Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
PowerPoint Lectures for
Biology, Seventh Edition
Neil Campbell and Jane Reece
Lectures by Chris Romero
Chapter 20
DNA Technology
and Genomics
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
La comprensión y la manipulación de los genomas
• La secuenciación del genoma humano se completó en
gran medida por 2003
• La secuenciación del ADN ha dependido de los avances
en la tecnología, comenzando con la fabricación de ADN
recombinante
• En el ADN recombinante, las secuencias de nucleótidos
de dos fuentes diferentes, a menudo dos especies, se
combinan in vitro en la misma molécula de ADN
• Los métodos para preparar ADN recombinante son
centrales a la ingeniería genética, la manipulación directa
de los genes para fines prácticos
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• Tecnología del ADN ha revolucionado la
biotecnología, la manipulación de organismos
vivos o sus componentes genéticos para hacer
productos útiles
Un ejemplo de la tecnología del ADN es el nivel
de expresión , una medición de la expresión
génica de miles de genes diferentes
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Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
La clonación del AND permite la producción de múltiples copias de un gen específico o de un fragmento de ADN
• Para trabajar directamente con los genes
específicos, los científicos preparan-gen de
tamaño trozos de ADN en copias idénticas, un
proceso llamado clonación de genes
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Clonación del AND y sus aplicaciones: peresentación preliminar
• La mayoría de los métodos para la clonación de
fragmentos de ADN en las características
generales de acciones de laboratorio, tales como
el uso de bacterias y sus plásmidos
• Genes clonados son útiles para hacer copias de
un gen particular y la producción de un producto
génico
LE 20-2Bacterium
Bacterialchromosome
Plasmid
Gene inserted intoplasmid
Célula que contieneEl gen de interés
Gene ofinterest DNA of
chromosome
RecombinantDNA (plasmid)
Plásmido introducidoEn la célula bacteriana
Recombinantbacterium
Célula huésped cultivadaPara formar un clon de célulasCon el gen “clonado
“proteína expresadaPor el gen de interés
Proteina recuperada
Gene ofinterest
Copies of gene
InvestigaciónBásicaSobre el gen
Investigación Básica sobreLa proteína
Investigación básicaY diversas aplicaciones
Gen para la resiste
Ncia contra las pla
Gas introducido en
Las plantas
Gen empleado para
Alterar las bacterias
Con el fin de que elim
Inen los descchod
tóxicos
Proteína que disuelve
Los coágulos de san
Gre como tratamiento
Del infarto
Hormona de crecimiento
Humana para el tratamiento
Del fretraso de crecimiento
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Utilización de las enzimas de restricción para producir ADN recombinante
• Las enzimas de restricción bacterianas cortan moléculas
de ADN en las secuencias de ADN llamadas sitios de
restricción
• Una enzima de restricción por lo general hace muchos
cortes, produciendo fragmentos de restricción
• La enzimas de restricción más útiles dividen los
esqueletos de azúcar y fosfato en ambas cadenas de ADN
de forma escalonada, produciendo fragmentos con
"extremos pegajosos“, estas regiones pueden unirse con
otros extremos pegajosos complementarios en otras
cadenas de ADN
• La ADN ligasa es una enzima que sella los enlaces entre
los fragmentos de restricción
LE 20-3Restriction site
DNA 53
35
La enzima de restricciónCorta los esqueletos de Azúcar-fosfato a la alturaDe cda flecha
One possible combination
Se agrega un fragmento de ADN de otra fuente. El apareaMiento de las bases de los Extremos cohesivos produceDiversas combinaciones
Fragmento de una moleculaDe ADN diferente cortadaPor la misma enzima de restricción
La ADN ligasa sellaLas cadenas.
