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MCDB 1041 Class 27
Making recombinant DNA and using it
Learning Goals
•  Explain why and how bacteria can be easily used to make copies of human
DNA.
•  Compare the two methods for making lots of copies of DNA: PCR and
bacterial amplification.
•  Given information about a bacterial plasmid and a piece of DNA, propose
how you would cut and combine the two to create recombinant DNA.
Quiz Friday
Review session: Thursday at 12:30 –will email location
18-1
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
PCR is good for making lots of copies of a specific
region of DNA
However, it is most effective for smallish pieces of
DNA (up to 10 Kb)
When scientists want to make copies of large pieces of
DNA, they often employ a different technique
The advantage of the next technique is that pieces of
DNA can be isolated, manipulated, and then amplified
18-2
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Making recombinant DNA
Restriction enzymes cut DNA at
particular locations
Scientists can put different pieces of
DNA together (by cutting with restriction
enzymes and then sealing the pieces back
together)
18-3
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Why make recombinant DNA?
•  Study how a gene (or its protein) normally work
•  delete genes
•  express a new protein in a chosen
location (“Mr. Green Genes” at right!)
•  introduce specific mutations into a gene
•  Produce a lot of a specific protein
medical uses (insulin,
human growth hormone)
•  Produce an altered protein not normally produced by an organism
genetically modified crops
gene therapy
18-4
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Putting two pieces of DNA together requires that they
have complementary sequences that can pair
Isolate gene of interest by
restriction enzyme digest (orange
piece of DNA)
Cut another piece of DNA with the
same restriction enzymes
(grey piece)
The two pieces of DNA
have complimentary sticky ends
Add the enzyme ligase, and the two
pieces of DNA become one piece
18-5
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
This circular piece of DNA is called
a “plasmid” (more in a minute)
They exist naturally in bacteria
They get replicated just like a
regular chromosome within the
bacterium
18-6
400 million people in the world are at risk of a vitamin A deficiency, which can lead to blindn
carrots, sweet potatoes, an
-carotene (precursor to vitamin A), but these foods are not always available in the developing
Copyright © The McGraw-Hill
Companies,
Inc. Permission
required
reproduction
display.
increase
in the severity
of infections
in for
young
children.orFoods
like
Imagine your goal is to generate engineered rice that would contain Vitamin A. By adding a g
Restriction enzymes and
DNA (review)
phytoene synthase (psy) from the daffodil plant, plus a promoter region that determines where
expressed, -carotene will accumulate in the rice grain.
picture below represents a piece of double-stranded DNA from daffodil. This DNA includ
This DNA sequence can beThe
cut
by 4 differentrestriction enzymes
phytoene synthase gene (psy), as well as additional sequences of DNA.
E=Eco RI
E P
B H
B
P=Pst1
B=BglII
Each
lineline
represent
1 Kilobase (1KB).
You can(1Kb)
amplify this 12 KB sequence from cells taken
Each
demarcates
1 Kilobase
H=HindIII
using PCR.
This DNA sequence can be cut by 4 different restriction enzymes (denoted by E, P, B, and H)
The restriction enzyme sites
are shown
below
of the nucleotide
sequencesin
thatdetail
occur at two
of these restriction enzyme sites, along with the
the REs. one with E and one with H, what can you
If you cut two piece of DNA,
Letter on the DNA
Restriction Enzyme Recognition
Name of Restriction
do next?
sequence
and cutting sequence
Enzyme
E
G*
A
A
T
T
C
Eco RI
a.  Paste the two pieces together
C T T A A *G
b.  Paste only E cut pieces
or H cutCpieces
together
P
T G C A*G
Pst I
G *A rejoined
C G T C
c.  Nothing—once cut, DNA cannot be
1.
G* A A T T C
C T T A A *G
C T G C A*G
G *A C G T C
18-7
Eco RI
In the strand of DNA shown below, find the restriction enzyme sites.
TATAAGATTGCGATGCCCTGCAGCTATTCGGCTGCCTAAAATCGGCCCCTAAGAA
ATATTCTAACGCTACGGGACGTCGATAAGCCGACGGATTTTAGCCGGGGATTCT
restriction
site
If this sequence (above) of DNA were cut with both enzymes, how many pieces would be crea
Hind III
These restriction enzymes produce sticky ends
where DNA nucleotides
restriction
site are not bound to their
pair. Thus, they can be easily hooked up to
another piece that has the complementary
unbound nucleotides:
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
The picture below represents a piece of double-stranded DNA from daffodil. This
phytoene synthase gene (psy), as well as additional sequences of DNA.
E
P
B H
B
Each
lineline
represent
1 Kilobase (1KB).
You can(1Kb)
amplify this 12 KB sequence from
Each
demarcates
1 Kilobase
using PCR.
If this sequence of DNAof the
were
with
nucleotidecut
sequences
that occur at two of these restriction enzyme sites, alo
the REs.
both E and H restrictionLetter
enzymes,
how Enzyme Recognition Name of Restriction
on the DNA
Restriction
sequence
and cutting sequence
Enzyme
E
G* A A T T C
Eco RI
many pieces would be created?
C T T A A *G
P
C T G C A*G
Pst I
a.  1
G *A C G T C
b.  2
1. In the strand of DNA shown below, find the restriction enzyme sites.
c.  3
TATAAGATTGCGATGCCCTGCAGCTATTCGGCTGCCTAAAATCGGCC
ATATTCTAACGCTACGGGACGTCGATAAGCCGACGGATTTTAGCCGG
d.  4
If this sequence (above) of DNA were cut with both enzymes, how many pieces w
This DNA sequence can be cut by 4 different restriction enzymes (denoted by E,
These restriction enzymes produce sticky ends
where DNA nucleotides are not bound to their
pair. Thus, they can be easily hooked up to
another piece that has the complementary
unbound nucleotides:
18-8
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
The picture below represents a piece of double-stranded DNA from daffodil. This
phytoene synthase gene (psy), as well as additional sequences of DNA.
E
P
B H
B
Each
lineline
represent
1 Kilobase (1KB).
You can(1Kb)
amplify this 12 KB sequence from
Each
demarcates
1 Kilobase
using PCR.
In
gel
below,
if the
gene
of
interest
isand
Thisisolate
DNAat
sequence
can
beat
cut2
byKB
4 different
enzymes
(denoted
by E,
base
andthe
learn
that
theyou
psy want
gene
you
want begins
to
and
endsinat
6 KB
in the
arn that
the
psy
gene
to
isolate
2begins
KB
ends
at
6restriction
KB
the
of
the
nucleotide
sequences
that
occur
at
two
of
these
restriction
enzyme
sites,
ded
DNA
in theabove.
diagram
you
cutthewith
the
DNA
withof
a variety
ofrestriction
different restriction alo
in the
you
the
DNA
aenzyme
variety
different
indiagram
between
the IfEabove.
andcutHIf
restriction
REs.
ow
with
the
letter
of band
the restriction
enzyme
thatonto
was
used
to cut the
DNA):
which
band
on
e letter
of
the
restriction
enzyme
that
was
used
the
DNA):
which
band
onName
Letter
thecut
DNA
Restriction
Enzyme
Recognition
of Restriction
sites,
which
on the
gel
represents
that
sequence
and cutting sequence
Enzyme
ns
gene of (write
interest?
thethe
lane
and
the band size)
ne your
of interest?
the (write
lane and
band
size)
piece?
H
B
E
H+
H E
B+PE
H+
Ladder(DNA of Ladder(DNA of
known sizes
P for known sizes for
referen
referen ce)
B+Pce)
12 KB
1.
11 KB
10 KB
G* A
C T
C T
G *A
A T T C
T A A *G
G C A*G
C G T C
Eco RI
Pst I
12 KB
In the strand
of DNA shown below, find the restriction enzyme sites.
