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NOTES - CH 20: DNA Technology
● BIOTECHNOLOGY: the use of
living organisms or their
components to do practical
tasks
-microorganisms to make
wine/cheese
-selective breeding of livestock
-production of antibiotics
**Practical goal of biotech =
improvement of human health
and food production
Recombinant DNA
● Recombinant DNA =
DNA in which genes
from 2 different
sources are linked
● Genetic engineering =
direct manipulation of
genes for practical
purposes
“Toolkit” for DNA technology involves:
-restriction enzymes
-DNA vectors
-host organisms
RESTRICTION ENZYMES
(a.k.a. ENDONUCLEASES) =
enzymes that recognize
short, specific nucleotide
sequences called
restriction sites;
● in nature, these enzymes
protect bacteria from intruding
DNA; they cut up the DNA
(restriction); very specific
Restriction Enzymes…
● restriction sites are symmetrical
(“palindromes”) in that the same sequence
of 4-8 nucleotides is found on both strands,
but run in opposite directions
● restriction enzymes usually cut
phosphodiester bonds of both strands in a
staggered manner producing single stranded
“sticky ends”
Restriction Enzymes (cont.)…
● “sticky ends” of restriction fragments are
used in the lab to join DNA pieces from
different sources (complementary base
pairing)
● unions of different DNA sources can be
made permanent by adding the enzyme
DNA ligase
CLONING VECTOR = DNA molecule that
can carry foreign DNA from test tubes
back into cells & replicate once there
-bacterial plasmids (small, circular DNA
molecules that replicate within bacterial
cells)
-viruses
HOST ORGANISMS:
bacteria are commonly
used as hosts in genetic
engineering because:
1) DNA can easily be isolated from &
reintroduced into bacterial cells;
2) bacterial cultures grow quickly, rapidly
replicating any foreign genes they carry.
Steps Involved in
Cloning a Human Gene:
1) Isolate human gene to clone;
plasmid
2) Isolate plasmid from bacterial cell;
Human gene
3) Add restriction endonuclease to cut out
human gene & add same R.E. to open up
bacterial plasmid (creates the same “sticky
ends”);
4) Add human gene to the open bacterial
plasmid and seal with DNA ligase;
Cloning a Human Gene (cont.)…
5) Insert recombinant DNA plasmid back into
bacterial cell;
6) As bacterial cell reproduces, it makes copies of
the desired gene;
7) Identify cell clones carrying the gene of
interest.
-HOW? Which ones took up the gene & are
making insulin?
Bacterial plasmids in gene cloning
DNA Analysis & Genomics
● PCR (polymerase chain
reaction)
● Gel electrophoresis
● Restriction fragment
analysis (RFLPs)
● Southern blotting
● DNA sequencing
● Human genome project
The Polymerase Chain Reaction
(PCR)
● allows any piece of DNA to be quickly amplified
(copied many times) in vitro.
● DNA is incubated under
appropriate conditions
with special primers &
DNA polymerase
molecules
PCR (continued)…
● BILLIONS of copies of DNA are produced in just a
few hours (enough to use for testing)
In 6 cycles of PCR:
cycle 1: 2 copies
cycle 2: 4 copies
cycle 3: 8 copies
cycle 4: 16 copies
cycle 5: 32 copies
cycle 6: 64 copies
cycle 20: 1,048,576!!
Polymerase Chain Reaction
(PCR)
● PCR is highly
specific; primers
determine the
sequence to be
amplified
● only tiny amounts of
DNA are needed
Remember
these?
Starting materials for
PCR:
● DNA to be copied
● Nucleotides
● Primers
● Taq polymerase
(DNA polymerase isolated from bacteria
living in hot springs…their enzymes can
withstand high temps!)
Steps of PCR:
1) Heat to separate DNA
strands (95ºC);
2) Cool to allow primers
to bind (55ºC);
3) Heat slightly so that
DNA polymerase
extends the 3’ end of
each primer (72ºC)
4) Repeat steps #1-3
many times!!!
