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Chapter 12
DNA Technology
PowerPoint® Lectures for
Campbell Essential Biology, Fourth Edition
– Eric Simon, Jane Reece, and Jean Dickey
Campbell Essential Biology with Physiology, Third Edition
– Eric Simon, Jane Reece, and Jean Dickey
Lectures by Chris C. Romero, updated by Edward J. Zalisko
© 2010 Pearson Education, Inc.
1. Recombinant DNA technology
1.Application
2.Techniques
2. DNA profiling and Forensic Science
1.Techniques
3. Genomics and proteomics
1.Human genome project
2.Comparing genomes
3.Genome mapping techniques
4.Proteomics
4. Human Gene Therapy
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DNA Technology
•
•
•
Modern laboratory techniques for studying and manipulating
genetic material.
Scientists can modify specific genes and move them between
organisms like bacteria, plants, and animals.
Biotechnology today means the use of DNA technology,
methods for:
Studying and manipulating genetic material,
Modifying specific genes, Moving genes between organisms
Recombinant DNA Technology
• When scientists combine pieces of DNA from two different sources
(different species) to form a single DNA molecule.
• Recombinant DNA technology is widely used in genetic
engineering, the direct manipulation of genes for practical purposes
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Cutting and Pasting DNA with Restriction Enzymes
• Recombinant DNA is produced by combining two ingredients
a bacterial plasmid and a gene of interest
• To combine these ingredients, a piece of DNA must be
spliced into a plasmid.
• This splicing process can be accomplished using restriction
enzymes which cut DNA at specific nucleotide sequences
– these cuts produce pieces of DNA called restriction
fragments with “sticky ends” important for joining
DNA from different sources
• DNA ligase connects the DNA pieces into continuous strands
by forming bonds between adjacent nucleotides.
Recombinant DNA Techniques
Bacteria represent the workhorses of modern biotechnology. To
manipulate genes in the laboratory, bacterial plasmids
- small, circular DNA molecules that are separate from the
much larger bacterial chromosome are used
- are ideal for gene cloning, the production of multiple identical
copies of a gene-carrying piece of DNA
• Can easily incorporate foreign
DNA
• Are readily taken up by bacterial
cells
• Can act as vectors, a DNA carriers Plasmids
that move genes from one cell to Bacterial
chromosome
another
Remnant of
bacterium
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Colorized TEM
Plasmids:
Cutting and pasting DNA
Recognition sequence
for a restriction enzyme
DNA
A restriction enzyme cuts
the DNA into fragments.
Restriction
enzyme
A DNA fragment is added
from another source.
Fragments stick together by
base pairing.
DNA ligase joins the
fragments into strands.
Recombinant DNA molecule
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DNA
ligase
Using recombinant DNA technology to produce useful products
Cut both DNAs
with same
enzyme.
Gene of Other
interest genes
Mix the DNAs and
Gene of interest
join them together.
Bacterial cell
DNA fragments
from cell
Isolate
DNA.
Cell containing
the gene of interest
Isolate
plasmids.
Recombinant DNA plasmids
Bacteria take up recombinant plasmids.
Plasmid
DNA
Bacterial clone
Recombinant bacteria
Clone the bacteria.
Find the clone with
the gene of interest.
Some uses
of genes
Gene for pest
resistance
Inserted
into plant
Some uses
of proteins
Protein for dissolving
clots in heart attack
therapy
Gene for
toxic-cleanup
bacteria
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Genes may be
inserted into
other organisms.
The gene and protein
of interest are isolated
from the bacteria.
Harvested
proteins may be
used directly.
Protein for
“stone-washing”
jeans
Applications of DNA Technology :
From Humulin to Foods to “Pharm” Animals
DNA technology is used to produce medically valuable
molecules, including:
•
•
•
Humulin is human insulin produced by genetically
modified bacteria
Human growth hormone (HGH)
The hormone EPO (Erythropoietin), which stimulates
production of red blood cells
Vaccines, harmless variant of a disease - causing microbe
(bacteria or virus) that is used to prevent an infectious disease.
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Applications of DNA Technology: Genetically Modified Foods
• DNA technology is improving productivity of agriculture
and quickly replacing traditional plant-breeding programs
• The modified organisms have acquired one or more genes
artificially.
