Don’t forget to count your flies!
Biotechnology and
Genetic Engineering
AP Biology
Chapter 20
Terminology
• Genetic engineering – direct
manipulation of genetic material for
practical purposes
• Biotechnology – use of living
organisms or their components to
make products for us
• Recombinant DNA – combining pieces
of DNA from different organisms
• Gene cloning – making copies of DNA
Making recombinant DNA
• Plasmids (small circular pieces of DNA
in bacterial cells) are used to insert
pieces of foreign DNA
The DNA is cut using restriction
enzymes
What are restriction enzymes?
• Restriction enzymes come from
bacteria and recognize a particular
pattern of DNA, often 4, 6 or 8 base
pairs long, and then cut the DNA within
this recognized sequence.
• Bacteria use these enzymes to kill off
other competing bacteria by cutting up
their DNA.
How do they cut?
STICKY ENDS
BLUNT ENDS
ACT GAA TTC CGG AAT GAA TTC
TGA CTT AAG GCC TTA CTT AAG
Where would the enzyme EcoRI cut?
ACT GAA TTC CGG AAT GAA TTC
TGA CTT AAG GCC TTA CTT AAG
There would be three pieces:
one 4 bases,
one 12 bases, and
one 5 bases.
How do bacteria protect it’s
own DNA from being cut by
the enzymes?
It methylates it’s own DNA.
Making recombinant DNA in
plasmids
http://glencoe.mcgrawhill.com/sites/9834092339/student_view0/chapter18/steps_in_cloning_a_gene.html
http://www.nearingzero.net/natural/screenres/natural039.jpg
Bacterial plasmids often contain
antibiotic resistance genes.
Genes can be
cloned into
vectors such
as plasmids
Fig. 20-2
Cell containing gene
of interest
Bacterium
1 Gene inserted into
plasmid
Bacterial
Plasmid
chromosome
Recombinant
DNA (plasmid)
Gene of
interest
DNA of
chromosome
2 Plasmid put into
bacterial cell
Recombinant
bacterium
3 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
Protein harvested
4 Basic research and
various applications
research
on gene
Gene for pest
resistance inserted
into plants
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
Fig. 20-2a
Cell containing gene
of interest
Bacterium
1 Gene inserted into
plasmid
Bacterial
chromosome
Plasmid
Recombinant
DNA (plasmid)
Gene of
interest
2
2 Plasmid put into
bacterial cell
Recombinant
bacterium
DNA of
chromosome
Fig. 20-2b
Recombinant
bacterium
3 Host cell grown in culture
to form a clone of cells
containing the “cloned”
gene of interest
Protein expressed
by gene of interest
Gene of
Interest
Copies of gene
Protein harvested
4 Basic research and
Basic
research
on gene
Gene for pest
resistance inserted
into plants
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
Fig. 20-4-1
Hummingbird
cell
TECHNIQUE
Bacterial cell
lacZ gene
Restriction
site
ampR gene
Bacterial
plasmid
Sticky
ends
Gene of interest
Hummingbird
DNA fragments
Fig. 20-4-2
Hummingbird
cell
TECHNIQUE
Bacterial cell
lacZ gene
Restriction
site
ampR gene
Sticky
ends
Bacterial
plasmid
Gene of interest
Hummingbird
DNA fragments
Nonrecombinant
plasmid
Recombinant plasmids
Fig. 20-4-3
Hummingbird
cell
TECHNIQUE
Bacterial cell
lacZ gene
Restriction
site
ampR gene
Sticky
ends
Bacterial
plasmid
Gene of interest
Hummingbird
DNA fragments
Nonrecombinant
plasmid
Recombinant plasmids
Bacteria carrying
plasmids
Fig. 20-4-4
Hummingbird
cell
TECHNIQUE
Bacterial cell
lacZ gene
Restriction
site
ampR gene
Sticky
ends
Bacterial
plasmid
Gene of interest
Hummingbird
DNA fragments
Nonrecombinant
plasmid
Recombinant plasmids
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
Steps
1. Plasmid and DNA of gene of interest
are isolated.
2. Both DNAs are cut with the same
restriction enzyme.
3. “new” DNA is ligated into plasmid
4. Recombinant plasmids are inserted
into bacterial cells.
5. Plate bacteria on agar. Bacteria will
express new genes.
Plasmid Maps
Plasmid Maps
Sometimes called restriction maps are graphical
representation of plasmids, that show the locations of
major identifiable landmarks on DNA like restriction
enzyme sites, genes of interest, plasmid length etc.
