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Chapter 12
DNA Technology and
Genomics
PowerPoint Lectures for
Biology: Concepts and Connections, Fifth Edition
– Campbell, Reece, Taylor, and Simon
Lectures by Chris Romero
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
DNA and Crime Scene Investigations
• DNA fingerprinting has provided a powerful
tool for crime scene investigators
– DNA is isolated from biological fluids left at
a crime scene
– The technique determines with near
certainty whether two samples of DNA are
from the same individual
• DNA technology—methods for studying and
manipulating genetic material—plays
significant roles in many areas of society
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
BACTERIAL PLASMIDS AND GENE CLONING
12.1 Plasmids are used to customize bacteria: An
overview
• Recombinant DNA technology: techniques for
combining genes from different sources
• Gene cloning: production of multiple identical
copies of gene-carrying DNA
• Genetic engineering: direct manipulation of
genes for practical purposes
• Biotechnology: use of organisms or their
components to make useful products
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• Recombinant DNA technology uses plasmids,
small, circular DNA molecules that replicate
separately from a bacterial chromosome
– Desired genes inserted into plasmids to
form recombinant DNA
– Plasmids inserted into bacteria
– Foreign genes copied when recombinant
bacteria multiply into a clone
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 12-01-3
Bacterium
Cell containing gene
of interest
Plasmid
isolated
DNA
isolated
Gene inserted
into plasmid
Bacterial
Plasmid
chromosome
Recombinant DNA
(plasmid)
DNA
Gene of
interest
Plasmid put into
bacterial cell
Recombinant
bacterium
Cell multiplies with
gene of interest
Copies of protein
Copies of gene
Clone of cells
Gene for pest
resistance
inserted into
plants
Gene used to alter bacteria
for cleaning up toxic waste
Protein used to
make snow
form at higher
temperature
Protein used to dissolve blood
clots in heart attack therapy
12.2 Enzymes are used to "cut and paste" DNA
• DNA from two sources cut by restriction
enzyme at specific restriction sites
• Resulting restriction fragments contain a
double-stranded sequence of DNA with
single-stranded "sticky ends"
• Fragments pair at their sticky ends by
hydrogen bonding
• DNA ligase pastes the strand into a
recombinant DNA molecule
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 12-02
Restriction enzyme
recognition sequence
G A AT T C
C T TAAG
DNA
Restriction enzyme
cuts the DNA into
fragments
G
C
G
Sticky end
Addition of a DNA
fragment from
another source
C
G
Two (or more)
fragments stick
together by
base-pairing
G A ATT C
C T T AA G
G A AT T C
C T T AA G
DNA ligase
pastes the strand
Recombinant DNA molecule
G
C
Animation: Restriction Enzymes
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
12.3 Genes can be cloned in recombinant
plasmids: a closer look
•
Bacteria take up recombinant plasmids from
their surroundings and reproduce, thereby
cloning the plasmids and the genes they
carry
1. Isolate DNA from two sources
2. Cut both DNAs with the same restriction
enzyme
3. Mix the DNAs, which join by basepairing
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
4. Add DNA ligase to bond the DNA
5. Put plasmid into bacterium by
transformation
6. Clone the bacterium
Animation: Cloning a Gene
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 12-03
Human cell
E. coli
Isolate DNA
from two sources
Cut both DNAs
with the same
restriction enzyme
Plasmid
DNA
Gene V
Sticky ends
Mix the DNAs;
they join by
base-pairing
Add DNA ligase
to bond the DNA covalently
Recombinant DNA
plasmid
Gene V
Put plasmid into bacterium
by transformation
Recombinant
bacterium
Clone the bacterium
Bacterial clone carrying many
copies of the human gene
12.4 Cloned genes can be stored in genomic
libraries
• Genomic library
– Set of cloned DNA fragments containing all
of an organism's genes
– Fragments can be constructed and stored in
cloned bacterial plasmids (plasmid library)
or phages (phage library)
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 12-04
Genome cut up with
restriction enzyme
Recombinant
plasmid
Recombinant
phage DNA
or
Bacterial
clone
Plasmid library
Phage
clone
Phage library
12.5 Reverse transcriptase helps make genes for cloning
•
Complementary DNA (cDNA), which contains only the
genes that are transcribed by a particular type of cell,
can be created using reverse transcriptase
1. Cell transcribes genes
2. RNA splicing removes introns
3. Single-strand DNA created from RNA with reverse
transcriptase
4. Enzymes added to break down RNA
5. Second DNA strand synthesized
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 12-05
