Chapter 12 DNA Technology and Genomics

PowerPoint Lectures for

Biology: Concepts & Connections, Sixth Edition
Campbell, Reece, Taylor, Simon, and Dickey

Lecture by Mary C. Colavito

Copyright 2009 Pearson Education, Inc.
Introduction: DNA and Crime Scene Investigations

DNA evidence was used to solve a double murder

in England
Showed that two murders could have been
committed by the same person
Showed the innocence of someone who confessed to
one of the murders
Showed the absence of a match in 5,000 men tested
when the murderer persuaded another man to
donate blood in his name
Showed a match with the murderer and DNA found
with both victims
Copyright 2009 Pearson Education, Inc.
 GENE CLONING

Copyright 2009 Pearson Education, Inc.

12.1 Genes can be cloned in recombinant plasmids

Genetic engineering involves manipulating

genes for practical purposes
Gene cloning leads to the production of multiple
identical copies of a gene-carrying piece of DNA
Recombinant DNA is formed by joining DNA
sequences from two different sources
One source contains the gene that will be cloned
Another source is a gene carrier, called a vector
Plasmids (small, circular DNA molecules
independent of the bacterial chromosome) are often
used as vectors

Copyright 2009 Pearson Education, Inc.

12.1 Genes can be cloned in recombinant plasmids

Steps in cloning a gene

1. Plasmid DNA is isolated
2. DNA containing the gene of interest is isolated
3. Plasmid DNA is treated with restriction enzyme that
cuts in one place, opening the circle
4. DNA with the target gene is treated with the same
enzyme and many fragments are produced
5. Plasmid and target DNA are mixed and associate with
each other

Copyright 2009 Pearson Education, Inc.

12.1 Genes can be cloned in recombinant plasmids

6. Recombinant DNA molecules are produced when

DNA ligase joins plasmid and target segments
together
7. The recombinant DNA is taken up by a bacterial cell
8. The bacterial cell reproduces to form a clone of
cells

Animation: Cloning a Gene

Copyright 2009 Pearson Education, Inc.

 E. coli bacterium
Plasmid

Cell with DNA

containing gene
Bacterial
1 Isolate
of interest
chromosome plasmid
2 Isolate
DNA

3 Cut plasmid DNA

with enzyme Gene of interest

4 Cut cells DNA

with same enzyme
Gene
of interest

5 Combine targeted fragment

and plasmid DNA

Examples of
gene use
6 Add DNA ligase,
which closes
the circle with Genes may be inserted
covalent bonds into other organisms
Recombinant
DNA Gene
plasmid of interest
9 Genes or proteins
7 Put plasmid are isolated from the
into bacterium cloned bacterium
by transformation
Recombinant
bacterium
Harvested
proteins Examples of
8 Allow bacterium may be protein use
to reproduce used directly

Clone
of cells
 E. coli bacterium
Plasmid

Cell with DNA

containing gene
Bacterial 1 Isolate
of interest
chromosome plasmid
2 Isolate
DNA

DNA
Gene of interest
 E. coli bacterium
Plasmid

Cell with DNA

containing gene
Bacterial 1 Isolate
of interest
chromosome plasmid
2 Isolate
DNA

3 Cut plasmid DNA

with enzyme Gene of interest
4 Cut cells DNA
with same enzyme
Gene
of interest
 E. coli bacterium
Plasmid

Cell with DNA

containing gene
Bacterial 1 Isolate
of interest
chromosome plasmid
2 Isolate
DNA

3 Cut plasmid DNA

with enzyme Gene of interest
4 Cut cells DNA
with same enzyme
Gene
of interest

5 Combine targeted fragment

and plasmid DNA
 E. coli bacterium
Plasmid

Cell with DNA

containing gene
Bacterial 1 Isolate
of interest
chromosome plasmid
2 Isolate
DNA

3 Cut plasmid DNA

with enzyme Gene of interest
4 Cut cells DNA
with same enzyme
Gene
of interest

5 Combine targeted fragment

and plasmid DNA

6 Add DNA ligase,

which closes
the circle with
covalent bonds
Recombinant
DNA Gene
plasmid of interest
Recombinant
DNA Gene
plasmid of interest

