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Chp 20: DNA Technology and
Genomics
Activity 20A
LE 20-2
Bacterium
Gene inserted into
plasmid
Cloning
Bacterial
chromosome
Cell containing gene
of interest
Plasmid
Recombinant
DNA (plasmid)
Gene of
interest
Plasmid put into
bacterial cell
DNA of
chromosome
Recombinant
bacterium
Host cell grown in culture
to form a clone of cells
containing the “cloned”
gene of interest
Gene of
interest
Protein expressed
by gene of interest
Copies of gene
Basic
research
on gene
Gene for pest
resistance inserted
into plants
Protein harvested
Basic research and
various applications
Gene used to alter
bacteria for cleaning
up toxic waste
Protein dissolves
blood clots in heart
attack therapy
Basic
research
on protein
Human growth hormone treats stunted
growth
LE 20-3
Restriction site
Recombinant
DNA
DNA 5
3
3
5
Restriction enzyme cuts
the sugar-phosphate
backbones at each arrow.
Sticky end
DNA fragment from another
source is added. Base pairing
of sticky ends produces
various combinations.
Fragment from different
DNA molecule cut by the
same restriction enzyme
RJ animation
One possible combination
DNA ligase
seals the strands.
Activity: Restriction
Enzymes
Recombinant DNA molecule
LE 20-4_3
Bacterial cell
Cloning
using a
bacterial
plasmid
Isolate plasmid DNA
and human DNA.
lacZ gene
(lactose
breakdown)
Human
cell
Restriction
site
ampR gene
(ampicillin
resistance)
Cut both DNA samples with
the same restriction enzyme.
Bacterial
plasmid
Gene of
interest
Sticky
ends
Human DNA
fragments
Mix the DNAs; they join by base pairing.
The products are recombinant plasmids
and many nonrecombinant plasmids.
Recombinant DNA plasmids
Introduce the DNA into bacterial cells
that have a mutation in their own lacZ
gene.
Recombinant
bacteria
Plate the bacteria on agar
containing ampicillin and X-gal.
Incubate until colonies grow.
Colony carrying nonrecombinant plasmid
with intact lacZ gene
Activity: Cloning a
gene in bacteria
Colony carrying
recombinant
plasmid with
disrupted lacZ gene
Bacterial
clone
RJ animation
LE 20-5
Nucleic Acid Hybridization
Master plate
Filter
Master plate
Probe
DNA
Radioactive
single-stranded
DNA
Solution
containing
probe
Colonies
containing
gene of
interest
Gene of
interest
Single-stranded
DNA from cell
Film
Filter lifted
and flipped over
Hybridization
on filter
A special filter paper
is pressed against
the master plate,
transferring cells to
the bottom side of
the filter.
The filter is treated to break
open the cells and denature
their DNA; the resulting
single-stranded DNA
molecules are treated so that
they stick to the filter.
The filter is laid
under photographic
film, allowing any
radioactive areas to
expose the film
(autoradiography).
After the
developed film is
flipped over, the
reference marks
on the film and
master plate are
aligned to locate
colonies carrying
the gene of
interest.
LE 20-6
Genomic Library
or
Bacterial
clones
Foreign genome
cut up with
restriction
enzyme
Recombinant
plasmids
Recombinant
phage DNA
Plasmid library
Phage
clones
Phage library
RJ animation
cDNA
LE 20-7
5
3
PCR
Target
sequence
Genomic DNA
Denaturation:
Heat briefly
to separate DNA
strands
Cycle 1
yields
2
molecules
Annealing:
Cool to allow
primers to form
hydrogen bonds
with ends of
target sequence
Extension:
DNA polymerase
adds nucleotides to
the 3 end of each
primer
Cycle 2
yields
4
molecules
Cycle 3
yields 8
molecules;
2 molecules
(in white boxes)
match target
sequence
3
5
5
3
3
5
Primers
RJ animation
New
nucleotides
3D video (amplification
techniques)
LE 20-8
Gel Electrophoresis
Cathode
Power
source
Mixture
of DNA
molecules
of different sizes
Longer
molecules
Shorter
molecules
Gel
Glass
plates
Anode
Activity: Gel Electrophoresis
Southern Blotting
Restriction
fragments
DNA + restriction enzyme
I
II
III
Heavy
weight
Nitrocellulose
paper (blot)
Gel
Sponge
I Normal
-globin
allele
II Sickle-cell III Heterozygote
allele
Preparation of restriction fragments.
