Download Recombinant Technology

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
• 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
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• 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)
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
BACTERIAL PLASMIDS AND GENE CLONING
12.1 Plasmids are used to customize bacteria: An
overview
• Gene cloning is one application of DNA
technology
– Methods for studying and manipulating
genetic material
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• Researchers can insert desired genes into
plasmids, creating recombinant DNA
– And insert those plasmids into bacteria
Bacterium
Cell containing gene
of interest
1 Plasmid
isolated
2 DNA
isolated
3 Gene inserted
into plasmid
Plasmid
Bacterial
chromosome
Recombinant DNA
(plasmid)
DNA
Gene of
4 Plasmid put into interest
bacterial cell
Recombinant
bacterium
5 Cell multiplies with
gene of interest
Copies of gene
Clone of cells
Gene for pest
resistance
inserted into
plants
Figure 12.1
Copies of protein
Gene used to alter bacteria
for cleaning up toxic waste
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
Protein used to
make snow form
at higher
temperature
Protein used to dissolve blood
clots in heart attack therapy
• If the recombinant bacteria multiply into a clone
– The foreign genes are also copied
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
12.2 Enzymes are 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
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• Creating recombinant DNA using restriction
enzymes and DNA ligase
Restriction enzyme
recognition sequence
1
GAATTC
CTTAAG
DNA
Restriction enzyme
cuts the DNA into
fragments
2
Sticky end
Addition of a DNA
fragment from
another source
3
Two (or more)
fragments stick
together by
base-pairing
4
G A AT T C
C T TA A G
G A AT T C
C T TA A G
DNA ligase
pastes the strand
5
Figure 12.2
Recombinant DNA molecule
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
12.3 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
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• Cloning a gene in a bacterial plasmid
E.coli
Human cell
Isolate DNA
1 from two sources
Cut both DNAs
2 with the same
restriction enzyme
Plasmid
DNA
Gene V
Sticky ends
3
Mix the DNAs;
they join by
base-pairing
4
Add DNA ligase
to bond the DNA covalently
Gene V
Recombinant DNA
plasmid
5
Put plasmid into bacterium
by transformation
6
Clone the bacterium
Recombinant
bacterium
Figure 12.3
Bacterial clone carrying many
copies of the human gene
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
12.4 Cloned genes can be stored in genomic
libraries
• Genomic libraries, sets of DNA fragments
containing all of an organism’s genes
–
Can be constructed and stored in cloned
bacterial plasmids or phages
Genome cut up with
restriction enzyme
Recombinant
plasmid
Recombinant
phage DNA
or
Bacterial
clone
Figure 12.4
Plasmid library
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
Phage
clone
Phage library
12.5 Reverse transcriptase helps make genes for cloning
• Reverse transcriptase can be used to make smaller,
complementary DNA (cDNA) libraries
–
Containing only the genes that are transcribed
by a particular type of cell
Cell nucleus
Exon Intron
DNA of
eukaryotic
gene
Exon
Intron Exon
1 Transcription
RNA
transcript
2 RNA splicing
(removes introns)
mRNA
Test tube
Reverse transcriptase
3 Isolation of mRNA
from cell and addition
of reverse transcriptase;
synthesis of DNA strand
cDNA strand
4
Breakdown of RNA
5 Synthesis of second
DNA strand
Figure 12.5
cDNA of gene
(no introns)
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CONNECTION
12.6 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
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• Different organisms, including bacteria, yeast,
and mammals
– Can be used for this purpose
Figure 12.6
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CONNECTION
12.7 DNA technology is changing the
pharmaceutical industry
• DNA technology
– Is widely used to produce medicines and
to diagnose diseases
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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
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
Diagnosis and Treatment of Disease
• DNA technology
– Is being used increasingly in disease
diagnosis
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Vaccines
• DNA technology
– Is also helping medical researchers
develop vaccines
Figure 12.7B
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
• DNA technology methods
– Can be used to identify specific pieces of
DNA
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• A nucleic acid probe
– Is a short, single-stranded molecule of
radioactively labeled or fluorescently
labeled DNA or RNA
– Can tag a desired gene in a library
Radioactive
probe (DNA)
Mix with singlestranded DNA from
various bacterial
(or phage) clones
Single-stranded
DNA
Figure 12.8
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
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
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• DNA microarray
DNA microarray
Each well contains DNA
from a particular gene
1 mRNA
isolated
Reverse transcriptase
and fluorescent DNA
nucleotides
2 cDNA made
from mRNA
Actual size
(6,400 genes)
4 Unbound
cDNA rinsed
away
Fluorescent
spot
3 cDNA applied
to wells
Nonfluorescent
spot
cDNA
DNA of an
expressed gene
Figure 12.9
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
DNA of an
unexpressed gene
12.10 Gel electrophoresis sorts DNA molecules