11 KB
10 KB
TATAAGATTGCGATGCCCTGCAGCTATTCGGCTGCCTAAAATCGGCC
9 KB
9 KB
ATATTCTAACGCTACGGGACGTCGATAAGCCGACGGATTTTAGCCGG
A
B
C
If6 this
KB sequence (above)
6 KB of DNA were cut with both enzymes, how many pieces w
These
restriction 3enzymes
produce sticky ends
3 KB
KB
where DNA nucleotides are not bound to their
pair. Thus, they can be easily hooked up to
1 KB
KB
another
piece that1has
the complementary
unbound nucleotides:
A band
out of
the out
gel and
purify
the purify
DNA from
the gel
using
specialized
kit.
dil
psy DNA
band
of the
gel and
the DNA
from
the agel
using a specialized
kit.
y gene into
plasmid
What
restriction
enzymes should
you
use toyou
cut use to cut
daffodil
psy the
gene
into thebelow.
plasmid
below.
What restriction
enzymes
should
18-9
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Features of Bacterial Plasmids
•  Bacterial plasmids are circular, have a site to initiate
replication, and can serve as a carrier for another piece of DNA
•  Can be introduced back into bacteria, which will make many
copies of the plasmid
Essential Features
Series of unique restriction
sites
Selectable Marker
AmpR – ampicillin resistance gene
Bacteria with AmpR will not die in the
presence of ampicillin, while other bacteria will
ORI – origin of replication
(for bacteria)
allows the plasmid to be replicated in bacteria
18-10
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Bacterial cells have to incorporate the plasmid
“Transformation”
+
AmpR
Plasmid+ gene
Competent bacteria
Chemically treated so they
will take up DNA
Few cells are
transformed
Transformation is a rare event,
so you need a way to kill off all
the cells that aren’t
transformed, leaving only the
cells that contain your gene.
18-11
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Select for transformed cells
•  the plasmid contains an ampicilin resistance gene AmpR
•  when a plasmid has this gene, it can make a protein that breaks
down ampicilin, preventing this antibiotic from killing the bacteria
Plate the bacteria on
media that contains
ampicilin
If you grow bacteria on a
plate that has ampicillin,
which bacteria will survive?
a.  All bacteria
b.  Bacteria that contain the
plasmid
c.  No bacteria
media + ampicilin
plasmid
media + ampicilin
18-12
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Finally, break open these bacterial cells
and remove the DNA from the plasmid
Or, allow the bacterial cells to make
protein from the DNA, then isolate
the protein, which can be used to
treat people who can’t make the
protein
This technique is used to make insulin
for diabetics
18-13
Copyright
© The
Companies,
required
forincludes
reproduction
or display.
The picture below represents
a piece
of McGraw-Hill
double-stranded
DNA Inc.
from Permission
daffodil. This
DNA
the daffodil
Imagine
your
goal
is
to
generate
engineered
rice
that
would
contain
Vitamin
phytoene synthase gene (psy), as well as additional sequences of DNA.
A. By adding a ge
phytoene synthase (psy) from the daffodil plant, plus a promoter region that determines where t
E P -carotene
B Hwill accumulate
B
expressed,
in the rice grain.
The picture below represents a piece of double-stranded DNA from daffodil. This DNA include
Each
linegene:
represent
1 Kilobase
(1KB).
KB sequencesequences
from cells takenoffrom
daffodil
Psy
Coding
from
E(psy),
toYou
H can
phytoene
synthase
gene
asamplify
well this
as 12
additional
DNA.
using PCR.
E can be
Pcut by 4 different
B restriction
H enzymes
Bby E, P, B, and H). Below is a list
B (denoted
E
B DNA P
This
sequence
of the nucleotide sequences that occur at two of these restriction enzyme sites, along with the actual names of
the REs.
Letter on the DNA
Restriction Enzyme Recognition
Name of Restriction
Crt
gene.
Coding
from
P
to
E.
Each line represent
1 Kilobase
can amplify this 12 KB sequence from cells taken f
sequence
and cutting
sequence (1KB). You
Enzyme
Eusing PCR.
G* A A T T C
Eco RI
C T T A A *G
P
C T G C A*G
Pst I
IfThis
youDNA
wantsequence
these
two
genes
to
be
hooked
together
in (denoted
sequence,
can
be
cut
by
4
different
restriction
enzymes
by E,crt,
P, B, and H).
