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Biotechnology
Pre-AP Biology
Ch.12
Ms. Haut
DNA technology has many useful
applications
– The Human Genome Project
– The production of vaccines, cancer drugs, and
pesticides
– Engineered
bacteria that
can clean up
toxic wastes
Copyright © 2003 Pearson Education, Inc. publishing Benjamin Cummings
•DNA and Crime Scene Investigations
– Many violent crimes go unsolved
•
For lack of enough evidence
– If biological fluids are left at a crime scene
•
DNA can be isolated from them
– DNA fingerprinting is a set of laboratory
procedures
•
•
That determines with near certainty whether two
samples of DNA are from the same individual
That has provided a powerful tool for crime
scene investigators
Investigator at one
of the crime scenes
(above), Narborough,
England (left)
BACTERIAL PLASMIDS AND
GENE CLONING
•Plasmids are used to customize bacteria:
An overview
– Gene cloning is one application of DNA
technology
•
Methods for studying and manipulating
genetic material
The Bacterial Chromosome
• One double-stranded,
circular molecule of
DNA
• Located in nucleoid
region, so
transcription and
translation can
occur simultaneously
• Many also contain
extrachromosomal
DNA in plasmids
Binary Fission
Plasmids
• Short, circular DNA
molecules outside the
chromosome
• Carry genes that are
beneficial but not
essential
• Replicate
independently of
chromosome
en.wikipedia.org/?title=Plasmid
R Plasmids
• Contain genes that
confer antibiotic
resistance
• Medical
consequences:
resistant strains of
pathogens due to
overuse of antibiotics
http://www.slic2.wsu.edu:82/hurlbert/micro101/images/AntibioticSelection.gif
Bacteria as Tools
• Bacterial Transformation—
– Uptake of DNA from the fluid surrounding the
cell
– Causes genetic
recombination
Transformation
• Biotech companies use this technique to
artificially introduce foreign genes into
bacterial genomes (human insulin, human
growth hormone)
– Researchers can insert desired genes into
plasmids, creating recombinant DNA
•
Figure 12.1
And insert those plasmids into bacteria
(transformation)
E. coli
– If the
recombinant
bacteria multiply
into a clone
• The foreign
genes are
also copied
1 Isolate DNA
Human cell
from two sources
2 Cut both
Plasmid
DNAs with
the same
restriction
enzyme
DNA
Gene V
Sticky ends
3 Mix the DNAs; they join
by base-pairing
4 Add DNA ligase
to bond the DNA covalently
Recombinant DNA
plasmid
Gene V
5 Put plasmid into bacterium
by transformation
6 Clone the bacterium
Bacterial clone carrying many
copies of the human gene
Restriction Enzymes
•Used to “cut and paste”
DNA
•The tools used to make
recombinant DNA are
– Restriction enzymes,
which cut DNA at
specific sequences
– DNA ligase, which
“pastes” DNA
fragments together
http://campus.queens.edu/faculty/jannr/Genetics/images/dnatech/bx15_01.jpg
Genes can be cloned in recombinant
plasmids: A closer look
– Bacteria take the
recombinant
plasmids from their
surroundings
– And reproduce,
thereby cloning the
plasmids and the
genes they carry
Cloned genes can be stored in
genomic libraries
•Genomic libraries, sets of
DNA fragments containing
all of an organism’s genes
Genome cut up with
restriction enzyme
Recombinant
plasmid
– Can be constructed and
stored in cloned
bacterial plasmids or
phages
Recombinant
phage DNA
or
Bacterial
clone
Plasmid library
Figure 12.4
Phage
clone
Phage library
Nucleic acid probes
Radioactive
probe (DNA)
•A short, singlestranded molecule of
radioactively labeled or
fluorescently labeled
DNA or RNA
– Can tag a desired
gene in a library
ATCCGA
Mix with singlestranded DNA from
various bacterial
(or phage) clones
Single-stranded
G
DNA
TC
T
TA
A
T
C
GC
G
C
T
G
A
AT
T
T
C
C
G
A
Master plate
A
A
T
G
G
C
G
C
TA
G
G
A
C
TA
Base pairing
indicates the
gene of interest
Master plate
Probe
DNA
Radioactive
single-stranded
DNA
Solution
containing
probe
Colonies
containing
gene of
interest
Gene of
interest
Single-stranded
DNA from cell
Filter
A
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.