7 Put plasmid
into bacterium
by transformation
Recombinant
bacterium
Recombinant
DNA Gene
plasmid of interest

7 Put plasmid
into bacterium
by transformation
Recombinant
bacterium

8 Allow bacterium
to reproduce

Clone
of cells
 Examples of
gene use

Genes may be inserted

Recombinant into other organisms
DNA Gene
plasmid of interest
9 Genes or proteins
7 Put plasmid are isolated from the
into bacterium cloned bacterium
by transformation
Recombinant
bacterium
Harvested
proteins
8 Allow bacterium may be
to reproduce used directly

Clone
of cells
Examples of
protein use
12.2 Enzymes are used to cut and paste DNA

Restriction enzymes cut DNA at specific

sequences
Each enzyme binds to DNA at a different restriction
site
Many restriction enzymes make staggered cuts that
produce restriction fragments with single-stranded
ends called sticky ends
Fragments with complementary sticky ends can
associate with each other, forming recombinant DNA

DNA ligase joins DNA fragments together

Animation: Restriction Enzymes

Copyright 2009 Pearson Education, Inc.

 Restriction enzyme
recognition sequence

1 DNA

Restriction enzyme
cuts the DNA into
fragments

2
Sticky end
 Restriction enzyme
recognition sequence

1 DNA

Restriction enzyme
cuts the DNA into
fragments

2
Sticky end

Addition of a DNA
fragment from 3
another source
 Restriction enzyme
recognition sequence

1 DNA

Restriction enzyme
cuts the DNA into
fragments

2
Sticky end

Addition of a DNA
fragment from 3
another source

Two (or more)

fragments stick
together by
base-pairing

4
 Restriction enzyme
recognition sequence

1 DNA

Restriction enzyme
cuts the DNA into
fragments

2
Sticky end

Addition of a DNA
fragment from 3
another source

Two (or more)

fragments stick
together by
base-pairing

DNA ligase
pastes the strands

Recombinant
5
DNA molecule
12.3 Cloned genes can be stored in genomic
libraries
A genomic library is a collection of all of the
cloned DNA fragments from a target genome
Genomic libraries can be constructed with
different types of vectors
Plasmid library: genomic DNA is carried by plasmids
Phage library: genomic DNA is incorporated into
bacteriophage DNA
Bacterial artificial chromosome (BAC) library:
specialized plasmids can carry large DNA sequences

Copyright 2009 Pearson Education, Inc.

 Genome cut up with
Recombinant restriction enzyme
plasmid
Recombinant
phage DNA
or

Bacterial Phage
clone clone

Plasmid library Phage library

12.4 Reverse transcriptase can help make genes
for cloning
Complementary DNA (cDNA) is used to clone
eukaryotic genes
mRNA from a specific cell type is the template
Reverse transcriptase produces a DNA strand
from mRNA
DNA polymerase produces the second DNA strand
Advantages of cloning with cDNA
Study genes responsible for specialized
characteristics of a particular cell type
Obtain gene sequences without introns
Smaller size is easier to handle
Allows expression in bacterial hosts
Copyright 2009 Pearson Education, Inc.
Cell nucleus
Exon Intron Exon Intron Exon
DNA of
eukaryotic
gene
1 Transcription

RNA
transcript
2 RNA splicing

mRNA

3 Isolation of mRNA
and addition of reverse
Test tube transcriptase; synthesis
Reverse transcriptase of DNA strand

cDNA strand
being synthesized 4 Breakdown of RNA

5 Synthesis of second
DNA strand
cDNA of gene
(no introns)
12.5 Nucleic acid probes identify clones carrying
specific genes
Nucleic acid probes bind to cloned DNA
Probes can be DNA or RNA sequences
complementary to a portion of the gene of interest
A probe binds to a gene of interest by base pairing
Probes are labeled with a radioactive isotope or
fluorescent tag for detection

Copyright 2009 Pearson Education, Inc.

