial host cells.
 Using
U i plasmids
l
id with
i h specific
ifi genetic
i
traits can help scientists determine
which bacterial cells have actually
absorbed the gene we wish to clone:
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Objective 9a
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Summary of procedure for gene cloning:
 Cut plasmids containing lac Z and amp
resistance genes with a restriction
enzyme. Use a restriction enzyme that
cuts the plasmid once, inside the lac Z
gene.
 Use the same restriction enzyme to cut
DNA containing the gene you wish to
clone.
Objective 9a
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Objective 9a
 Mix
DNA from both sources together.
Some plasmids will simply reclose. Other
plasmids will join with a piece of foreign
DNA to form a recombinant plasmid.
 Incubate bacterial cells with the plasmids.
Some cells will absorb no plasmid, some
will absorb a reclosed plasmid, and some
will absorb a recombinant plasmid.
 When
plated on media containing
ampicillin and XX-gal, how do we know
which bacterial cells absorbed no
plasmid?
 These cells will not survive because
they lack the gene for ampicillin
resistance. Therefore no colonies are
formed.
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Objective 9a
Objective 9a
 When
plated on media containing
ampicillin and XX-gal, how do we know
which bacterial cells absorbed a
reclosed (non
(non--recombinant) plasmid?
 These cells have a functional laclac-Z
gene. Therefore they will make the
enzyme β-galactosidase and will form
blue colonies.
 When
plated on media containing
ampicillin and XX-gal, how do we know
which bacterial cells absorbed a
recombinant plasmid?
 The inserted foreign DNA will
inactivate the laclac-Z gene. Therefore
these cells do not make β-galactosidase
and will form white colonies.
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6
Objective 9a
do we know which white
colonies contain the specific gene of
interest?
 The white colonies can be screened for
the specific gene of interest using a
genetic probe
probe.. A genetic probe is a
radioactive molecule of RNA or
single--stranded DNA that is
single
complementary to the gene of interest.
Using a Genetic Probe to Screen for the Gene
of Interest
 How
1. Colonies of bacteria, each grown
from cells taken from a white colony.
2. A replica of the plate
is made by pressing
a filter against the
colonies. Some cells
from each colony
adhere to the filter.
5. A comparison with the original
plate identifies the colony
containing the gene.
Filter
Film
3. The filter is washed with a solution
that denatures the DNA and contains
the radioactively labeled probe. The
probe contains nucleotide sequences
complementary to the gene of interest
and binds to cells containing the gene.
4. Only those colonies
containing the gene will
retain the probe and emit
radioactivity on film placed
over the filter.
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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Objective 9b
b) A second method for producing
multiple copies of a gene is PCR
 With PCR, we create the
conditions needed for DNA
replication inside a test tube that
contains a copy of the gene:
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Objective 9b
Summary of procedure for
polymerase chain reaction (PCR):
1) Denaturation – a solution containing
primers and the DNA
RNA p
fragment to be amplified is heated so
that the DNA dissociates into single
strands.

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Objective 9b
2) Annealing of primers – the solution
is cooled, and the primers bind to
complementary sequences on the
DNA flanking the gene to be
amplified.
3) Primer extension – DNA
polymerase then copies the
remainder of each strand, beginning
at the primer.
Objective 9b
 Repeat
steps 1 – 3 many times, each
time doubling the number of copies,
until a sufficient number of copies are
produced.
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Objective # 10
Objective 10
Explain the difference between
the following types of DNA
libraries:
a) Genomic libraries
b) cDNA libraries
A
DNA library is a collection of DNA
fragments representing all the DNA of
an organism.
r im
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Objective 10a
 The
simplest kind of DNA library is a
genomic library.
library.
 To create a genomic library, the entire
ggenome of an organism
g
is fragmented.
g
The fragments are then inserted into a
vector, such as a plasmid or phage, and
introduced into a host:
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Plasmid Library
Phage Library
DNA fragments
from source DNA
DNA fragments
from source DNA
DNA inserted
into plasmid vector
DNA inserted
into phage vector
Transformation
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Each cell contains a
single fragment. All cells
together are the library.
Phages infect E. coli
Each phage contains a
single fragment. All phage
together are the library.
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Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
8
Objective 10b
Objective 10b
 Another
type of DNA library is a
cDNA library.
 A cDNA library includes only DNA
fragments
g
that actually code for
proteins rather than all DNA
fragments. This means that introns
and other non
non--coding sections of the
genome are not included.