by size
Mixture of DNA
molecules of
different sizes
–
–
Longer
molecules
Power
source
Gel
+
Shorter
molecules
+
Figure 12.10
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Completed gel
12.11 Restriction fragment length polymorphisms
can be used to detect differences in DNA
sequences
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How Restriction Fragments Reflect DNA Sequence
• Restriction fragment length polymorphisms (RFLPs)
–
Reflect differences in the sequences of DNA
samples
Crime scene
Suspect
w
Cut
C
C
G
G
G
G
C
C
z
A
C
G
G
T
G
C
C
C
C
G
G
G
G
C
C
x
Cut
y
Figure 12.11A
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
C
C
G
G
G
G
C
C
Cut
y
DNA from chromosomes
• After digestion by restriction enzymes
– The fragments are run through a gel
1
–
2
Longer
fragments
z
x
w
Shorter
fragments
Figure 12.11B
y
+
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
y
Using DNA Probes to Detect Harmful Alleles
• Radioactive probes
– Can reveal DNA bands of interest on a
gel
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• Detecting a harmful allele using restriction
fragment analysis
1 Restriction fragment
preparation
I
II
III
Restriction
fragments
2 Gel electrophoresis
I II III
3 Blotting
Filter paper
4 Radioactive probe
Radioactive, singlestranded DNA (probe)
Probe
5 Detection of radioactivity
(autoradiography)
I
II
III
Film
Figure 12.11C
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
I
II
III
CONNECTION
12.12 DNA technology is used in courts of law
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• DNA fingerprinting can help solve crimes
Defendant’s
blood
Blood from
defendant’s clothes
Victim’s
blood
Figure 12.12A
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
Figure 12.12B
CONNECTION
12.13 Gene therapy may someday help treat a variety of
diseases
• Gene therapy
–
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
3 Viral DNA inserts
into chromosome
Bone marrow
cell from patient
Bone
marrow
Figure 12.13
4 Inject cells
into patient
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• Gene therapy
– May one day be used to treat both
genetic diseases and nongenetic
disorders
• Unfortunately, progress is slow
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 clone a small sample of
DNA quickly, producing enough copies
for analysis
Initial
DNA
segment
1
Figure 12.14
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
2
4
8
Number of DNA molecules
GENOMICS
CONNECTION
12.15 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
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• 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
• The haploid human genome contains about
25,000 genes
– And a huge amount of noncoding DNA
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• Much of the noncoding DNA consists of
repetitive nucleotide sequences
– And transposons 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
• The sequencing of many prokaryotic and
eukaryotic genomes
– Has produced data for genomics, the
study of whole genomes
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• Besides being interesting themselves
– Nonhuman genomes can be compared
with the human genome
Table 12.17
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• Proteomics
– Is the study of the full sets of proteins
produced by organisms
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
GENETICALLY MODIFIED ORGANISMS
CONNECTION
12.18 Genetically modified organisms are
transforming agriculture
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• Recombinant DNA technology
– Can be used to produce new genetic
varieties of plants and animals,
genetically modified (GM) organisms
Agrobacterium tumefaciens
DNA containing
gene for desired trait
Ti
plasmid
1
Insertion of gene
into plasmid using
restriction enzyme
and DNA ligase
T DNA
Restriction
site
Plant cell
Recombinant
Ti plasmid
2
Introduction
Regeneration
into plant
of plant
cells in
culture
T DNA carrying new
Plant with new trait
gene within plant chromosome
Figure 12.18A
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
3
• Transgenic organisms
– Are those that have had genes from
other organisms inserted into their
genomes
Figure 12.18B
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• A number of important crops and plants
– Are genetically modified
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
CONNECTION
12.19 Could GM organisms harm human health
or the environment?
• Development of GM organisms
– Requires significant safety measures
Figure 12.19A
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• Genetic engineering involves risks
– Such as ecological damage from GM
crops
Figure 12.19B
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
CONNECTION
12.20 Genomics researcher Eric Lander
discusses the Human Genome Project
• Genomics pioneer Eric Lander
– Points out that much remains to be
learned from the Human Genome Project
Figure 12.20
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
12.17 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
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
HOMEWORK
Read Cohen’s work for plasmids and answer the
questions on the following website
http://www.stanford.edu/class/bio11n/html/week1/
papertopic.htm
Read Villa-Komaroff et al. work and answer the
questions on the following website
http://www.stanford.edu/class/bio11n/html/week2/
papertopic.htm
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
READING
http://www.dnaftb.org/dnaftb/
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings