7. Figure 20.2 A preview of gene cloning and some uses of cloned genes.
Bacterium
1 Gene inserted into
plasmid
Bacterial
Plasmid
chromosome
Recombinant
DNA (plasmid)
Cell containing gene
of interest
Gene of
interest
DNA of
2 Plasmid put into chromosome
(“foreign” DNA)
bacterial cell
Recombinant
bacterium
3 Host cell grown in culture to
form a clone of cells containing
the “cloned” gene of interest
Protein expressed from
gene of interest
Gene of
interest
Copies of gene
Basic
research
on gene
Protein harvested
4 Basic research
and various
applications
Basic
research
on protein
Gene for pest
Gene used to alter Protein dissolves
Human growth
resistance inserted bacteria for cleaning blood clots in heart hormone treats
into plants
up toxic waste
attack therapy
stunted growth
9. Figure 20.3 Using a restriction enzyme and DNA ligase to make recombinant DNA.
Restriction site
5′
3′
GAATTC
CTTAAG
DNA
3′
5′
1 Restriction enzyme
cuts sugar-phosphate
backbones.
3′
5′
G
CTTAA
end
2 DNA fragment added
from another molecule
cut by same enzyme.
Base pairing occurs.
5′
3 DNA ligase
3′
AATTC
G
5′ Sticky 3′
3′
3′
5′
3′ 5′
G AATT C
C TTAA G
5′3′
5′
5′
3′
AATTC
G
G
CTTAA
3′
5′
3′ 5′
G AATT C
C TTAA G
5′ 3′
One possible combination
3′
5′
seals strands
5′
3′
3′
Recombinant DNA molecule
5′
15. Figure 20.5 Genomic libraries.
Foreign genome
Cut with restriction enzymes into either
small
large
or
Bacterial artificial
fragments
fragments
chromosome (BAC)
Large
insert
with
many
genes
Recombinant
plasmids
(b) BAC clone
Plasmid
clone
(a) Plasmid library
(c) Storing genome libraries
18. Figure 20.6 Making complementary DNA (cDNA) from eukaryotic genes.
DNA in
nucleus
mRNAs in
cytoplasm
Reverse
Poly-A tail
mRNA transcriptase
A A A A A A 3′
5′
3′
T T T T T 5′
DNA Primer
strand
A A A A A A 3′
T T T T T 5′
5′
3′
5′
3′
3′
DNA
polymerase
5′
3′
cDNA
5′
3′
5′
22. Figure 20.7 Research Method: Detecting a Specific DNA Sequence by Hybridization with a
Nucleic Acid Probe
Radioactively
labeled probe
molecules
TECHNIQUE
Gene of
interest
Probe
DNA
Multiwell plates
holding library
clones
Nylon membrane
5′
3′
⋅⋅⋅ CTCATCACCGGC⋅⋅⋅
GAGTAGTGGCCG
5′
3′
Singlestranded
DNA from
cell
Location of
DNA with the
complementary
sequence
Film
Nylon
membrane
35. Figure 20.11 Research Method: Southern Blotting of DNA Fragments
TECHNIQUE
DNA + restriction enzyme
Restriction
fragments
I
II III
Heavy
weight
Nitrocellulose
membrane (blot)
Gel
Sponge
I Normal II Sickle-cell III Heterozygote
β-globin allele
allele
1 Preparation of
restriction fragments
I
II III
Radioactively labeled
probe for β-globin
gene
Nitrocellulose blot
4 Hybridization with labeled probe
Alkaline
solution
2 Gel electrophoresis
Paper
towels
3 DNA transfer (blotting)
Probe base-pairs
with fragments
Fragment from
sickle-cell
β-globin allele
Fragment from
normal β- globin
allele
I
II III
Film
over
blot
5 Probe detection
37. Figure 20.12 Research Method: Dideoxy Chain Termination Method for Sequencing DNA
TECHNIQUE
DNA
(template strand)
5′ C
3′
5′
3′
T
G
A
C
T
T
C
G
A
C
A
A
Primer Deoxyribonucleotides Dideoxyribonucleotides
T 3′
(fluorescently tagged)
G
T
T
dATP
ddCTP
dTTP
ddTTP
dGTP
DNA
polymerase
ddATP
dCTP
5′
ddGTP
P P P
P P P
G
OH
DNA (template
C
T strand)
G
A
C
T
T
C
ddG
C
G
ddC
A
T
T
C
G
G
A
T
T
T
A
T
H
Labeled strands
ddA
G
C
T
G
T
T
ddA
A
G
C
T
G
T
T
ddG
A
A
G
C
T
G
T
T
ddT
G
A
A
G
C
T
G
T
T
ddC
T
G
A
A
G
C
T
G
T
T
Shortest
Direction
of movement
of strands
Longest labeled strand
Detector
Laser
Shortest labeled strand
RESULTS
Last nucleotide
of longest
labeled strand
Last nucleotide
of shortest
labeled strand
G
A
C
T
G
A
A
G
C
G
ddA
C
T
G
A
A
G
C
T
G
T
T
ddG
A
C
T
G
A
A
G
C
T
G
T
T
3′
5′
Longest
45. Figure 20.15
TECHNIQUE
1 Isolate mRNA.
2 Make cDNA by reverse
transcription, using
fluorescently labeled
nucleotides.
3 Apply the cDNA mixture to a
microarray, a different gene
in each spot. The cDNA hybridizes
with any complementary DNA on
the microarray.
Tissue sample
mRNA molecules
Labeled cDNA molecules
(single strands)
DNA fragments
representing a
specific gene
DNA microarray
4 Rinse off excess cDNA; scan microarray
for fluorescence. Each fluorescent spot
(yellow) represents a gene expressed
in the tissue sample.
DNA microarray
with 2,400
human genes
59. Figure 20.22 Impact: The Impact of Induced Pluripotent Stem (iPS) Cells on Regenerative Medicine
1 Remove skin cells
from patient.
2 Reprogram skin cells
so the cells become
induced pluripotent
stem (iPS) cells.
Patient with
damaged heart
tissue or other
disease
3 Treat iPS cells so
that they differentiate
into a specific
cell type.
4 Return cells to
patient, where
they can repair
damaged tissue.
63. Figure 20.23 Gene therapy using a retroviral vector.
Cloned gene
1 Insert RNA version of normal allele
into retrovirus.
Viral RNA
Retrovirus
capsid
2 Let retrovirus infect bone marrow cells
that have been removed from the
patient and cultured.
3 Viral DNA carrying the normal
allele inserts into chromosome.
Bone
marrow
cell from
patient
4 Inject engineered
cells into patient.
Bone
marrow
69. Figure 20.25 STR analysis used to release an innocent man from prison.
(a) This photo shows
Washington just before
his release in 2001,
after 17 years in prison.
Source of
sample
STR
marker 1
STR
marker 2
STR
marker 3
Semen on victim
17,19
13,16
12,12
Earl Washington
16,18
14,15
11,12
Kenneth Tinsley
17,19
13,16
12,12
(b)These and other STR data exonerated Washington
and led Tinsley to plead guilty to the murder.
73. Figure 20.26 Research Method: Using the Ti Plasmid to Produce Transgenic Plants
TECHNIQUE
Agrobacterium tumefaciens
Ti
plasmid
Site where
restriction
enzyme cuts
T DNA
DNA with
the gene
of interest
RESULTS
Recombinant
Ti plasmid
Plant with new trait