G *A C G T C
of the
nucleotide
sequences
that occur at two of these restriction enzyme sites, along with the ac
then
psy,
what can
you do?
the
1. REs.
In the strand of DNA shown below, find the restriction enzyme sites.
a. Letter
Mix
the DNA all together,
cut with E, P and H.Name
Separate
on gel,
on the DNA
Restriction Enzyme Recognition
of Restriction
TATAAGATTGCGATGCCCTGCAGCTATTCGGCTGCCTAAAATCGGCCCCTAAGAATTCTTATCG
ligate pieces
sequence
and cutting sequence
Enzyme
ATATTCTAACGCTACGGGACGTCGATAAGCCGACGGATTTTAGCCGGGGATTCTTAAGAATAGC
G* ACut
A with
T T C
RI P for other.
b. E Keep DNA separate.
E and H for one, Eco
E and
Ccut T
Aenzymes,
A *Ghow many pieces would be created?
If thisSeparate
sequence (above)on
of DNA
wereLigate.
with T
both
gel.
P
C T G C A*G
Pst I
c. TheseRun
gelenzymes
to separate
DNA.
Cut
with
E
and
H
for
one, E and P for
restriction
produce sticky
ends
G *A C G T C
where DNA nucleotides are not bound to their
other. Ligate
pair. Thus, they can be easily hooked up to
1.piecegel
In
strand
of DNA
shownMix
below,
the restriction
enzyme
sites. then
d. another
Run
tothe
separate
DNA.
all find
together,
cut with
E,P,H,
thatthe
has
complementary
unbound nucleotides:
ligate.
TATAAGATTGCGATGCCCTGCAGCTATTCGGCTGCCTAAAATCGGCCCCTAAGAA
ATATTCTAACGCTACGGGACGTCGATAAGCCGACGGATTTTAGCCGGGGATTCTT
If this sequence (above) of DNA were cut with both enzymes, how many pieces would be creat
18-14
These restriction enzymes produce sticky ends
Copyright
© The
Companies,
required
forincludes
reproduction
or display.
The picture below represents
a piece
of McGraw-Hill
double-stranded
DNA Inc.
from Permission
daffodil. This
DNA
the daffodil
Imagine
your
goal
is
to
generate
engineered
rice
that
would
contain
Vitamin
phytoene synthase gene (psy), as well as additional sequences of DNA.
A. By adding a ge
phytoene synthase (psy) from the daffodil plant, plus a promoter region that determines where t
E P -carotene
B Hwill accumulate
B
expressed,
in the rice grain.
Eco RI9:/$..$
The picture below represents a piece of double-stranded
DNA from daffodil. This DNA include
%&'()&'*+,,'(-$
Each
linegene:
represent
1 Kilobase
(1KB).
KB sequence
from cells takenoffrom
daffodil
.('*-"+(-$
Psy
Coding
from
E(psy),
toYou
H can
phytoene
synthase
gene
asamplify
well this
as 12
additional
sequences
DNA.
using PCR.
Amp
R
(-/-01+2/-$*+"3-"$
4#,5$...$
60'$7.$
%(1$8$
9:/$..$
E can be
Pcut by 4 different
B restriction
H enzymes
Bby E, P, B, and H). Below is a list
B (denoted
E
B DNA P
This
sequence
of the nucleotide sequences that occur at two of these restriction enzyme sites, along with the actual names of
the REs.
Letter on the DNA
Restriction Enzyme Recognition
Name of Restriction
Crt
gene.
Coding
from
P
to
E.
Each line represent
1 Kilobase
can amplify this 12 KB sequence from cells taken f
sequence
and cutting
sequence (1KB). You
Enzyme
Eusing PCR.
G* A A T T C
Eco RI
C T T A A *G
!"#$
P
C T G C A*G
Pst I
This DNA sequence
can be cut by 4 different restriction enzymes (denoted by E, P, B, and H).
G *A C G T C
of the nucleotide sequences that occur at two of these restriction enzyme sites, along with the ac
the
1. REs.
In the strand of DNA shown below, find the restriction enzyme sites.
