Download Chapter 20: Biotechnology 11/18/2015

11/18/2015
Chapter 20:
Biotechnology
1. DNA Sequencing
2. DNA Cloning
3. Studying Gene Expression
4. Manipulating Genomes
5. Therapeutic & Diagnostic
Techniques
1. DNA Sequencing
Chapter Reading – pp. 409-412
DNA Sequencing…
TECHNIQUE
DNA
(template strand)
5
3
C
T
G
A
C
T
T
C
G
A
C
A
A
Primer
T
G
T
T
Deoxyribonucleotides
3
5
DNA
polymerase
Dideoxyribonucleotides
(fluorescently tagged)
dATP
ddATP
dCTP
ddCTP
dTTP
ddTTP
dGTP
ddGTP
P P P
G
OH
P P P
G
H
• DNA replication in vitro using 1 of 4 different “chainterminating” dideoxynucleotides (ddNTPs)
• results in a set of DNA fragments ending in all positions
with A, C, G, or T that can be resolve by length on a gel
1
11/18/2015
DNA Sequencing uses
Chain Terminators
Normal nucleotide
(dNTP)
Dideoxynucleotide
(ddNTP)
*
DNA synthesis is carried out in reactions
containing the following:
• DNA template to be sequenced
• dNTP’s
• DNA primer
• DNA polymerase
• ddATP, ddCTP, ddGTP or ddTTP
• each ddNTP is labeled with a different
fluorescent tag
The color reveals the identity of the ddNTP that
terminated DNA synthesis at each position,
thus revealing the sequence!
…DNA Sequencing…
TECHNIQUE (continued)
5
3
DNA (template
C strand)
T
G
A
C
T
T
C
ddG
G
C
ddC
A
T
T
G
C
G
A
T
T
T
A
T
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
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
Longest labeled strand
Detector
Laser
Shortest labeled strand
2
11/18/2015
…DNA Sequencing
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
“Next Generation”
Sequencing
2. DNA Cloning
Chapter Reading – pp. 409-417
3
11/18/2015
What is “Recombinant DNA”?
The joining of DNA from different sources.
This can happen in nature (in vivo)…
• the transfer of DNA involving bacteria or viruses
…or in the laboratory (in vitro)
• the cutting & splicing of DNA fragments by
molecular biologists
The term “recombinant DNA” generally refers to the in
vitro kind which is commonly called “gene cloning”…
Gene Cloning…
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
• gene of interest is inserted into a plasmid vector
• bacterial host transformed with recombinant plasmid
• bacterial clone with recombinant plasmid is identified
…Gene Cloning
3 Host cell grown in
culture to form a clone
of cells containing the
“cloned” gene of interest
Cloned gene can
then be used in a
variety of ways.
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
4
11/18/2015
Restriction site
5
Restriction
Enzymes
3
GAATTC
CTTAAG
DNA
5
3
1 Restriction enzyme
cuts sugar-phosphate
backbones.
5
3
5
5 Sticky 3
3
5
end
5
3
2 DNA fragment added
from another molecule
cut by same enzyme.
Base pairing occurs.
5
3
5
e.g. EcoRI cuts at:
3 5
3 5
G AATT C
C TTAA G
G AATT C
C TTAA G
53
5 3
3
Gene cloning usually
involves the use of
restriction enzymes
that cut DNA at very
specific sequences:
3
..GAATTC..
..CTTAAG..
3
5
One possible combination
3 DNA ligase
seals strands
5
3
3
5
Recombinant DNA molecule
There are many different
restriction enzymes,
each cutting a different
sequence
The Gene Cloning Technique
Hummingbird cell
TECHNIQUE
Bacterial plasmid
ampR gene
lacZ gene
Restriction
site
• cut plasmid &
DNA to be
cloned with
same RE
Sticky
ends
Gene of
interest
Hummingbird DNA
fragments
• ligate vector &
DNA insert with
DNA ligase
Recombinant plasmids
• transform bacteria
with ligated DNA
Nonrecombinant
plasmid
Bacteria carrying
plasmids
Selection for Bacterial Clones
Antibiotic resistance allows selection for bacterial
clones containing the plasmid vector.
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
Blue/white
selection via
lacZ gene
disruption
allows
identification
of clones
containing a
DNA insert.
5
11/18/2015
TECHNIQUE
1
Power
source
 Cathode
Anode 
Mixture of
DNA molecules of
different
sizes
Wells
Gel
Power
source
2


Longer
molecules
Shorter
molecules
Gel
Electrophoresis
Separation of DNA
fragments through a
porous gel matrix:
• gel is either agarose or
polyacrylamide
• electric current pulls
negatively charged DNA
toward the positive pole
RESULTS
• rate of movement is
inversely proportional to
DNA fragment size
DNA Libraries…
A collection of cloned genes from an
organism is called a DNA library.