The collection of thousands of
clones of bacteria containing
recombinant plasmids is called a
genomic library.
• In molecular biology, plasmid (or restriction)
maps are used as a reference to engineer
plasmids.
• The plasmids are digested by enzymes
chosen and the resulting samples are
subsequently run on an electrophoresis gel.
Our experiment: to transform E.coli with
pGLO plasmid containing the jellyfish gene
GFP to make them have the ability to glow
• To isolate only the cells containing the pGLO
DNA, the plasmid contains the beta-lactamase
gene which encodes for an ampicillin
resistance (Ampr) protein.
• After the transformation, the cells are grown
on a solid medium called an agar plate. This
medium will contain the antibiotic ampicillin.
• In the presence of the ampicillin, only the
bacteria containing the pGLO plasmid will
have the Ampr protein which will break down
the antibiotic, and be able to grow.
• This process is called antibiotic selection.
GFP results in E.coli
This plate shows bacteria expressing six
different types of flourescent proteins
GRP has been used as tracers to see
if the plasmid has been taken up by
the bacteria.
Expression of eukaryotic
genes in prokaryotes
• Use an expression vector with a
prokaryotic promoter upstream from
the location of the gene (ie operon)
• Create artificial genes without introns
since bacteria do not have the
machinery for eliminating introns.
Ways to
introduce
new
genes into
bacteria.
Conjugation – through tubes between bacteria
Transformation – negative DNA taken up
Transduction by bacteriophages or other viruses
Mutation
ALL of these introduce GENETIC VARIATION!
What about transferring DNA
into eukaryotic cells
• Electroporation - injecting DNA into
eukaryotic cells
Plants can have genes transferred
through bacterial plasmids.
Storing Cloned Genes in DNA
Libraries
These can be also be used as vectors to
transfer genes to other organisms.
1) Plasmid libraries plasmids that
contain genes of interest
2) Phage library that is made using
bacteriophages which store genes of
interest
Fig. 20-5a
Foreign genome
cut up with
restriction
enzyme
or
Recombinant
phage DNA
Bacterial
clones
(a) Plasmid library
Recombinant
plasmids
(b) Phage library
Phage
clones
Viruses
used
as vectors
3)BACs (bacterial artificial chromosome)
a plasmid that can carry a large DNA
insert
Fig. 20-5
Formation of BAC Clones
Foreign genome
cut up with
restriction
enzyme
Large insert
Large plasmid with many genes
or
BAC
clone
Recombinant
phage DNA
Bacterial
clones
(a) Plasmid library
Recombinant
plasmids
(b) Phage library
Phage
clones
(c) A library of bacterial artificial
chromosome (BAC) clones
4) YACS
• Yeast artificial chromosomes that carry
foreign DNA.
• Yeast cells have plasmids that can act
as vectors.
5) Complementary DNA (cDNA) library is
made by cloning DNA made in vitro by
reverse transcription of all the mRNA
produced by a particular cell
• A cDNA library represents only part of
the genome—only the subset of genes
transcribed into mRNA in the original
cells
http://glencoe.mcgrawhill.com/sites/9834092339/student_view0/chapter18/cdna.html
Fig. 20-6-1
DNA in
nucleus
mRNAs in
cytoplasm
Fig. 20-6-2
DNA in
nucleus
mRNAs in
cytoplasm
mRNA
Reverse
transcriptase Poly-A tail
DNA Primer
strand
Fig. 20-6-3
DNA in
nucleus
mRNAs in
cytoplasm
mRNA
Reverse
transcriptase Poly-A tail
Degraded
mRNA
DNA Primer
strand
Fig. 20-6-4
DNA in
nucleus
mRNAs in
cytoplasm
mRNA
Reverse
transcriptase Poly-A tail
Degraded
mRNA
DNA
polymerase
DNA Primer
strand
Fig. 20-6-5
DNA in
nucleus
mRNAs in
cytoplasm
mRNA
Reverse
transcriptase Poly-A tail
DNA Primer
strand
Degraded
mRNA
http://glencoe.mcgrawhill.com/sites/9834092339/
student_view0/chapter18/f
ish.html
DNA
polymerase
cDNA
In Summary, libraries of
cloned genes can be made by
•
•
•
•
•
Plasmids
Bacteriophage viruses
BACS
YACS
cDNA (with reverse transcriptase)
Other biotechniques
•
•
•
•
Nucleic acid hybridization
PCR
Gel electrophoresis
DNA fingerprinting (Southern Blotting)
Nucleic Acid Hybridization
• Used to detect specific sequences in DNA
fragments
Steps:
1) Cut DNA with restriction enzymes and
electrophorese.