 To
produce a cDNA library, scientists
first isolate the mature mRNA from an
organism.
 An enzyme called reverse transcriptase
p
is then used to make a complementary
DNA copy of each mature mRNA
molecule:
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Objective 10b
 If
you want to make bacterial cells that
can manufacture a particular human
protein, why is it important to insert
cDNA rather than the original
genomic DNA into the bacterial cells?
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Objective # 11
Objective 11
A
DNA fragment containing a
particular nucleotide sequence can
be isolated and identified from a
sample containing many different
DNA fragments using a procedure
developed by E.M. Southern called
the Southern blot procedure:
Explain how a DNA fragment
containing a particular
nucleotide sequence can be
isolated and identified from a
sample containing many
different DNA fragments.
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Objective # 12
Explain the process and
importance of RFLP analysis
and
nd DNA fin
fingerprinting.
rprintin
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Objective 12
Objective 12
 If
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DNA from 2 individuals is different,
then the location of recognition sites
for a particular restriction enzyme may
also be different.
 If we cut DNA from both individuals
with the same restriction enzyme, we
may get different size fragments.
 This is called a restriction fragment
length polymorphism (RFLP).
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 As
we have seen, restriction enzymes
can be used to cut DNA into
fragments called restriction fragments.
fragments.
 However, these cuts are not made at
random, each restriction enzyme cuts
the DNA only where a particular
sequence of bases occurs. These are
called recognition sites.
sites.
Objective 12
 How
can we determine the length of
the fragments that are produced when
we treat DNA from 2 individuals with
the same restriction enzyme?
 Gel
Electrophoresis
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Objective 12
Objective 12
 RFLP
analysis is a powerful technique
that is being in the field of forensics:
 small amounts of DNA collected at a
crime scene can be amplified
p
usingg PCR
 the DNA is cut into fragments with a
restriction enzyme, and the fragments
are separated using gel electrophoresis
 the
resulting banding pattern is then
compared with the banding pattern
produced by DNA samples from
different suspects
 the banding pattern for each individual
is essentially unique, and is referred to
as a DNA fingerprint
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Objective 12
Objective 12
 RFLPs
can also be used to distinguish
between DNA that contains different
alleles if different recognition sites
occurr within
ithi orr very
r close
l
tto th
the
different alleles.
 This is referred to as genetic screening:
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Objective 13
Objective # 13
 DNA
sequencing involves determining
the actual sequence of base pairs in a
DNA molecule.
 This is the ultimate level of g
genetic
analysis.
 A method of sequencing called
enzymatic sequencing was developed
by Fredrick Sanger
Explain the process and
p
of DNA
importance
sequencing.
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Dideoxynucleotides have an
H in place of an OH at both
the 2′ position and the 3′
position of the sugar.
Objective 13
 Sanger’s
method uses modified
nucleotides called dideoxynucleotides.
dideoxynucleotides.
 Dideoxynucleotides have an H in place
of an OH at both the 2′ position and the
33′ position of the sugar
sugar. As a result
result, if a
dideoxynucleotide is incorporated into a
growing nucleotide chain, no additional
nucleotides can be added and chain
elongation is terminated.
NH2
N
O
–O
P
N
O
CH2
5´
O
O–
4´
1´
3´
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N
2´
H
H
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Objective # 14
Describe at least 4 ways genetic
technology can be used for
h
human
b
benefit.
fi
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Objective 14
Objective 14
1) Introduce genes coding for proteins
with commercial or medical value into
other organisms, such as bacteria, in
order to mass produce the proteins.
Proteins produced in this way include:
 Human insulin – helps regulate blood
sugar level
 Interferons – assist the immune
response by inhibiting viral replication
 Human
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growth hormone – stimulates
cell division and growth
 Erythropoietin – stimulates red blood
cell p
production
 Atrial peptides – may be a new way to
treat high blood pressure and kidney
failure
 Tissue plasminogen activator (TPA) –
dissolves blood clots
Objective 14
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Objective 14
2) Produce vaccines that provide
protection against disease. Genetic
technology has been used to develop
two types of vaccines : subunit vaccines
and DNA
N vvaccines.
 A subunit vaccine is developed using a
small portion (or subunit) of the
pathogen - for example, a protein in the
coat or envelope that surrounds a
harmful virus.