12.5 Nucleic acid probes identify clones carrying
specific genes
Screening a gene library
Bacterial clones are transferred to filter paper
Cells are lysed and DNA is separated into single
strands
A solution containing the probe is added, and binding
to the DNA of interest is detected
The clone carrying the gene of interest is grown for
further study

Copyright 2009 Pearson Education, Inc.

 Radioactive
DNA probe
Mix with single-
stranded DNA from
genomic library

Single-stranded
DNA

Base pairing
indicates the
gene of interest
 GENETICALLY MODIFIED
ORGANISMS

Copyright 2009 Pearson Education, Inc.

12.6 Recombinant cells and organisms can
mass-produce gene products
Cells and organisms containing cloned genes are
used to manufacture large quantities of gene
products
Capabilities of the host cell are matched to the
characteristics of the desired product
Prokaryotic host: E. coli
Can produce eukaryotic proteins that do not require post-
translational modification
Has many advantages in gene transfer, cell growth, and
quantity of protein production
Can be engineered to secrete proteins

Copyright 2009 Pearson Education, Inc.

12.6 Recombinant cells and organisms can
mass-produce gene products
Capabilities of the host cell are matched to the
characteristics of the desired product
Eukaryotic hosts
Yeast: S. cerevisiae
Can produce and secrete complex eukaryotic
proteins
Mammalian cells in culture
Can attach sugars to form glycoproteins
Pharm animals
Will secrete gene product in milk

Copyright 2009 Pearson Education, Inc.

12.7 CONNECTION: DNA technology has
changed the pharmaceutical industry and
medicine
Products of DNA technology
Therapeutic hormones
Insulin to treat diabetes
Human growth hormone to treat dwarfism

Diagnosis and treatment of disease

Testing for inherited diseases
Detecting infectious agents such as HIV

Copyright 2009 Pearson Education, Inc.

12.7 CONNECTION: DNA technology has
changed the pharmaceutical industry and
medicine
Products of DNA technology
Vaccines
Stimulate an immune response by injecting
Protein from the surface of an infectious agent
A harmless version of the infectious agent
A harmless version of the smallpox virus containing
genes from other infectious agents

Copyright 2009 Pearson Education, Inc.

12.7 CONNECTION: DNA technology has
changed the pharmaceutical industry and
medicine
Advantages of recombinant DNA products
Identity to human protein
Purity
Quantity

Copyright 2009 Pearson Education, Inc.

12.8 CONNECTION: Genetically modified
organisms are transforming agriculture
Genetically modified (GM) organisms contain
one or more genes introduced by artificial means
Transgenic organisms contain at least one
gene from another species
GM plants
Resistance to herbicides
Resistance to pests
Improved nutritional profile
GM animals
Improved qualities
Production of proteins or therapeutics
Copyright 2009 Pearson Education, Inc.
Agrobacterium tumefaciens

DNA containing
gene for desired trait

1
Ti Recombinant
plasmid Insertion of gene Ti plasmid
into plasmid

Restriction site
Agrobacterium tumefaciens

DNA containing Plant cell

gene for desired trait

1 2
Ti Recombinant
plasmid Insertion of gene Ti plasmid Introduction
into plasmid into plant
cells
DNA carrying new gene
Restriction site
Agrobacterium tumefaciens

DNA containing Plant cell

gene for desired trait

1 2 3
Ti Recombinant
plasmid Insertion of gene Ti plasmid Introduction Regeneration
into plasmid into plant of plant
cells
DNA carrying new gene
Plant with new trait
Restriction site
12.9 Genetically modified organisms raise concerns
about human and environmental health
Scientists use safety measures to guard against
production and release of new pathogens
Concerns related to GM organisms
Can introduce allergens into the food supply
FDA requires evidence of safety before approval
Exporters must identify GM organisms in food shipments
May spread genes to closely related organisms
Hybrids with native plants may be prevented by modifying
GM plants