Cell nucleus
DNA of
eukaryotic
gene
Exon Intron
Exon
Intron Exon
Transcription
RNA
transcript
RNA splicing
(removes introns)
mRNA
Test tube
Reverse transcriptase
Isolation of mRNA
from cell and addition
of reverse transcriptase;
synthesis of DNA strand
cDNA strand
Breakdown of RNA
Synthesis of second
DNA strand
cDNA of gene
(no introns)
CONNECTION
12.6 Recombinant cells and organisms can
mass-produce gene products
• Recombinant cells and organisms constructed
by DNA technology are used to manufacture
many useful products, chiefly proteins
– Bacteria are usually the best vectors
– Some eukaryotic cells are used
• Saccharomyces cerevisiae fungus for
brewing and baking
• Mammalian cells for pharmaceuticals
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
CONNECTION
12.7 DNA technology is changing the
pharmaceutical industry
• DNA technology is widely used to produce
medicines and to diagnose diseases
– Therapeutic hormones
• Example: humulin, human insulin produced
by bacteria
– Diagnosis and treatment of disease
• Example: analysis to identify HIV
– Development of vaccines
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Video: Biotechnology Lab
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
RESTRICTION FRAGMENT ANALYSIS AND
DNA FINGERPRINTING
12.8 Nucleic acid probes identify clones carrying
specific genes
• Detecting genes depends on base pairing
between the gene and a complementary
sequence on another nucleic acid molecule
• A nucleic acid probe
– Is a short, single-stranded molecule of
radioactively or fluorescently labeled DNA
or RNA
– Can base pair with a desired gene in a
library, thus tagging it
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 12-08
Radioactive
probe (DNA)
Mix with singlestranded DNA from
various bacterial
(or phage) clones
Single-stranded
DNA
Base pairing
indicates the
gene of interest
CONNECTION
12.9 DNA microarrays test for the expression of
many genes at once
•
DNA microarray assays can reveal patterns of
gene expression in different kinds of cells
1. Isolate mRNA
2. Make cDNA from mRNA using reverse
transcriptase
3. Apply cDNA (single-stranded) to wells
4. cDNA binds to corresponding gene;
unbound cDNA is rinsed away; remaining
DNA produces a glow
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 12-09
DNA microarray
Each well contains DNA
from a particular gene
mRNA
isolated
Reverse transcriptase
and fluorescent DNA
nucelotides
cDNA made
from mRNA
Actual size
(6,400 genes)
Unbound
cDNA rinsed
away
Fluorescent
spot
cDNA applied
to wells
Nonfluorescent
spot
cDNA
DNA of an
expressed gene
DNA of an
unexpressed gene
12.10 Gel electrophoresis sorts DNA molecules
by size
• Gel electrophoresis uses a gel as a molecular
sieve to separate nucleic acids by size or
electrical charge
– Longer macromolecules move through the
gel more slowly than shorter
macromolecules, resulting in a pattern of
bands on the gel
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 12-10
Mixture of DNA
molecules of
different sizes
Longer
molecules
Power
source
Gel
Shorter
molecules
Completed gel
12.11 Restriction fragment length polymorphisms
can be used to detect differences in DNA
sequences
•
Differences in DNA sequences on homologous
chromosomes produce sets of restriction
fragments that differ between individuals
– Are called restriction fragment length
polymorphisms (RFLPs)
– Are of different lengths and will migrate
different distances in an electrophoretic gel
– Can be used as genetic markers
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 12-11a
Crime scene
Suspect
w
Cut
C
C
G
G
G
G
C
C
A
C
G
G
T
G
C
C
C
C
G
G
G
G
C
C
z
x
Cut
y
C
C
G
G
G
G
C
C
Cut
y
DNA from chromosomes
LE 12-11b
1
2
Longer
fragments
z
x
w
Shorter
fragments
y
y
• Restriction fragments can be used as DNA
probes to detect harmful alleles
– Patterns of normal and harmful alleles
identified
– Banding patterns compared with probe
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 12-11c-3
Restriction fragment preparation
I
II
III
Restriction
fragments
Gel electrophoresis
I
II III
Blotting
Filter paper
Radioactive probe
Radioactive, singlestranded DNA (probe)
Probe
I
Detection of radioactivity
(autoradiography)
II
III
Film
I
II
III
CONNECTION
12.12 DNA technology is used in courts of law
•
Forensic science is the scientific analysis of evidence
for criminal and other legal investigations
•
DNA fingerprinting requires only about 1,000 cells
– Radioactive probes mark electrophoresis bands
that contain certain markers
– Produces a specific pattern of bands to compare to
those of accused person
– Highly reliable because odds of two people having
identical DNA fingerprints are extremely small
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 12-12a
Defendant’s