Recombinant DNA molecule
Extremo cohesivo
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Animation: Restriction Enzymes
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Clonación de un gen eucarionte en un plásmido bacteriano
• En la clonación de genes, el plásmido original se
llama un vector de clonación
• Un vector de clonación es una molécula de ADN
que puede transportar el ADN extraño en una
célula y replicarse
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Producción de clones de células
• La clonación de un gen humano en un plásmido bacteriano se puede dividir en cinco pasos: 1. Se aisla el ADN del plásmido y el ADN de las células humanas
• 2. Se .cortan ambas muestras de ADN con la misma enzima de restricción
• 3. Se mezclan los plásmidos cortados con los fragmentos de ADN. Se agrega ADN ligasa para sellar las uniones
• 4. Se introduce el ADN en las células bacterianas que tienen una mutación en su propio gen IacZ
•Animation: Cloning a Gene
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• 5. Se siembran las bacterias sobre agar que
contiene ampilcilina y X – gal. se incuban hasta
que proliferen las colonias
LE 20-4_1
Se aisla el ADN del plás
Mido y el ADN de las células
humanas
Se .cortan ambas muestras de
ADN con la misma enzima de
restricción
Se mezclan los plásmidos cortados con los
Fragmentos de ADN. Se agrega ADN ligasa
Para sellar las uniones
Bacterial cell lacZ gene
(ruptura de la
lactosa)
Human
cell
Restriction
site
ampR gene
(ampicillin
resistance)
Bacterial
plasmid Gene of
interest
Extremos
cohesivosHuman DNA
fragments
Recombinant DNA plasmids
LE 20-4_2
Isolate plasmid DNA
and human DNA.
Cut both DNA samples with
the same restriction enzyme.
Mix the DNAs; they join by base pairing.
The products are recombinant plasmids
and many nonrecombinant plasmids.
Bacterial cell lacZ gene
(lactose
breakdown)
Human
cell
Restriction
site
ampR gene
(ampicillin
resistance)
Bacterial
plasmid Gene of
interest
Sticky
endsHuman DNA
fragments
Recombinant DNA plasmids
Se introduce el ADN en las células
Bacterianas que tienen una mutación
En su propio gen IacZ
Recombinant
bacteria
LE 20-4_3
Isolate plasmid DNA
and human DNA.
Cut both DNA samples with
the same restriction enzyme.
Mix the DNAs; they join by base pairing.
The products are recombinant plasmids
and many nonrecombinant plasmids.
Bacterial cell lacZ gene
(rupture de
La lactosa)
Human
cell
Restriction
site
ampR gene
(ampicillin
resistance)
Bacterial
plasmid Gene of
interest
Sitio de
restricciónHuman DNAfragments
Recombinant DNA plasmids
Introduce the DNA into bacterial cells
that have a mutation in their own lacZ
gene.
Recombinant
bacteria
Se siembran las bacterias sobre
Agar que contiene ampilcilina
Y X – gal. se incuban hasta que
Proliferenlas colonias
Colony carrying non-
recombinant plasmid
with intact lacZ gene
Colony carrying
recombinant
plasmid with
disrupted lacZ gene
Bacterial
clone
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Identifying Clones Carrying a Gene of Interest
• A clone carrying the gene of interest can be
identified with a nucleic acid probe having a
sequence complementary to the gene
• This process is called nucleic acid hybridization
• An essential step in this process is denaturation of
the cells’ DNA, separation of its two strands
LE 20-5
Master plate
Filter
Solution
containing
probe
Filter lifted
and flipped over
Radioactive
single-stranded
DNA
Probe
DNA
Gene of
interest
Single-stranded
DNA from cell
Film
Hybridization
on filter
Master plate
Colonies
containing
gene of
interest
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.