Genomic DNA Library
• a collection of chromosomal DNA fragments cloned
into a particular vector
• essentially cloned pieces of the organism’s genome
cDNA Library
• a collection of DNA fragments produced from
messenger RNA (mRNA) cloned into a vector
• produced from mRNA by reverse transcriptase
• a collection of expressed genes with NO intron DNA
…DNA Libraries
Foreign genome
Cut with restriction enzymes into either
small
large
or
fragments
fragments
Bacterial artificial
chromosome (BAC)
Large
insert
with
many
genes
Recombinant
plasmids
(b) BAC clone
Plasmid
clone
(a) Plasmid library
(c) Storing genome libraries
6
11/18/2015
Producing cDNA
DNA in
nucleus
mRNAs in
cytoplasm
mRNA
5
Reverse
transcriptase
cDNA (DNA complementary to
mRNA) is produced as follows:
• purify mRNA fr. desired cell type
Poly-A tail
A A A A A A 3
T T T T T 5
3
• treat with reverse transcriptase
and oligo-dT primer
DNA Primer
strand
• results in double-stranded DNA
copies of all mRNA from the cell
A A A A A A 3
T T T T T 5
5
3
5
3
DNA
polymerase
3
• clone cDNA fragments into
vector such as a plasmid
• transform bacterial hosts
5
Results in a collection of cloned
cDNA corresponding to coding
sequences of all expressed proteins
3
5
5
3
cDNA
Screening DNA Libraries
Radioactively
labeled probe
molecules
TECHNIQUE
5
3
 CTCATCACCGGC
GAGTAGTGGCCG
5
3
Gene of
interest
Probe
DNA
Film
Singlestranded
DNA from
cell
Multiwell plates
holding library
clones
Nylon membrane
Nylon
membrane
Location of
DNA with the
complementary
sequence
• DNA from library clones immobilized on a membrane
• radioactively labeled DNA similar in sequence to desired
clone is added as a probe
• hybridization of probe to complementary DNA IDs clone
PCR
TECHNIQUE
• essentially DNA replication
in vitro
3
3
5
Target
sequence
Genomic DNA
The Polymerase Chain
Reaction is a technique
to selectively amplify a
desired DNA sequence:
5
1 Denaturation
5
3
3
5
2 Annealing
Cycle 1
yields
2
molecules
Primers
3 Extension
New
nucleotides
• results in exponential
amplification of target DNA
sequence
• artificial primers specific
for target DNA sequence
limit replication to DNA
complementary to primers
Cycle 2
yields
4
molecules
Cycle 3
yields 8
molecules;
2 molecules
(in white boxes)
match target
sequence
7
11/18/2015
Overview of PCR
Every PCR reaction requires the following:
1) source of target DNA template
2) artificial primers “flanking” DNA of interest
3) heat-stable DNA polymerase (from hyperthermophile)
4) dNTP’s
5) automated thermocycler to facilitate repeated:
• denaturation of DNA (separating the 2 strands)
• hybridization of primers to template
• DNA synthesis
Resulting PCR products can then be
analyzed by gel electrophoresis.
“PCR Cloning” involves
designing restriction
sites into the primers to
facilitate cloning
3. Studying Gene Expression
Chapter Reading – pp. 417-419, 421-422
8
11/18/2015
TECHNIQUE
1 cDNA synthesis
mRNAs
cDNAs
2 PCR amplification
Primers
-globin
gene
3 Gel electrophoresis
RESULTS
Embryonic stages
2
1
3 4 5 6
RT-PCR
• produce cDNA
with reverse
transcriptase
• carry out PCR
using specific
primers for
desired gene
• gel electrophoresis
of resulting PCR
fragments
Amount of DNA
reflects amount of
original mRNA
sequence.
in situ Hybridization
…in situ Hybridization
How is in situ hybridization carried out?
• fluorescently labeled anti-sense RNA probes
added to tissue sample
• hybridization with complementary mRNA
sequences
• location of specific gene expression within the
tissue (i.e., in situ) is revealed
9
11/18/2015
DNA Microarrays
A DNA microarray is a solid surface containing a
precise array of single-stranded DNA sequences
from 1000s of different genes in an organism.
• labeled cDNA is produced from test cells and
allowed to hybridize with sequences in the
array
• intensity of signals reveal expression of
specific genes within the test cells.