Regulatory agencies address the safe use of

biotechnology
Copyright 2009 Pearson Education, Inc.
12.10 CONNECTION: Gene therapy may
someday help treat a variety of diseases
Gene therapy aims to treat a disease by
supplying a functional allele
One possible procedure
Clone the functional allele and insert it in a retroviral
vector
Use the virus to deliver the gene to an affected cell
type from the patient, such as a bone marrow cell
Viral DNA and the functional allele will insert into the
patients chromosome
Return the cells to the patient for growth and division

Copyright 2009 Pearson Education, Inc.

12.10 CONNECTION: Gene therapy may
someday help treat a variety of diseases
SCID (severe combined immune deficiency) was
the first disease treated by gene therapy
First trial in 1990 was inconclusive
Second trial in 2000 led to the development of
leukemia in some patients due to the site of gene
insertion
Challenges
Safe delivery to the area of the body affected by the
disease
Achieving a long-lasting therapeutic effect
Addressing ethical questions
Copyright 2009 Pearson Education, Inc.
Cloned gene
(normal allele) 1 Insert normal gene
into virus

Viral nucleic acid

Retrovirus

2 Infect bone marrow

cell with virus

3 Viral DNA inserts

into chromosome

Bone marrow
cell from patient

Bone
marrow
4 Inject cells
into patient
 DNA PROFILING

Copyright 2009 Pearson Education, Inc.

12.11 The analysis of genetic markers can
produce a DNA profile
DNA profiling is the analysis of DNA fragments
to determine whether they come from a particular
individual
Compares genetic markers from noncoding regions
that show variation between individuals
Involves amplification (copying) of markers for
analysis
Sizes of amplified fragments are compared

Copyright 2009 Pearson Education, Inc.

 Crime scene Suspect 1 Suspect 2
1 DNA isolated

2 DNA of selected
markers amplified

3 Amplified DNA
compared
12.12 The PCR method is used to amplify DNA
sequences
Polymerase chain reaction (PCR) is a method
of amplifying a specific segment of a DNA
molecule
Relies upon a pair of primers
Short DNA molecules that bind to sequences at each
end of the sequence to be copied
Used as a starting point for DNA replication
Repeated cycle of steps for PCR
Sample is heated to separate DNA strands
Sample is cooled and primer binds to specific target
sequence
Target sequence is copied with heat-stable DNA
polymerase
Copyright 2009 Pearson Education, Inc.
12.12 The PCR method is used to amplify DNA
sequences
Advantages of PCR
Can amplify DNA from a small sample
Results are obtained rapidly
Reaction is highly sensitive, copying only the target
sequence

Copyright 2009 Pearson Education, Inc.

 Cycle 1 Cycle 2 Cycle 3
yields 2 molecules yields 4 molecules yields 8 molecules

Genomic
DNA
3 5 3 5 3 5
5
1 Heat to 2 Cool to allow 3 DNA
3 5 separate primers to form polymerase adds
DNA strands hydrogen bonds nucleotides
5 3 with ends of to the 3 end
Target target sequences of each primer
sequence 5
5 3 5 3 5 3
Primer New DNA
 Cycle 1
yields 2 molecules

Genomic
DNA
3 5 3 5 3 5
5
1 Heat to 2 Cool to allow 3 DNA
3 5 separate primers to form polymerase adds
DNA strands hydrogen bonds nucleotides
5 3 with ends of to the 3 end
Target target sequences of each primer
sequence 5
5 3 5 3 5 3
Primer New DNA
 Cycle 2 Cycle 3
yields 4 molecules yields 8 molecules
12.13 Gel electrophoresis sorts DNA molecules
by size
Gel electrophoresis separates DNA molecules
based on size
DNA sample is placed at one end of a porous gel
Current is applied and DNA molecules move from the
negative electrode toward the positive electrode
Shorter DNA fragments move through the gel pores
more quickly and travel farther through the gel
DNA fragments appear as bands, visualized through
staining or detecting radioactivity or fluorescence
Each band is a collection of DNA molecules of the
same length
Video: Biotechnology Lab

Copyright 2009 Pearson Education, Inc.