CONNECTION
•Recombinant cells and
organisms can massproduce gene products
– Applications of gene
cloning include
•
The mass
production of gene
products for
medical and other
uses
Table 12.6
Genetically modified organisms
are transforming agriculture
• New genetic varieties of animals and plants are
being produced
– A plant with a new trait can be created using the
Ti plasmid
• Biotech companies can artificially
induce transformation of bacteria
• “Golden rice” has been genetically modified
to contain beta-carotene
– This rice could help prevent vitamin A
deficiency
Figure 12.18B
Drought resistant corn
Flavr Savr Tomato (CalGene)
•Transgenic organisms
– Are those that have had genes from
other organisms inserted into their
genomes
– Different organisms, including bacteria,
yeast, and mammals
•
Can be used for this purpose
These sheep carry a
gene for a human
blood protein that is a
potential treatment for
cystic fibrosis
Figure 12.6
CONNECTION
•DNA technology is changing the
pharmaceutical industry
– DNA technology
•
Is widely used to produce medicines and
to diagnose diseases
DNA technology is changing the
pharmaceutical industry and medicine
• Hormones, cancer-fighting drugs, and new
vaccines are being produced using DNA
technology
– This lab equipment
is used to produce
a vaccine against
hepatitis B
Figure 12.17
•Therapeutic hormones
– In 1982, humulin, human insulin produced
by bacteria
•
Became the first recombinant drug
approved by the Food and Drug
Administration
Figure 12.7A
•Diagnosis and Treatment of Disease
– DNA technology
•
Is being used increasingly in disease
diagnosis
•Vaccines
– DNA technology
•
Is also helping medical researchers
develop vaccines
Figure 12.7B
DNA Fingerprinting
Dr. Alec Jeffreys
• A method of
developing a
person’s DNA
“profile,” similar to a
fingerprint.
• Pioneered in
England in 1984 by
Dr. Alec Jeffreys
First Forensic Use
• First used by law
enforcement in
England in the mid1980’s.
• DNA evidence
exonerated one man,
and convicted
another.
• Described in The
Blooding, by Joseph
Wambaugh
How does it work?
• 99.9% of your DNA is the same as
everyone else’s.
• The 0.1% that differs are a combination of:
– Gene differences (Differences in the genes
themselves)
– Differences in “polymorphic regions” between
the genes on the DNA.
How does it work?
• Certain points between the genes on the
DNA have repeating base sequences.
– For example:
ATTACGCGCGCGCGCGCGCTAGC
– These are called short tandem repeats (STRs
for short)
How does it work?
• Everyone has STRs at the same place in
their DNA, but they are different lengths
for different people.
– For example:
Person 1: ATTACGCGCGCGCGCGCGTAGC
(7 repeats)
Person 2: ATTACGCGCGCGCGTAGC
(5 repeats)
To Make a DNA Fingerprint…
• First, we use restriction enzymes to
chop the DNA up into millions of
fragments of various lengths.
– Some of the fragments contain STRs;
some do not. The ones that do are different
lengths for different people.
Restriction Fragment Length
Polymorphisms (RFLPs)
• Polymorphisms are slight differences in
DNA sequences as seen in individuals
of the same species
To Make a DNA Fingerprint…
• Next, we use gel
electrophoresis to sort
the DNA fragments by
size.
Gel Electrophoresis
• Method for sorting
proteins or nucleic
acids on the basis of
their electric charge
and size
Gel Electrophoresis
Electrical current
carries negativelycharged DNA through
gel towards positive
electrode
• Agarose gel sieves
DNA fragments
according to size
•
–
Small fragments move
farther than large
fragments
Gel Electrophoresis
1
To Make a DNA
Fingerprint…
Restriction fragment
preparation
Restriction
fragments
2
Gel electrophoresis
3
Blotting
4
Radioactive probe
• Finally, a radioactive
probe attaches to our
STRs. Only the
fragments with our
STRs will show up on
the gel.
Filter paper
Probe
5
Detection of radioactivity
(autoradiography)
Film
Figure 12.11C
To Make a DNA Fingerprint…
• Since STRS are
different lengths in
different people, this
creates a DNA
Fingerprint.
Two uses for DNA
Fingerprints...
• Forensics
DNA taken from crime
scenes (blood, semen, hair,
etc.) can be compared to
the DNA of suspects.
Real-life CSI!
Two uses for DNA
Fingerprints...
• Forensics
This is an example of a
gel that might be used to
convict a rape suspect.