• If the new gene is from another organism (another
species) the recombinant organism is called a transgenic
organisms.
• In the United States today, roughly
one-half of the corn crop and over
three-quarters of the soybean and
cotton crops are genetically modified
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Applications of DNA Technology: GM Foods II
“Golden rice” has been genetically modified to contain
beta-carotene.
– Our bodies use beta-carotene to make vitamin A.
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Applications of DNA Technology: “Pharm” Animals
• While transgenic plants are used today as
commercial products, transgenic whole animals
are currently only in the testing phase.
• These transgenic sheep carry a gene for a
human blood protein.
– This protein may help in the treatment of
cystic fibrosis.
• While transgenic animals are currently used
to produce potentially useful proteins, none
are yet found in our food supply.
• It is possible that DNA technology will
eventually replace traditional animal
breeding.
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Transgenic animals raised for
the purposes of producting
pharmaceuticals are called
pharm animals
DNA Profiling and Forensic Science
•
•
•
Forensics the scientific analysis of evidence from crime
scenes, has been revolutionized by DNA technology.
DNA profiling is used to determine whether two DNA samples
come from the same individual.
To produce a DNA profile, scientists compare genetic
markers, sequences in the genome that vary from person to
person
Investigating Murder, Paternity, and Ancient DNA
DNA profiling (DNA fingerprinting) can be used to establish
innocence or guilt of a criminal suspect, identity victims,
determine paternity, identify illegally exported animals products,
trace the evolutionary history of organisms and contribute to
research.
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Overview of DNA profiling
1.
Collect cells
2.
Extract DNA
3.
Cut the DNA in
fragments using the
same restriction
enzyme
1 DNA isolated
Crime scene
2 DNA amplified
4. Separate the fragments
using gel
electrophoresis
3 DNA compared
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Suspect 1 Suspect 2
Profiling Techniques
The Polymerase Chain Reaction (PCR)-specific segment of
DNA is targeted and copied. Scientists can obtain enough DNA
in blood or tissues to allow profiling.
Short Tandem Repeat (STR) Analysis- used to compare
genomes in two samples to prove they came from the same
person. This is used by a series of short sequences (Repetitive
DNA) that is repeated many times one after another.
Gel Electrophoresis - Comparing length of DNA fragments by
sorting macromolecules.
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Profiling Techniques: The Polymerase Chain
Reaction (PCR)
The polymerase chain reaction (PCR):
• Is a technique to copy quickly and precisely any
segment of DNA and
• Can generate enough DNA, from even minute amounts
of blood or other tissue, to allow DNA profiling
• a single DNA molecule can be replicated in a test tube
to make 30 million identical copies in a few hours
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Polymerase Chain Reaction:
DNA Replication in a Test Tube
Exponential Increase in the Number of DNA Molecules each Cycle
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Profiling Techniques:
Short Tandem Repeat (STR) Analysis
How do you test if two samples of DNA come from the same person?
•Repetitive DNA:
Compares dozen of short segment of repetitive sequences, present
in multiple copies, that makes up much of the DNA that lies
between genes in humans
•Short tandem repeats (STRs) are short sequences of DNA that
are repeated many times, tandemly (one after another), in the genome
Used as a method of DNA profiling by comparing the lengths of
STR sequences at certain sites in the genome
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Short tandem repeat (STR) sites
STR site 1
AGAT
STR site 2
GATA
Crime scene DNA
Different numbers of
short tandem repeats
Same number of
short tandem repeats
Suspect’s DNA
AGAT
GATA
STR sites contain tandem repeats of 4-nucleotide sequences. The
number of repetitions at each site vary from individual to individual.
Here, both the samples have same number of repeats (7) at first site
but different numbers in the second site
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Gel Electrophoresis
STR analysis compares the lengths of DNA fragments
• Uses gel electrophoresis, a method for sorting macromolecules usually proteins or nucleic acids—primarily by their electrical
charge and Size
• The DNA fragments are visualized as “bands” on the gel.
• The differences in the locations of the bands reflect the different
lengths of the DNA fragments.