When cDNA from different sources is labeled differently,
gene expression from each source can be compared in a
single microarray (as shown on the slide after next).
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
Analyzing
gene
expression
using a DNA
microarray.
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
Comparing Gene Expression
10
11/18/2015
Single Nucleotide
Polymorphisms (SNPs)
SNPs are single nucleotide differences that correspond with
specific disease-causing alleles.
PCR and hybridization techniques (e.g., microarrays) can
reveal the presence of such alleles (genetic testing).
DNA
T
Normal allele
SNP
C
Disease-causing
allele
4. Manipulating Genomes
Chapter Reading – pp. 422-425, 429-430
Cloning Plants
Some plants can be cloned from a single adult plant cell.
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
11
11/18/2015
Frog embryo
EXPERIMENT
Frog egg cell
Frog tadpole
UV
This
experiment
produced
the first
artificially
cloned
animal.
Fully differentiated
(intestinal) cell
Less differentiated cell
Donor
nucleus
transplanted
Enucleated
egg cell
Egg with donor nucleus
activated to begin
development
Donor
nucleus
transplanted
RESULTS
Cloning
Animals
TECHNIQUE
Mammary
cell donor
Egg cell
donor
1
2
Egg
cell from
ovary
Cultured
mammary
cells
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
Most stop developing
before tadpole stage.
Most develop
into tadpoles.
The first cloned
mammal, “Dolly
the sheep”, was
cloned by this
method.
Other mammals
such as cats
have since been
cloned and
presumably
humans could be
cloned this way
as well.
Lamb (“Dolly”) genetically
identical to mammary cell donor
Transgenic Organisms
Transgenic organisms have a foreign gene (e.g. from
another species) inserted into their genome.
• also known as “genetically modified organisms” (GMOs)
12
11/18/2015
5. Therapeutic & Diagnostic
Techniques
Chapter Reading – pp. 425-431
Stem Cells
Adult
stem cells
Embryonic
stem cells
Cells generating
some cell types
Cells generating
all embryonic
cell types
Cultured
stem cells
Different
culture
conditions
Liver
cells
Nerve
cells
Different
types of
differentiated
cells
Blood
cells
Stem cells are
undifferentiated cells
that can give rise to
multiple differentiated
cell types.
Embryonic stem cells
are considered more
pluripotent than adult
stem cells, though
techniques are being
developed to expand
the potential of adult
stem cell.
Stem Cell Therapy
Techniques are
being developed to
treat a person’s
own cells in vitro to
produce a needed
cell type which is
then reintroduce
into the patient to
repair damaged
tissue.
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.
13
11/18/2015
Gene Therapy
Cloned gene
1 Insert RNA version of normal allele
into retrovirus.
Viral RNA
2 Let retrovirus infect bone marrow cells
that have been removed from the
patient and cultured.
Retrovirus
capsid
3 Viral DNA carrying the normal
allele inserts into chromosome.
Bone
marrow
cell from
patient
Bone marrow
stem cells can
be genetically
modified in vitro
to fix a genetic
defect and then
reintroduced back
into the patient.
Bone
marrow
4 Inject engineered
cells into patient.
Restriction Fragment Length
Polymorphisms (RFLPs)
Normal -globin allele
175 bp
DdeI
Sickle-cell
allele
Large fragment
201 bp
DdeI
Normal
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
• DNA from different sources cut with the same RE will
result in different size DNA fragments (RFLPs)
Short Tandem Repeat Analysis
Many parts of the
human genome
contain short DNA
sequences repeated
many times in a row
(in tandem)
• the number of repeats
in these regions is
highly variable from
person to person
• RFLP analysis can
produce a DNA
fingerprint that is
unique for each
individual
(a) This photo shows
Washington just before
his release in 2001,
after 17 years in prison.
Source of
sample
STR
marker 1
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
STR
marker 2
STR
marker 3
(b) These and other STR data exonerated Washington
and led Tinsley to plead guilty to the murder.
14
11/18/2015
Southern Blotting
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
Alkaline
solution
2 Gel electrophoresis
II III
Radioactively labeled
probe for -globin
gene
Nitrocellulose blot
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
4 Hybridization with labeled probe
Key Terms for Chapter 20
• recombinant DNA, gene cloning, plasmid vector
• transformation, ligation, restriction enzymes
• genomic & cDNA libraries, hybridization, probe
• gel electrophoresis, PCR, RT-PCR
• dideoxynucleotide, RFLP, STR, SNP
• in situ hybridization, DNA microarrays
• transgenic, stem cell, southern blot
• in vivo, in vitro, in situ
Relevant
Chapter
Questions
1-10, 12
15