blood
Blood from
defendant’s clothes
Victim’s
blood
CONNECTION
12.13 Gene therapy may someday help treat a
variety of diseases
• Gene therapy is the alteration of an afflicted
individual's genes
– Where a disorder is due to a single gene, it
is sometimes possible to replace the
defective gene with a normal allele
– To be permanent, the normal allele must be
transferred to cells that multiply throughout
a person's life, such as bone marrow cells
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 12-13
Cloned gene
(normal allele)
Insert normal gene
into virus
Viral nucleic
acid
Retrovirus
Infect bone marrow
cell with virus
Viral DNA inserts
into chromosome
Bone marrow
cell from patient
Bone
marrow
Inject cells
into patient
• Gene therapy
– May one day be used to treat both genetic
diseases and nongenetic disorders, but
progress is slow
– Raises both technical and ethical issues
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
12.14 The PCR method is used to amplify DNA
sequences
• The polymerase chain reaction (PCR) can be
used to quickly clone a very large number of
DNA copies for analysis
– DNA sample mixed with DNA polymerase,
nucleotide monomers, other ingredients
– Mixture exposed to cycles of heating to
separate the DNA strands
– During each cycle, DNA replicates, doubling
the amount
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 12-14
Initial
DNA
segment
1
2
4
Number of DNA molecules
8
GENOMICS CONNECTION
12.15 The Human Genome Project is an
ambitious application of DNA technology
• The Human Genome Project was begun in
1990 and is now largely completed
– Initially involved three stages: genetic
(linkage) and physical mapping of
chromosomes, followed by DNA sequencing
– Superseded by "shotgun" approach, going
directly to stage 3
• The data are providing insight into
development, evolution, and many diseases
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
12.16 Most of the human genome does not
consist of genes
• Surprisingly, the haploid human genome
contains only about 25,000 genes
• About 97% of the human genome consists of
noncoding DNA
– Gene-control sequences
– Introns
– Noncoding DNA located between genes
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
– Repetitive DNA-nucleotide sequences
present in many copies
• Teleomeres found at chromosome ends
• Transposons ("jumping genes") that can
move about within the genome
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
CONNECTION
12.17 The science of genomics compares whole
genomes
• Genomics is the study of whole sets of genes
and their interactions
– As of 2005, the genomes of about 150
species had been sequenced
– Besides being interesting in themselves,
nonhuman genomes provide understanding
of the human genome
• Proteomics is the study of the full protein sets
encoded by genomes
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
GENETICALLY MODIFIED ORGANISMS
CONNECTION
12.18 Genetically modified organisms are
transforming agriculture
• Recombinant DNA technology can produce
new varieties of plants and animals for use in
agriculture
– Genetically modified (GM) organisms have
acquired genes by artificial means
– Transgenic organisms have had genes from
other organisms inserted into their genomes
• A number of important crop plants are
genetically modified using the Ti plasmid
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 12-18a
Agrobacterium tumefaciens
DNA containing
gene for desired trait
Ti
plasmid
T DNA
Restriction site
Insertion of gene
into plasmid using
restriction enzyme
and DNA ligase
Recombinant
Ti plasmid
Plant cell
Introduction
Regeneration
into plant
of plant
cells in
culture
T DNA carrying new
Plant with new trait
gene within plant chromosome
• Transgenic animals have also been
engineered to be pharmaceutical "factories"
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
CONNECTION
12.19 Could GM organisms harm human health or the
environment?
•
Scientists have developed safety guidelines to
minimize the risks involved in genetic engineering
– Laboratory safety procedures
– Organisms altered so they cannot live outside the
lab
•
Exported GM organisms must be identified
•
Today most concern focuses on transgenic crop plants
passing their genes to wild relatives
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
CONNECTION
12.20 Genomics researcher Eric Lander
discusses the Human Genome Project
• Dr. Eric Lander founded the Broad Institute of
MIT and Harvard
– Uses genomics to develop new methods to
investigate and treat diseases
• The Human Genome Project
– Results will give researchers the opportunity
to examine the human genome from a "big
picture" approach
– Revolutionizing evolutionary biology
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings