Download Gene Technology

Avery, MacLeod, and McCarty
1944
• Used bacteria from Griffith’s mouse
experiment
• Denatured proteins in membrane and
discovered that the DNA still could
make other bacteria pathogenic
Biotechnology – pg. 140 in Cliffs
Ch. 20 in text





Recombinant DNA – a combination of
DNA segments from two different sources
Can occur through transduction,
conjugation, transformation
Can also occur during crossing over during
meiosis in eukaryotes
Biotechnology – use of biological systems
to produce products like medicine
Often use bacteria and viruses in
experiments and production of products
Recombinant DNA technology
 Set of lab techniques for combining genes
from different sources.
 Requires the “cutting” of DNA using
restriction enzymes
 Restriction enzymes cut DNA at very
specific sequences called restriction sites
 Using bacterial plasmids we can clone
specific genes to produce proteins of
interest
 Ex. Medicine, farming, oil clean up
Creating Sticky Ends
Animation: Restriction Enzymes
Right-click slide / select “Play”
© 2011 Pearson Education, Inc.
Using Restriction Enzymes to cut DNA
• Restriction Enzyme Video
Figure 20.3-3
Restriction site
5
3
GAATTC
CTTAAG
DNA
5
3
1 Restriction enzyme
cuts sugar-phosphate
backbones.
5
3
5
3
5 Sticky 3
3
5
end
5
2 DNA fragment added
3
3
5
from another molecule
cut by same enzyme.
Base pairing occurs.
5
3 5
3
3 DNA ligase
3 5
G AATT C
C TTAA G
G AATT C
C TTAA G
53
5 3
3
5
One possible combination
seals strands
5
3
3
Recombinant DNA molecule
5
Recombinant DNA
Figure 20.6-5
DNA in
nucleus
mRNAs in
cytoplasm
Reverse
transcriptase Poly-A tail
mRNA
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
DNA
polymerase
3
5
3
5
5
3
cDNA
Figure 20.2
Bacterium
1 Gene inserted into
plasmid
Bacterial
Plasmid
chromosome
Recombinant
DNA (plasmid)
Cell containing gene
of interest
Gene of
interest
2 Plasmid put into
bacterial cell
DNA of
chromosome
(“foreign” DNA)
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
Protein harvested
Copies of gene
Basic
research
on gene
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
DNA cloning
1. Use restriction enzyme to cut a sample of DNA in
test tube – this will create fragments with sticky
ends, some will have our gene of interest
2. Cut a plasmid (cloning vector) with one restriction
site for the restriction enzyme – the plasmid will
now have the same sticky ends (plasmid should
also be resistant to antibiotic like ampicillin)
3. Mix the foreign DNA with the plasmids
4. Apply DNA ligase
Transformation Time
 Place the engineered plasmid into bacterial
culture (in test tube)
 Heat shock and let transformation occur
 Plate the bacteria and those that grow on
ampicillin will have “transformed” with the
foreign gene of interest
Genomic Library
 At the end, the bacteria now contain our
gene of interest – genomic library
 Now the gene can be transcribed and
translated to make the protein of interest
 This DNA is without introns because it was
made from mRNA using reverse
transcriptase before the experiment. cDNA
Figure 20.4
TECHNIQUE
Bacterial plasmid
R
amp gene
Hummingbird cell
lacZ gene
Restriction
site
Sticky
ends
Gene of
interest
Hummingbird DNA
fragments
Recombinant plasmids Nonrecombinant
plasmid
Bacteria carrying
plasmids
RESULTS
Colony carrying nonrecombinant plasmid
with intact lacZ gene
Colony carrying
recombinant
plasmid
with disrupted
lacZ gene
One of many
bacterial
clones
Figure 20.5
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
Storing Cloned Genes in DNA Libraries
• A genomic library that is made using bacteria is
the collection of recombinant vector clones
produced by cloning DNA fragments from an
entire genome
• A genomic library that is made using
bacteriophages is stored as a collection of phage
clones
• A clone carrying the gene of interest can be
identified with a nucleic acid probe having a
sequence complementary to the gene
• This process is called nucleic acid hybridization
© 2011 Pearson Education, Inc.
• A probe can be synthesized that is
complementary to the gene of interest
• For example, if the desired gene is
5
 CTCATCACCGGC
3
– Then we would synthesize this probe
3 GAGTAGTGGCCG
© 2011 Pearson Education, Inc.
5
Figure 20.7
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
Finding specific mutations
Gel Electrophoresis
• In humans, researchers analyze the genomes of
many people with a certain genetic condition to try to
find nucleotide changes specific to the condition
• Genetic markers called SNPs (single nucleotide
polymorphisms) occur on average every 100–300
base pairs
© 2011 Pearson Education, Inc.