Compare the “Sperm
DNA” to the “Suspect
DNA.” Which suspect
committed the rape?
Two uses for DNA
Fingerprints...
• Paternity Testing
Since all of our DNA
markers came from
either mommy or
daddy, we can use
DNA fingerprints to
determine whether a
child and alleged
father are
related…just like on
Maury Povich!
Two uses for DNA
Fingerprints...
• Look at the two “Child” markers
on this gel. Can they both be
matched up to either the
mother or the “alleged father?”
• Yes. This is a “positive” test for
paternity.
Two uses for DNA
Fingerprints...
• How about this gel? Do both
of the child’s markers match
either the mother or the
“alleged father.”
• No! The “alleged father” is not
this child’s biological parent.
Interpreting DNA Fingerprints
• Which child is not
related to the
mother?
• Son 2
• Which children
are not related to
the father?
• Daughter 2 and
Son 2
Interpreting DNA Fingerprints
• A blood stain was found
at a murder scene. The
blood belongs to which of
the seven possible
suspects?
Suspect 3
Interpreting DNA Fingerprints
• Who committed this
sexual assault?
Suspect 1
Interpreting DNA Fingerprints
• These DNA fingerprints
are from a mother, a
child, and two possible
biological fathers. Which
one is the daddy?
2nd alleged father
Interpreting DNA Fingerprints
• Mother, father, and
four children. Which
child is from a
different father?
Child 2
The Polymerase Chain
Reaction (PCR)
• The polymerase chain reaction, PCR, can
produce many copies of a specific target
segment of DNA
• A three-step cycle—heating, cooling, and
replication—brings about a chain reaction that
produces an exponentially growing population of
identical DNA molecules
5
3
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
New
nucleotides
Polymerase Chain
Reaction (PCR)
• Quick method of
cloning DNA in vitro
(without using cells)
• Can be used to
increase small
amounts of DNA for
analysis
GENOMICS CONNECTION
•The Human Genome Project is an
ambitious application of DNA technology
– The Human Genome Project, begun in 1990
and now largely completed, involved
•
Genetic and physical mapping of
chromosomes, followed by DNA sequencing
Figure 12.15
CONNECTION
•The science of genomics compares whole
genomes
– The sequencing of many prokaryotic and
eukaryotic genomes
•
Has produced data for genomics, the study of
whole genomes
•Besides being interesting themselves
– Nonhuman genomes can be compared
with the human genome
Table 12.17
Proteomics
•The study of the full
sets of proteins
produced by organisms
Biotechnology Explorer™
Protein Fingerprinting
Instruction Manual
Biotechnology
Explorer™
Protein
Fingerprinting
Instruction
Manual
Gene therapy may someday help
treat a variety of diseases
• Techniques for manipulating
DNA have potential for
treating disease by altering
an afflicted individual’s genes
• Is the alteration of an afflicted
individual’s genes
Cloned gene (normal allele)
1 Insert
normal gene
into virus
Viral nucleic
acid
Retrovirus
2 Infect bone
marrow cell
with virus
– Progress is slow, however
– There are also ethical
questions related to gene
therapy
3 Viral DNA
inserts into
chromosome
Bone marrow
cell from patient
Bone
marrow
4 Inject cells
Figure 12.19
into patient
DNA technology raises important
ethical questions
• Our new genetic knowledge
will affect our lives in
many ways
• The deciphering of the
human genome, in particular,
raises
profound ethical issues
– Many scientists have
counseled that we
must use the
information wisely
Figure 12.21A-C
Could GM organisms harm human
health or the environment?
• Genetic engineering involves
some risks
– Possible ecological damage
from pollen transfer between
GM and wild crops
– Pollen from a transgenic variety
of corn that contains a pesticide
may stunt or kill monarch
caterpillars
Figure 12.20A, B
Acknowledgements
• BIOLOGY: CONCEPTS AND CONNECTIONS 4th Edition, by
Campbell, Reece, Mitchell, and Taylor, ©2003. These images have
been produced from the originals by permission of the publisher.
These illustrations may not be reproduced in any format for any
purpose without express written permission from the publisher.
• Unless otherwise noted, illustrations are credited to Pearson
Education which have been borrowed from BIOLOGY: CONCEPTS
AND CONNECTIONS 4th Edition, by Campbell, Reece, Mitchell,
and Taylor, ©2001. These images have been produced from the
originals by permission of the publisher. These illustrations may not
be reproduced in any format for any purpose without express written
permission from the publisher.