 Mixture of DNA
fragments of
different sizes

Longer
(slower)
Power molecules
source
Gel
Shorter
(faster)
molecules
Completed gel
12.14 STR analysis is commonly used for DNA
profiling
Short tandem repeats (STRs) are genetic
markers used in DNA profiling
STRs are short DNA sequences that are repeated
many times in a row at the same location
The number of repeating units can differ between
individuals
STR analysis compares the lengths of STR
sequences at specific regions of the genome
Current standard for DNA profiling is to analyze 13
different STR sites

Copyright 2009 Pearson Education, Inc.

 STR site 1 STR site 2

Crime scene DNA

Number of short tandem Number of short tandem

repeats match repeats do not match

Suspects DNA
Crime scene Suspects
DNA DNA
12.15 CONNECTION: DNA profiling has
provided evidence in many forensic
investigations
Forensics
Evidence to show guilt or innocence

Establishing family relationships

Paternity analysis

Identification of human remains

After tragedies such as the September 11, 2001, attack on the
World Trade Center

Species identification
Evidence for sale of products from endangered species

Copyright 2009 Pearson Education, Inc.

12.16 RFLPs can be used to detect differences in
DNA sequences
Single nucleotide polymorphism (SNP) is a
variation at one base pair within a coding or
noncoding sequence
Restriction fragment length polymorphism
(RFLP) is a variation in the size of DNA fragments
due to a SNP that alters a restriction site
RFLP analysis involves comparison of sizes of
restriction fragments by gel electrophoresis

Copyright 2009 Pearson Education, Inc.

 Restriction
enzymes added

DNA sample 1 DNA sample 2

Cut

z
x

Cut Cut
y y

Longer
fragments
z

w
Shorter
y y
fragments
 GENOMICS

Copyright 2009 Pearson Education, Inc.

12.17 Genomics is the scientific study of whole
genomes
Genomics is the study of an organisms complete
set of genes and their interactions
Initial studies focused on prokaryotic genomes
Many eukaryotic genomes have since been
investigated

Evolutionary relationships can be elucidated

Genomic studies showed a 96% similarity in DNA
sequences between chimpanzees and humans
Functions of human disease-causing genes have
been determined by comparisons to similar genes in
yeast
Copyright 2009 Pearson Education, Inc.
12.18 CONNECTION: The Human Genome
Project revealed that most of the human
genome does not consist of genes
Goals of the Human Genome Project (HGP)
To determine the nucleotide sequence all DNA in the
human genome
To identify the location and sequence of every
human gene

Copyright 2009 Pearson Education, Inc.

12.18 CONNECTION: The Human Genome
Project revealed that most of the human
genome does not consist of genes
Results of the Human Genome Project
Humans have 21,000 genes in 3.2 billion nucleotide
pairs
Only 1.5% of the DNA codes for proteins, tRNAs, or
rRNAs
The remaining 88.5% of the DNA contains
Control regions such as promoters and enhancers
Unique noncoding DNA
Repetitive DNA
Found in centromeres and telomeres
Found dispersed throughout the genome, related to
transposable elements that can move or be
copied from one location to another
Copyright 2009 Pearson Education, Inc.
Exons (regions of genes coding for protein
or giving rise to rRNA or tRNA) (1.5%)

Repetitive Introns and

DNA that regulatory
includes sequences
transposable (24%)
elements
and related
sequences
(44%) Unique
noncoding
DNA (15%)
Repetitive
DNA
unrelated to
transposable
elements
(15%)
12.19 The whole-genome shotgun method of
sequencing a genome can provide a wealth
of data quickly
Three stages of the Human Genome Project
A low-resolution linkage map was developed using
RFLP analysis of 5,000 genetic markers
A physical map was constructed from nucleotide
distances between the linkage-map markers
DNA sequences for the mapped fragments were
determined

Copyright 2009 Pearson Education, Inc.