Figure 20.16
DNA
T
Normal allele
SNP
C
Disease-causing
allele
Figure 20.10
Normal -globin allele
175 bp
DdeI
Large fragment
201 bp
DdeI
Normal Sickle-cell
allele
allele
DdeI
DdeI
Large
fragment
Sickle-cell mutant -globin allele
376 bp
376 bp
DdeI
201 bp
175 bp
Large fragment
DdeI
DdeI
(a) DdeI restriction sites in normal and
sickle-cell alleles of the -globin gene
(b) Electrophoresis of restriction
fragments from normal and
sickle-cell alleles
Figure 20.11
TECHNIQUE
DNA  restriction enzyme
Restriction
fragments
I
II III
Heavy
weight
Nitrocellulose
membrane (blot)
Gel
Sponge
I Normal II Sickle-cell III Heterozygote
-globin allele
allele
1 Preparation of
restriction fragments
I
II III
Radioactively labeled
probe for -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
-globin allele
Fragment from
normal - globin
allele
I
II III
Film
over
blot
5 Probe detection
Gel Box
Applications of Gene Technology
 DNA Fingerprint
DNA Fingerprinting
• DNA fingerprinting
Making copies of DNA - PCR
 Polymerase chain reaction (PCR) makes
copies of DNA in order to have enough
sample to run many tests on.
 You take the sample of DNA, and heat them
along with DNA polymerase and A,T,C,G
“primers”
 They will make millions of copies of the
sample.
Figure 20.8
5
TECHNIQUE
3
Target
sequence
Genomic DNA
1 Denaturation
3
5
5
3
3
5
2 Annealing
Cycle 1
yields
2
molecules
Primers
3 Extension
New
nucleotides
Cycle 2
yields
4
molecules
Cycle 3
yields 8
molecules;
2 molecules
(in white boxes)
match target
sequence
Studying the Expression of
Interacting Groups of Genes
 Automation has allowed scientists to measure
the expression of thousands of genes at one
time using DNA microarray assays
 DNA microarray assays compare patterns of
gene expression in different tissues, at different
times, or under different conditions
© 2011 Pearson Education, Inc.
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
Using reverse transcriptase in
gene therapy
 Isolate mRNA and use an enzyme called
reverse transcriptase to create DNA
 These artificial DNA molecules can be
inserted via a virus into a patient’s cells,
then into the patient.
Gene Therapy in humans
Gene technology in Farming
Golden Rice
 Rice injected with DNA
that codes for betacarotene that we use
to make vitamin A
DNA injection
Cloning Plants: Single-Cell Cultures
• One experimental approach is to see whether a
differentiated cell can generate a whole organism
• A totipotent cell is one that can generate a
complete new organism
• Plant cloning is used extensively in agriculture
© 2011 Pearson Education, Inc.
Figure 20.17
Cross
section of
carrot root
2-mg
fragments
Fragments were
cultured in nutrient medium;
stirring caused
single cells to
shear off into
the liquid.
Single cells
free in
suspension
began to
divide.
Embryonic
plant developed
from a cultured
single cell.
Plantlet was
cultured on
agar medium.
Later it was
planted in soil.
Adult
plant
Reproductive Cloning of Mammals
• In 1997, Scottish researchers announced the birth
of Dolly, a lamb cloned from an adult sheep by
nuclear transplantation from a differentiated
mammary cell
• Dolly’s premature death in 2003, as well as her
arthritis, led to speculation that her cells were not
as healthy as those of a normal sheep, possibly
reflecting incomplete reprogramming of the
original transplanted nucleus
© 2011 Pearson Education, Inc.
TECHNIQUE
Mammary
cell donor
Egg cell
donor
1
Figure 20.19
Cultured
mammary
cells
2
Egg
cell from
ovary
3 Cells fused
4 Grown in culture
Nucleus
removed
Nucleus from
mammary cell
Early embryo
5 Implanted in uterus
of a third sheep
Surrogate
mother
6 Embryonic
development
RESULTS
Lamb (“Dolly”) genetically
identical to mammary cell donor
DNA Sequencing
• Relatively short DNA fragments can be sequenced
by the dideoxy chain termination method, the first
automated method to be employed
• Modified nucleotides called
dideoxyribonucleotides (ddNTP) attach to
synthesized DNA strands of different lengths
• Each type of ddNTP is tagged with a distinct
fluorescent label that identifies the nucleotide at
the end of each DNA fragment
• The DNA sequence can be read from the resulting
spectrogram
© 2011 Pearson Education, Inc.
Figure 20.12
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
5
DNA
polymerase
dATP
ddATP
dCTP
ddCTP
dTTP
ddTTP
dGTP
ddGTP
P P P
P P P
G
OH
DNA (template
C strand)
T
G
A
C
T
T
C
ddG
C
G
ddC
T
A
T
C
G
G
A
T
T
T
A
T
ddA
G
C
T
G
T
T
ddA
A
G
C
T
G
T
T
ddG
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
H
Labeled strands
ddT
G
A
A
G
C
T
G
T
T
G
A
C
T
G
A
A
G
C
G
ddC
T
G
A
A
G
C
T
G
T
T
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
Gene Sequencing
• Sanger Method of Sequencing
• DNA sequencing machine ad