To prepare a subunit vaccine against a
harmful virus, a gene coding for a
protein in the coat or envelope of the
harmful virus is spliced into the
genome of a harmless virus like
vaccinia.
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Construction of a subunit vaccine against
herpes simplex:
Objective 14
 Next,
the modified vaccinia virus,
which contains surface proteins from
the harmful virus, is injected into
uninfected people.
 The immune system detects the
proteins from the harmful virus on the
surface of vaccinia and makes
antibodies against any virus with those
proteins.
2. Herpes simplex
gene is isolated.
1. DNA is
extracted.
3. Vaccinia DNA
is extracted
and cleaved.
Herpes simplex virus
Human immune
response
6. Antibodies directed
against herpes
simplex viral coat
are made.
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Gene
specifying
herpes
simplex
surface
protein
Harmless vaccinia
(cowpox) virus
4. Fragment containing
surface gene
combines with
cleaved vaccinia DNA.
5. Harmless engineered
virus (the vaccine)
with surface like
herpes simplex is
injected into the
human body.
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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Objective 14
prepare a DNA vaccine
vaccine,, a gene
from a pathogen is artificially replicated
and then injected directly into
uninfected people. If human cells take
up the gene, some may use it to make
the protein encoded by the gene.
 The presence of the foreign protein in
the body triggers an immune response
against the pathogen.
Objective 14
 To
 Unlike
subunit vaccines, DNA
vaccines do not stimulate the
production of antibodies against the
pathogen. Instead, they stimulate the
activity of killer TT-cells, which are
another component of the body’s
immune response.
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Objective 14
3) Alter the human genome to cure
genetic disease or give people
certain desirable traits.


Manyy genetic
g
disorders are caused
by a single defective allele.
In gene therapy
therapy,, scientists try to
supply a copy of the normal allele to
those cells that need it but lack it.
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Objective 14
 There
83
are some serious obstacles to
successful gene therapy:
 How do you get a copy of the gene
into enough of the cells that need it?
 Will the gene function normally once it
is inserted into a cell?
 Will inserting a new gene into a cell
damage or alter the expression of any
other genes?
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Objective 14
Objective 14
 During
gene therapy, there is always
the concern that insertion of a normal
allele into a cell could inactivate
another essential gene or turn on a
gene inappropriately.
 Gene therapy was first used
successfully to treat SCID (severe
combined immunodeficiency).
 The
procedure involved removing
white blood cells from the patient,
using a virus to insert the necessary
gene into the cells, and then returning
the cells to the bloodstream.
 Although the treatment was successful,
about 15% of patients developed a
rare form of leukemia.
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Objective 14
Objective 14
 Scientists
determined that the vector
used to introduce the normal allele
into white blood cells integrated into
the genome next to a proto
proto--oncogene
called LM02.
 Activation of this gene caused the
leukemias.
Beans
Ferritin gene
is transferred
into rice from
beans.
Fe
Ferritin protein
increases iron
content of rice.
Transgenic Rice
Aspergillus fungus
Wild rice
Phytase gene is
transferred into
rice from a
fungus.
Metallothionin
gene is
transferred into
rice from wild
rice.
Pt
Rice
chromosome
Phytate, which
inhibits iron
reabsorption,
is destroyed by the
phytase enzyme.
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4) Genetically alter organisms, including
crops and livestock, to give them certain
desirable traits such as disease
resistance, frost resistance, faster growth
rate, or higher nutritional value.
 Organisms that have genes introduced
without the use of conventional
breeding are called transgenic:
transgenic:
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Daffodil
Enzymes for
-carotene
synthesis are
transferred into
rice from daffodils.
S
Metallothionin
protein supplies
extra sulfur to
increase iron
uptake.
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
A1 A2 A3 A4
-carotene, a
precursor to
vitamin A, is
synthesized.
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Objective # 15
Objective 15
1) Some question the safety of eating
genetically modified organisms. So
far, no negative effects have been
documented.
2) Some worry that genes from
genetically modified organisms may
spread into the gene pools of wild
organisms and modify them. There
is no evidence this has occurred.
Discuss some potential problems
associated with genetic technology.
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Objective 15
3) Another concern is that genetically
altered organisms may escape into
the environment and replace natural
organisms or upset the balance of
nature
nature.
4) There are also moral and ethical
questions associated with controlling
the genetic makemake-up and evolution
of existing life forms, including
humans.
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