Mixture of DNA
fragments of
different sizes
Band of
longest
(slowest)
fragments
Power
source
Gel
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Band of
shortest
Completed gel (fastest)
fragments
Visualizing STR fragment patterns
• Difference in the
locations of the band
reflect the different
length of DNA
fragments
• Provide evidence that
crime scene DNA did
not come from the
suspect
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Amplified
crime scene
DNA
Amplified
suspect’s
DNA
Longer
fragments
Shorter
fragments
Genomics and Proteomics
Genomics the study of complete sets of genes. Bacteria were
the first targets of genomics. As of 2009, the genomes of
nearly 1000 species have been published, including Baker’s
yeast, Mice, Fruit flies, Rice and Sorghum
Genome - Mapping Techniques
The whole- genome shotgun method involves sequencing
DNA fragments from an entire genome and then
assembling the sequences.
Proteomics
The systematic study of the full set of proteins found in
organisms.
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The Human Genome Project
• In 1990 an international consortium of government-funded the
Human Genome Project. The goal was to sequence the human
genome so scientists could have roadmap for finding genes
• Completed in 2004:
– Over 99% of the genome had been determined to 99.999% accuracy
– 3.2 billion nucleotide pairs were identified, about 21,000 genes were
found and about 98% of the human DNA was identified as noncoding
• The Human Genome Project can
help map the genes for specific
diseases such as:
– Alzheimer’s disease
– Parkinson’s disease
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Genome-Mapping Techniques
Genomes are most often sequenced using
whole-genome shotgun method in which:
– The entire genome is chopped into
fragments using restriction enzymes
– The fragments are cloned and sequenced
– Computers running specialized mapping
software reassemble the millions of
overlapping short sequences into a single
continuous sequence for every
chromosome—an entire genome
• Begun in 2006, the Human Variome
Project:
– Seeks to collect information on all of the
genetic variations that affect human
health
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Chromosome
Chop up with
restriction enzyme
DNA fragments
Sequence
fragments
Align
fragments
Reassemble
full sequence
Human Gene Therapy
What is it?
Human Gene Therapy a recombinant DNA procedure
intended to treat disease by altering an afflicted person's
genes.
How?
Some cases, a mutant version of a gene is replaced or
supplemented with the normal allele. Or replaced
long enough to fix the medical problem.
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HUMAN GENE THERAPY
• Human gene therapy:
– Is a recombinant DNA
procedure
Normal human
gene isolated
and cloned
– Seeks to treat disease
by altering the genes of
the afflicted person
– Often replaces or
supplements the mutant
version of a gene with a
properly functioning
one
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Normal human
gene inserted
into virus
Harmless
virus (vector)
Virus containing
normal human gene
Bone
marrow
Healthy person
Virus injected
into patient with
abnormal gene
Bone of person
with disease
Ethics of DNA Technology
Controversy over Genetically Modified Food
• The debate about genetically modified crops centers on whether
they might harm humans or damage the environment by
transferring genes through cross-pollination with other species.
Ethical Questions raised by DNA Technology
• Some ethical questions are how far should we take technology?
• Some controversies are whether or not it is morally right to know
your DNA when you are born and have a DNA profile, and how
private that would be.
• Also whether or not parents should be able to give their child who
has dwarfism a growth hormone to make them grow.
• Scientist are still weighing out the positive and negative effects of
DNA technology.
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The diagram here summarizes
_____.
A) how a gene is cloned
B) how a human could be cloned
C) human gene therapy
D) how a vaccine is made
E) how viruses can cause disease
Answer: C
Skill: Knowledge/Comprehension
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Please read the following scenario then answer the following question(s).
Molecular biologists have perfected DNA fingerprinting so that it is possible to
use the technique to provide evidence to solve crimes and even identify a
child's parents. Recently, a U.S. immigrant asked the U.S. Citizenship and
Immigration Services for permission to have her young daughter who was
living with grandparents in their homeland join her. Her request was denied
because there was an apparent mix-up with the child's birth certificate and it
could not be used as proof of maternity. Proof is required in cases such as
this. The mother requested DNA fingerprinting to make her case. Samples of
DNA were taken from the mother (Mom) and daughter (D1) as well as from
another daughter (D2) and a son (S) living in the United States with her.
Tandem repeat analysis was run on the four samples, and the results are
shown here.
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1) The results indicate that ______.
A) she is not the mother of the son
B) she is the mother of daughter D1
C) she is not the mother of daughter D1
D) daughter D1 and daughter D2 are identical twins
E) the mother could not be the mother of both daughter D1 and daughter
D2
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