2) The DNA is denatured to produce single stranded
DNA.
2) The radioactive probe will hybridize (bond) with
complementary bases if present.
• Probes can be radioactive isotopes or
flourescent dyes.
Looking for this sequence
The radioactive probe is made
by determining a short
segment of the protein
sequence, then "back
translating" to the possible
short DNA sequence.
The DNA sequence is
radiolabeled, and applied to
the blotted clones.
They should hybridize only to
sequences that would identify
that particular piece of DNA.
How does this work?
• Protein segment
gly – gly – ser – glutamic acid
• Look on Genetic Codon chart, find mRNA
codons
GGU – GGU – UCU – GAA
• Make a radioactive DNA
(oligos)probe
CCA – CCA – AGA - CTT
PCR Polymerase Chain Reaction
• Used to amplify DNA
• Discovered by Kary Mullis (GT grad)
A Thermocycler
Steps of PCR?
• Denature DNA (94-96 C)
• Anneal (base pair) primers (50 – 65 C)
• Extend primers (72 for polymerase to
work)
• Machines called thermocyclers do this.
http://glencoe.mcgrawhill.com/sites/9834092339/student_view0/chapter18/polymerase_chain_reactio
n.html
http://www.dnalc.org/ddnalc/resources/shockwave/pcranwhole.html
Fig. 20-8b
1 Denaturation
5
3
3
5
2 Annealing
Cycle 1
yields
2
molecules
Primers
3 Extension
New
nucleotides
Fig. 20-8
5
TECHNIQUE
3
Target
sequence
3
Genomic DNA
1 Denaturation
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
• In PCR, a heat-stable DNA polymerase is
used, most commonly Taq Polymerase
from the thermophilic microbe Thermus
aquaticus.
• Thomas Brock discovered T.
aquaticus from a hot spring at
Yellowstone National Park.
Applications of PCR
PCR has replaced cloning for many
purposes, particularly the sequencing of
DNA.
• It is faster and requires no vectors, which
can mutate as they reproduce.
• It can be used forensically, to amplify tiny
amounts of DNA from criminal evidence; or
clinically, to detect DNA sequences linked to
inherited disorders.
What is gel electrophoresis?
• A technique to separate DNA based on
the movement of DNA fragments from
neg to pos (DNA is neg).
• Smaller fragments travel farther.
• Samples are placed in gels.
Gel Electrophoresis
• DNA can be digested by restriction enzymes
and the resulting samples are run on an
electrophoresis gel.
• They can be compared to a reference ladder
(a DNA ladder) or a sample with known sized
pieces.
You can compare your pieces to
a marker as shown below.
Or you can make a graph from a known
sample to determine the sizes.
Line of
best fit
Southern Blotting
“DNA Fingerprinting”
- named for Edwin Southern
- used to identify DNA fragments
1. Isolate DNA
2. Cut DNA into fragments with restriction enzymes.
3. Electrophorese.
4. Blot onto nylon membrane.
5. Apply radioactive probes.
6. Wash to remove unbonded probes.
http://highered.mcgrawhill.com/sites/0072556781/student_view0/chapter14/animation_quiz_5.html
In DNA fingerprinting
• Single nucleotide polymorphisms
(SNPs) are useful genetic markers
• These are single base-pair sites that
vary in a population (most of our DNA
is identical
http://glencoe.mcgrawhill.com/sites/9834092339/student_view0/chapter18/restriction_fragment_length_polym
orphisms.html
Fig. 20-21
Many genetic diseases are the result of a
polymorphism at a single locus.
DNA
T
Normal allele
SNP
C
Disease-causing
allele
http://glencoe.mcgrawhill.com/sites/9834092339/student_view0/chapter18/video_quiz__world_trade_center_dna.html
• When a restriction enzyme is added, SNPs
result in DNA fragments with different
lengths, or restriction fragment length
polymorphisms (RFLP)
• Some polymorphisms cause disease,
while some do not. Others indicate a
predisposition to disease.
Too many abbreviations!
•SNP –
different size pieces due to different
nucleotide sequences
•RFLP -
fragments of DNA from restriction
enzyme procedure
RFLP-s differ in sizes due to SNP’s in a
population.