Radioactively
labeled probe
for -globin
gene is added
to solution in
a plastic bag
I
II
III
Paper
towels
Alkaline
solution
Gel electrophoresis.
Probe hydrogenbonds to fragments
containing normal
or mutant -globin
Blotting.
I
II
Fragment from
sickle-cell
-globin allele
Paper blot
Hybridization with radioactive probe.
III
Film over
paper blot
Fragment from
normal -globin
allele
Autoradiography.
LE 20-9
Normal -globin allele
Restriction
Fragment
Analysis
175 bp
Ddel
201 bp
Ddel
Large fragment
Ddel
Ddel
Sickle-cell mutant -globin allele
376 bp
Ddel
Large fragment
Ddel
Ddel
Ddel restriction sites in normal and sickle-cell alleles of
-globin gene
Normal
allele
Sickle-cell
allele
Large
fragment
376 bp
201 bp
175 bp
Electrophoresis of restriction fragments from normal
and sickle-cell alleles
RJ animation
LE 20-11
Chromosome
bands
Cytogenetic map
Mapping
Genes located
by FISH
Genetic (linkage)
mapping
Genetic
markers
Physical mapping
Overlapping
fragments
DNA sequencing
LE 20-12
DNA
(template strand)
5
Primer
3
Deoxyribonucleotides
Dideoxyribonucleotides
(fluorescently tagged)
Dideoxymethod
(Sanger) DNA
sequencing
5
DNA
polymerase
3
5
DNA (template
strand)
Labeled strands
3
Direction
of movement
of strands
Laser
Detector
3
LE 20-13
Cut the DNA from
many copies of an
entire chromosome
into overlapping fragments short enough
for sequencing
Clone the fragments
in plasmid or phage
vectors
Sequence each fragment
Order the
sequences into one
overall sequence
with computer
software
Table 20-1
LE 20-14
DNA
Microarray
Assay
Tissue sample
Isolate mRNA.
Make cDNA by reverse
transcription, using
fluorescently labeled
nucleotides.
Apply the cDNA mixture to a
microarray, a microscope slide
on which copies of singlestranded DNA fragments from
the organism’s genes are fixed,
a different gene in each spot.
The cDNA hybridizes with any
complementary DNA on the
microarray.
Rinse off excess cDNA; scan
microarray for fluorescent.
Each fluorescent spot
(yellow) represents a gene
expressed in the tissue
sample.
mRNA molecules
Labeled cDNA molecules
(single strands)
DNA
microarray
Size of an actual
DNA microarray
with all the genes
of yeast (6,400 spots)
Figure 20-1
DNA Arrays & Gene Chips
RJ animation
LE 20-15
RFLP marker
analysis
RFLP marker
DNA
Restriction
sites
Disease-causing
allele
Normal allele
LE 20-16
Cloned gene
Insert RNA version of normal allele
into retrovirus.
Viral RNA
Retrovirus
capsid
Let retrovirus infect bone marrow cells
that have been removed from the
patient and cultured.
Viral DNA carrying the normal
allele inserts into chromosome.
Bone
marrow
cell from
patient
Inject engineered
cells into patient.
Bone
marrow
LE 20-17
Defendant’s
blood (D)
Blood from defendant’s
clothes
Victim’s
blood (V)
Activity: DNA Fingerprinting
Video: DNA Forensics
Figure 20-18
Insulin &
Genentech
Transgenics video
Figure 20.20 “Golden” rice contrasted with ordinary rice
Activity: Golden Rice
LE 20-19
Agrobacterium tumefaciens
Ti
plasmid
Site where
restriction
enzyme cuts
T DNA
DNA with
the gene
of interest
Recombinant
Ti plasmid
RJ animation
Plant with
new trait