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
5
TECHNIQUE
3
Target
sequence
Genomic DNA
3
5
Figure 20.8b
1 Denaturation
5
3
3
5
2 Annealing
Cycle 1
yields
2
molecules
Primers
3 Extension
New
nucleotides
Figure 20.8c
Cycle 2
yields
4
molecules
Figure 20.8d
Cycle 3
yields 8
molecules;
2 molecules
(in white boxes)
match target
sequence
Applications of PCR:
● DNA / forensic analysis of tiny amounts of tissue
or semen found at crime scene;
● DNA from single embryonic cells for prenatal
diagnosis;
● DNA or viral genes from cells infected with
difficult-to-detect viruses such as HIV;
● used extensively in Human Genome Project to
produce linkage maps without the need for large
family pedigree analysis.
PCR works
like a
copying
machine for
DNA!
DNA Analysis
●
Gel electrophoresis: separates nucleic acids or
proteins on the basis of size or electrical charge
creating DNA bands of the same length
Restriction fragment analysis
Restriction fragment length polymorphisms
(RFLPs)
● Southern blotting: process that reveals
sequences and the RFLPs in a DNA sequence
● DNA Fingerprinting
●
DNA Sequencing
● Determination of
nucleotide sequences
(Sanger method,
sequencing machine)
● Human Genome
Project
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
T 3
G
T
T
Deoxyribonucleotides
5
DNA
polymerase
Dideoxyribonucleotides
(fluorescently tagged)
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
G
ddC
T
A
C
G
A
T
T
A
ddG
C
T
G
T
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
Figure 20.12a
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
Figure 20.12b
TECHNIQUE (continued)
5
3
DNA (template
C strand)
T
G
A
C
T
T
C
G
A
C
A
A
ddC
T
G
T
T
ddG
C
T
G
T
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
Shortest
Direction
of movement
of strands
3
5
Longest
Longest labeled strand
Detector
Laser
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
Shortest labeled strand
Figure 20.12c
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
Analyzing Gene Expression
● Nucleic acid probes can hybridize with mRNAs
transcribed from a gene
● Probes can be used to identify where or when a
gene is transcribed in an organism
© 2011 Pearson Education, Inc.
● In situ hybridization uses fluorescent dyes
attached to probes to identify the location of
specific mRNAs in place in the intact organism
© 2011 Pearson Education, Inc.
Figure 20.14
50 m
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
Figure 20.15a
DNA microarray
with 2,400
human genes
Determining Gene Function
● One way to determine function is to disable the
gene and observe the consequences
● Using in vitro mutagenesis, mutations are
introduced into a cloned gene, altering or
destroying its function
● When the mutated gene is returned to the cell, the
normal gene’s function might be determined by
examining the mutant’s phenotype
© 2011 Pearson Education, Inc.
● Gene expression can also be silenced using RNA
interference (RNAi)
● Synthetic double-stranded RNA molecules
matching the sequence of a particular gene are
used to break down or block the gene’s mRNA
© 2011 Pearson Education, Inc.
● 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
● SNPs can be detected by PCR, and any SNP
shared by people affected with a disorder but
NOT among unaffected people may pinpoint the
location of the disease-causing gene
© 2011 Pearson Education, Inc.
Figure 20.16
DNA
T
Normal allele
SNP
C
Disease-causing
allele
20.3: Cloning organisms may lead to
production of stem cells for research
and other applications
● Organismal cloning produces one or more
organisms genetically identical to the “parent” that
donated the single cell
© 2011 Pearson Education, Inc.
Cloning Plants: Single-Cell Cultures
● 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
Cloning Animals: Nuclear
Transplantation
● In nuclear transplantation, the nucleus of an
unfertilized egg cell or zygote is replaced with the
nucleus of a differentiated cell
● Experiments with frog embryos have shown that a
transplanted nucleus can often support normal
development of the egg
● However, the older the donor nucleus, the lower
the percentage of normally developing tadpoles
© 2011 Pearson Education, Inc.
Figure 20.18
EXPERIMENT Frog embryo
Frog egg cell
Frog tadpole
UV
Less differentiated cell
Fully differentiated
(intestinal) cell
Donor
nucleus
transplanted
Donor
nucleus
transplanted
Enucleated
egg cell
Egg with donor nucleus
activated to begin
development
RESULTS
Most develop
into tadpoles.