12.19 The whole-genome shotgun method of
sequencing a genome can provide a wealth
of data quickly
Whole-genome shotgun method
Restriction enzymes were used to produce fragments
that were cloned and sequenced
Computer analysis assembled the sequence by
aligning overlapping regions

Copyright 2009 Pearson Education, Inc.

Chromosome
Chop up with
restriction enzyme

DNA fragments
Sequence
fragments

Align
fragments

Reassemble
full sequence
12.20 Proteomics is the scientific study of the full
set of proteins encoded by a genome
Proteomics
Studies the proteome, the complete set of proteins
specified by a genome
Investigates protein functions and interactions

The human proteome may contain 100,000

proteins

Copyright 2009 Pearson Education, Inc.

12.21 EVOLUTION CONNECTION: Genomes
hold clues to the evolutionary divergence of
humans and chimps
Comparisons of human and chimp genomes
Differ by 1.2% in single-base substitutions
Differ by 2.7% in insertions and deletions of larger
DNA sequences
Human genome shows greater incidence of
duplications
Genes showing rapid evolution in humans
Genes for defense against malaria and tuberculosis
Gene regulating brain size
FOXP2 gene involved with speech and vocalization

Copyright 2009 Pearson Education, Inc.

 Bacterial
clone
Cut
Bacterium
DNA
fragments

Cut Recombinant
DNA
plasmids
Recombinant
bacteria
Plasmids
Genomic library
Mixture of DNA
fragments Longer
fragments
move slower
A band is a Power
collection of DNA source
fragments of one Shorter
particular length fragments
move faster

DNA attracted to +
pole due to PO4 groups
 DNA
amplified (a)
via

Bacterial
plasmids

DNA
sample

treated with treated with

(b)

DNA
fragments
sorted by size via

(c)

Recombinant plasmids
are inserted
into bacteria

Add

(d)
Particular
DNA
sequence are copied via
highlighted

(e)

Collection
(f) is called a
 DNA
amplified (a)
via

Bacterial
plasmids

DNA
sample

treated with treated with

(b)
 (b)

DNA
fragments
sorted by size via

(c)

Recombinant plasmids
are inserted
into bacteria

Add

(d)

Particular
DNA
sequence are copied via
highlighted

(e)

Collection
(f)
is called a
You should now be able to

1. Distinguish between terms in the following

groups: restriction enzymeDNA ligase; GM
organismtransgenic organism; SNPRFLP;
genomicsproteomics
2. Define the following terms: cDNA, gel
electrophoresis, gene cloning, genomic library,
pharm animal, plasmid, probe, recombinant
DNA, repetitive DNA, reverse transcriptase, STR,
Taq polymerase, vector, whole-genome shotgun
method
3. Describe how genes are cloned
Copyright 2009 Pearson Education, Inc.
You should now be able to

4. Describe how a probe is used to identify a gene of

interest
5. Describe how gene therapy has been attempted
and identify challenges to the effectiveness of this
treatment approach
6. Distinguish between the use of prokaryotic and
eukaryotic cells in producing recombinant DNA
products
7. Identify advantages to producing pharmaceuticals
with recombinant DNA technology
Copyright 2009 Pearson Education, Inc.
You should now be able to

8. Describe the basis for DNA profiling and explain

how it is used to provide evidence in forensic
investigations
9. Explain how PCR provides copies of a specific
DNA sequence
10. Identify ethical concerns related to the use of
recombinant DNA technology
11. Describe how comparative information from
genome projects has led to a better
understanding of human biology
Copyright 2009 Pearson Education, Inc.