Hemoglobin S is missing a restriction
site due to a polymorphism
RFLP Analysis in Paternity Cases
RFLP Analysis in Paternity Cases
Short Tandem Repeats
• Even more sensitive is the use of
genetic markers called short tandem
repeats (STRs), which are variations in
the number of repeats of specific DNA
sequences
• The Human Genome Project has
shown that there are tens of
thousands of STR loci in human
DNA.
• An individual inherits one copy of
an STR from each parent,
D7S280 is one of the 13 core CODIS STR genetic loci. This DNA is
found on human chromosome 7. The DNA sequence of a representative
allele of this locus is shown below. The tetrameric repeat sequence of
D7S280 is "gata". Different alleles of this locus have from 6 to 15
tandem repeats of the "gata" sequence. How many tetrameric repeats
are present in the DNA sequence shown below?
1 aatttttgta ttttttttag agacggggtt tcaccatgtt ggtcaggctg actatggagt
61 tattttaagg ttaatatata taaagggtat gatagaacac ttgtcatagt
ttagaacgaa121 ctaacgatag atagatagat agatagatag atagatagat
agatagatag atagacagat181 tgatagtttt tttttatctc actaaatagt
ctatagtaaa catttaatta ccaatatttg241 gtgcaattct gtcaatgagg
ataaatgtgg aatcgttata attcttaaga atatatattc301 cctctgagtt tttgatacct
cagattttaa ggcc
If the genotypes of both parents are known, we use a Punnett
Square to predict the possible phenotypes of their offspring. Each
child inherits one allele of a given locus from each parent. Panel
(a) - At the D21S11 locus, the children of Bob Blackett and wife
Anne can have four different genotypes. Son David is 28, 31.
Daughter Katie is 29, 30. Panel (b) - Bob Blackett inherited the 31
allele from his mother, Norma. Therefore the 29 allele is paternal. If
Bob's paternal was not 29, what would be your conclusion?
Huntington’s Disease can be diagnosed
by the number of CAG repeats
The data below shows the results of electrophoresis of PCR fragments amplified
using probes for the site which has been shown to be altered in Huntington's
disease. The male parent, as shown by the black box, got Huntington's disease
when he was 40 years old. His children include 6 (3,5,7,8,10,11) with Huntington's
disease, and the age at which the symptoms first began is shown by the number
above the band from the PCR fragment.
What is the
prognosis for
the normal
children 4, 6,
and 9?
Sanger Sequencing
• Used to sequence short segments of DNA
• Single-stranded fragments are incubated
with fluorescent-tagged short segments for
DNA hybridization.
• When fragments hybridize with the tagged
nucleotide, the hybridization stops.
• Fragments are electrophoresed and
analyzed.
http://glencoe.mcgrawhill.com/sites/9834092339/student_view0/chapter18/sang
er_sequencing.html
Analyzing Expression of Genes
• Northern Blotting – using radioactive
probes to look for mRNA being
produced
• RT-PCR – Reverse transcriptasepolylmerase chain reaction – makes
cDNA from mRNAs and then PCRs the
DNA for electrophoresis
• in situ hybridization – can locate
specific mRNA’s in cells
• Micro – arrays - Isolate mRNA from
cells, make cDNA using reverse
transcriptase, then uses cDNA to
explore collections of genomic DNA to
see if they hybridize
http://glencoe.mcgrawhill.com/sites/9834092339/student_view0/chapter18/using_a_dna_microarray.html
• Microarrays are useful in discerning
gene expression in different tissues
AND at different stages of
development.
• Different brightness and
colors signify rates of
expression.
http://www.dnalc.org/resources/3d/2
6-microarray.html
Google Image Result for
http://www.g2conline.info/content/1178/1
178_what_microarray_thumb.jpg
An
example
DNA Microarray Methodology
Animation
Determining Gene Function
• In vitro mutagenesis – changes made
to cloned gene, gene returned to cell
and it “knocks out” the normal gene.
Then look for abnormalities.
• RNA interference (RNAi) – uses RNA to
block translation of mRNA and see
what happens.
Cloning Organisms
• Organismal cloning – producing
genetically identical individuals from a
single somatic cell of a multicellular
organism
In plants
• Steward demonstrated
genomic equivalence in
plants by growing
carrot plants from
differentiated root cells.
• Most plant cells remain
totipotent, retaining the
ability to give rise to a
complete new
organisms.
In Animals
• Briggs and all transplanted nuclei from
embryonic frog cells into enucleated
egg cells and produced cloned frogs
• Nuclear transplantation – name of
process
• Whether normal development occurred
depended on developmental age of the
transplanted nucleus.
Fig. 20-17
EXPERIMENT
Frog egg cell Frog tadpole
Frog embryo
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
Nuclear Transplantation
And then Dolly came along in
1997
Fig. 20-18
TECHNIQUE
Mammary
cell donor
Egg cell
donor
2
1
Egg cell
from ovary
3 Cells fused
Cultured
mammary cells 3
4 Grown in
Nucleus
removed
Nucleus from
mammary cell
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
Why Dolly died young 6 yrs
• Dolly's telomeres were found to be
approximately 80% of the length they
should be for a sheep her age.
• Also there is the concern of damaged
DNA being carried into the clone
Cloned animals do not look exactly like
the transplanted nucleus due to
cytoplasmic affects.
Rainbow
Hi Mrs.
Smith!
CC and her
Surrogate mom
CC
• In most nuclear transplantation studies,
only a small percentage of cloned
embryos have developed normally to
birth
• 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
Stem Cells
• Relatively unspecialized cells that
continue to reproduce themselves and
can be induced to form specialized
cells
• Embryonic cells are more totipotent
than adult stem cells
http://cbm.msoe.edu/stupro/so/SOStemCellVideo2010.html
• Therapeutic cloning – using stem cells to
replace organs and tissues
• Reproductive cloning – using stem cells to
reproduce new organisms
• Both raise ethical
debates
Research points to a new direction
in Stem Cell Research
Induced
Pluripotent
Stem Cells
The Nobel Prize in Medicine 2012 was awarded to
two biologists for their breakthroughs in the field of
stem-cell research — two discoveries that happened
44 years apart. The honors go to Britain's Sir John
B. Gurdon and Japan's Shinya Yamanaka for their
pioneering work with the life-shaping cells, which
can be reprogrammed to create any kind of tissue in
the body.
A mouse embryo injected with cells
made pluripotent through stress, tagged
with a fluorescent protein.
The latest…
Acid bath
offers easy
path to stem
cells;
just squeezing
or bathing
cells in acidic
conditions can
readily
reprogram
them into an
embryonic
state.
Acid bath offers easy path to
stem cells
Benefits of DNA technology
• Medical Applications
• identification of human genes in
which mutation plays a role in
genetic diseases
Huntington’s Disease can be diagnosed
by the number of CAG repeats
The data below shows the results of electrophoresis of PCR fragments amplified
using probes for the site which has been shown to be altered in Huntington's
disease. The male parent, as shown by the black box, got Huntington's disease
when he was 40 years old. His children include 6 (3,5,7,8,10,11) with Huntington's
disease, and the age at which the symptoms first began is shown by the number
above the band from the PCR fragment.
What is the
prognosis for
the normal
children 4, 6,
and 9?
Human Gene Therapy
• Gene therapy is the alteration of an
afflicted individual’s genes
• Vectors, such as viruses, are used for
delivery of genes into specific types of
cells, for example bone marrow
• It may be difficult to target cells.
• Gene therapy raises ethical questions,
such as whether human germ-line cells
should be treated to correct the defect
in future generations
Fig. 20-22
Cloned
gene
1
Insert RNA version of normal allele
into retrovirus.
Viral RNA
2
Retrovirus
capsid
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
Pharmaceutical Products
• Advances in DNA technology and
genetic research are important to the
development of new drugs to treat
diseases
• In particular “pharm” animals and
plants can be used to produce
certain products
Fig. 20-23
Forensic Evidence and Genetic
Profiles
• An individual’s unique DNA sequence, or
genetic profile, can be obtained by
analysis of tissue or body fluids
Fig. 20-24
(a) This photo shows Earl
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.
Environmental Cleanup
• Some modified microorganisms can
be used to extract minerals from the
environment or degrade potentially
toxic waste materials
• Biofuels make use of crops such as
corn, soybeans, and cassava to
replace fossil fuels
Genetic Engineering in Plants
• Agricultural scientists have endowed
a number of crop plants with genes
for desirable traits
• The Ti plasmid is the most
commonly used vector for
introducing new genes into plant
cells
Is this safe?
• Most public concern about possible
hazards centers on genetically
modified (GM) organisms used as
food
• Some are concerned about the
creation of “super weeds” from the
transfer of genes from GM crops to
their wild relatives
Fig. 20-25
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
• Guidelines are in place in the United
States and other countries to ensure safe
practices for recombinant DNA
technology
http://ecowatch.com/2
014/10/15/bill-nyescience-guy-gmofoods/