Most stop developing
before tadpole stage.
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.
Figure 20.19
TECHNIQUE
Mammary
cell donor
Egg cell
donor
1
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
Figure 20.19a
TECHNIQUE
Mammary
cell donor
Egg cell
donor
1
Egg
cell from
ovary
Cultured
mammary
cells
2
Nucleus
removed
3 Cells fused
Nucleus from
mammary cell
Figure 20.19b
Nucleus from
mammary cell
4 Grown in culture
Early embryo
5 Implanted in uterus
of a third sheep
Surrogate
mother
6 Embryonic
development
RESULTS
Lamb (“Dolly”) genetically
identical to mammary cell donor
● Since 1997, cloning has been demonstrated in
many mammals, including mice, cats, cows,
horses, mules, pigs, and dogs
● CC (for Carbon Copy) was the first cat cloned;
however, CC differed somewhat from her female
“parent”
● Cloned animals do not always look or behave
exactly the same
© 2011 Pearson Education, Inc.
Figure 20.20
Problems Associated with Animal
Cloning
● In most nuclear transplantation studies, only a
small percentage of cloned embryos have
developed normally to birth, and many cloned
animals exhibit defects
● Many epigenetic changes, such as acetylation of
histones or methylation of DNA, must be reversed
in the nucleus from a donor animal in order for
genes to be expressed or repressed appropriately
for early stages of development
© 2011 Pearson Education, Inc.
Stem Cells of Animals
● A stem cell is a relatively unspecialized cell that
can reproduce itself indefinitely and differentiate
into specialized cells of one or more types
● Stem cells isolated from early embryos at the
blastocyst stage are called embryonic stem (ES)
cells; these are able to differentiate into all cell
types
● The adult body also has stem cells, which replace
nonreproducing specialized cells
© 2011 Pearson Education, Inc.
Figure 20.21
Embryonic
stem cells
Adult
stem cells
Cells generating
some cell types
Cells generating
all embryonic
cell types
Cultured
stem cells
Different
culture
conditions
Different
types of
differentiated
cells
Liver
cells
Nerve
cells
Blood
cells
● Researchers can transform skin cells into ES cells
by using viruses to introduce stem cell master
regulatory genes
● These transformed cells are called iPS cells
(induced pluripotent cells)
● These cells can be used to treat some diseases
and to replace nonfunctional tissues
© 2011 Pearson Education, Inc.
Figure 20.22
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.
Applications of DNA Technology…
● Medicine / Pharmaceutical
1) Diagnosis of disease
2) Human gene therapy
3) Pharmaceutical products
-insulin, growth hormone, TPA
(dissolves blood clots), proteins that
mimic cell surface receptors for viruses
like HIV
Applications of DNA Technology…
● Forensic uses (PCR, DNA fingerprinting
to match a suspect to DNA found at the
scene of the crime)
● Environmental uses: microorganisms
engineered to break down sewage, oil
spills, etc.
O.J. Simpson capital murder case,1/95-9/95
● Odds of blood in Ford Bronco not being R. Goldman’s:
● 6.5 billion to 1
● Odds of blood on socks in bedroom not being N. Brown-Simpson’s:
● 8.5 billion to 1
● Odds of blood on glove not being from R. Goldman, N. Brown-Simpson, and O.J.
Simpson:
● 21.5 billion to 1
● Number of people on planet earth:
● 6.1 billion
● Odds of being struck by lightning in the U.S.:
● 2.8 million to 1
● Odds of winning the Powerball lottery:
● 76 million to 1
● Odds of getting killed driving to the gas station to buy a lottery ticket
● 4.5 million to 1
● Odds of seeing 3 albino deer at the same time:
● 85 million to 1
● Odds of having quintuplets:
● 85 million to 1
● Odds of being struck by a meteorite:
● 10 trillion to 1
Applications of DNA Technology…
● Agricultural uses
1) livestock (bGH to enhance
milk prod.)
2) genetically engineered
plants (resistant to herbicides
& pests, prevent spoilage, etc.)