Once
joined Restriction
your genes
of Recognition
interest together,
Letter you’ve
on the DNA
Enzyme
Name of Restriction
TATAAGATTGCGATGCCCTGCAGCTATTCGGCTGCCTAAAATCGGCCCCTAAGAATTCTTATCG
sequence
and cutting
sequence
you
need to get them
into this
plasmid. How Enzyme
should
ATATTCTAACGCTACGGGACGTCGATAAGCCGACGGATTTTAGCCGGGGATTCTTAAGAATAGC
E
G* A A T T C
Eco RI
you
cut the
soT
can
incorporate
the
DNA
T
A
A *G
If this sequence
(above)plasmid
of DNA wereCcut
withit
both
enzymes,
how many pieces would
be created?
P be grown in bacteria
C T G C
A*Gampicillin in thePst I
and
with
These restriction enzymes produce sticky
ends
G *A C G T C
where DNA nucleotides are not bound to their
environment?
pair. Thus, they can be easily hooked up to
1.pieceIn
strand
a. 
With
Phasand
Eof DNA shown below, find the restriction enzyme sites.
another
thatthe
the
complementary
unbound nucleotides:
b. 
With H and E
TATAAGATTGCGATGCCCTGCAGCTATTCGGCTGCCTAAAATCGGCCCCTAAGAA
c. ATATTCTAACGCTACGGGACGTCGATAAGCCGACGGATTTTAGCCGGGGATTCTT
With B
d.  With P and H
If this sequence (above) of DNA were cut with both enzymes, how many pieces would be creat
18-15
These restriction enzymes produce sticky ends
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Practice:
Here is a bacterial plasmid and a piece of human DNA that contains a
gene called tol. You want to join the tol gene and the bacterial plasmid
together, and you want all of the tol gene to be present. You also
want to use Ampicillin (Amp) to select for bacteria that take up this
plasmid. What enzyme(s) should you use to cut both the plasmid and
the genomic DNA?
Amp
resistance
gene
Ori
Unique
restriction
sites
tol gene
BamHI
EglI
A.  XhoI
B.  BamHI
C.  EglI and BamHI
D.  EcoRII
18-16
E.  BamHI and XhoI
BamHI
EcoRII
XhoI
EglI
EcoRII
XhoI
XhoI
XhoI
EcoRII
BamHI
Human genomic DNA
Plasmid DNA
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Another one:
Here is a bacterial plasmid and a piece of human DNA that contains a
gene called qt. You want to join the qt gene and the bacterial plasmid
together and introduce them into bacteria. You also want to use
Ampicillin (Amp) to select for bacteria that take up this plasmid.
What enzyme(s) should you use to cut both the plasmid and the
genomic DNA?
Amp
resistance
gene
Ori
Unique
restriction
sites
qt gene
EcoRII
A.  XhoI
EglI
B.  BamHI and XhoI
BamHI
C.  EglI
D.  EcoRII
18-17
E.  BamHI and EcoRII
EcoRII
XhoI
EglI
EglI
XhoI
XhoI
BamHI
Human genomic DNA
Plasmid DNA
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
An organism that contains DNA from
another organism in its genome is called a
“transgenic organism”
Thus, a bacterium with a piece of human DNA
in it is a transgenic bacterium!
A plant engineered to contain a piece of DNA
from another organism is also transgenic: we
usually call these “GMOs”
18-18
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Genetically Modified Foods
• 
• 
• 
• 
higher yield
improved quality
pest or disease resistance
tolerance to heat, cold and drought.
Plants have been bred for years to specifically
yield these desirable qualities
Transgenic technology allows the introduction of
genes from OTHER organisms into a plant or
animal to yield a specific outcome
18-19
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
One example: Insect Resistance
Bt corn
Normal corn
Bacillus thuringiensis
(Bt) (a bacterium):
Makes a protein that
causes paralysis and
death to some
insects (corn weevil).
18-20
Use this rather than an applied
pesticide, which often kills beneficial
insects as well!