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Storing Cloned Genes in DNA Libraries
• A genomic library that is made using bacteria is
the collection of recombinant vector clones
produced by cloning DNA fragments from an
entire genome
• A genomic library that is made using
bacteriophages is stored as a collection of phage
clones
LE 20-6
Bacterial
clonesRecombinant
plasmidsRecombinant
phage DNA
or
Foreign genome
cut up with
restriction
enzyme
Phage
clones
Plasmid library Phage library
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• A complementary DNA (cDNA) library is made by
cloning DNA made in vitro by reverse transcription
of all the mRNA produced by a particular cell
• A cDNA library represents only part of the
genome—only the subset of genes transcribed
into mRNA in the original cells
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Cloning and Expressing Eukaryotic Genes
• As an alternative to screening a DNA library,
clones can sometimes be screened for a desired
gene based on detection of its encoded protein
• After a gene has been cloned, its protein product
can be produced in larger amounts for research
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Bacterial Expression Systems
• Several technical difficulties hinder expression of
cloned eukaryotic genes in bacterial host cells
• To overcome differences in promoters and other
DNA control sequences, scientists usually employ
an expression vector, a cloning vector that
contains a highly active prokaryotic promoter
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Eukaryotic Cloning and Expression Systems
• The use of cultured eukaryotic cells as host cells
and yeast artificial chromosomes (YACs) as
vectors helps avoid gene expression problems
• YACs behave normally in mitosis and can carry
more DNA than a plasmid
• Eukaryotic hosts can provide the posttranslational modifications that many proteins require
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• One method of introducing recombinant DNA into
eukaryotic cells is electroporation, applying a brief
electrical pulse to create temporary holes in
plasma membranes
• Alternatively, scientists can inject DNA into cells
using microscopic needles
• Once inside the cell, the DNA is incorporated into
the cell’s DNA by natural genetic recombination
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Amplificación del ADN in vitro: reacción en cadena de la polimerasa (PCR)
• La reacción en cadena de la polimerasa, PCR,
puede producir muchas copias de un segmento
diana específica de ADN
• Un ciclo de calentamiento de tres pasos, de
refrigeración, y la replicación da lugar a una
reacción en cadena que produce una población
en crecimiento exponencial de moléculas de ADN
idénticas
LE 20-7
Genomic DNA
Secuencia
objetivo
5
3
3
5
5
3
3
5
Primers
Desnaturalización:Se calienta brevementePara separar las Cadenas de ADN
Hibridación: se enfríaPara permitir que los Cebadores formrnEnlaces de H con Los extremos de laSecuencia diana
Extensión: la ADNPolimerasa agregaNucleotidos en el eExtremo 3’de cada cebador
Cycle 1yields
2molecules
New
nucleo-
tides
Ciclo 2:Se obtienen4 moléculas
Ciclo 3:Se obtienen8 moléculas2 moléculas
(en los recuadros Blancos) coincidenCon la secuencia
diana
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Concept 20.2: Restriction fragment analysis detects DNA differences that affect restriction sites
• Restriction fragment analysis detects differences
in the nucleotide sequences of DNA molecules
• Such analysis can rapidly provide comparative
information about DNA sequences
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Gel Electrophoresis and Southern Blotting
• One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis
• This technique uses a gel as a molecular sieve to
separate nuclei acids or proteins by size
Video: Biotechnology Lab
LE 20-8
Cathode
Powersource
Anode
Mixtureof DNAmoleculesof differ-ent sizes
Gel
Glassplates
Longermolecules
Shortermolecules
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• In restriction fragment analysis, DNA fragments
produced by restriction enzyme digestion of a
DNA molecule are sorted by gel electrophoresis
• Restriction fragment analysis is useful for
comparing two different DNA molecules, such as
two alleles for a gene
LE 20-9Normal b-globin allele
175 bp 201 bp Large fragment
Sickle-cell mutant b-globin allele
376 bp Large fragment
Ddel Ddel Ddel Ddel
Ddel Ddel Ddel
Ddel restriction sites in normal and sickle-cell alleles ofb-globin gene
Normalallele
Sickle-cellallele
Largefragment
376 bp201 bp
175 bp
Electrophoresis of restriction fragments from normaland sickle-cell alleles
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• A technique called Southern blotting combines gel electrophoresis with nucleic acid hybridization
• Specific DNA fragments can be identified by
Southern blotting, using labeled probes that
hybridize to the DNA immobilized on a “blot” of gel
LE 20-10
DNA + restriction enzyme Restriction
fragments
I Normalb-globinallele
II Sickle-cellallele
III Heterozygote
Preparation of restriction fragments. Gel electrophoresis. Blotting.
I II III Nitrocellulose
paper (blot)
Gel
Sponge
Alkaline
solution
Paper
towels
Heavy
weight
Hybridization with radioactive probe.
I II III
Radioactivelylabeled probefor b-globingene is addedto solution ina plastic bag
Paper blot
Probe hydrogen-bonds to fragmentscontaining normalor mutant b-globin
Fragment fromsickle-cellb-globin allele
Fragment fromnormal b-globinallele
Autoradiography.
I II III
Film overpaper blot
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Restriction Fragment Length Differences as Genetic Markers
• Restriction fragment length polymorphisms
(RFLPs, or Rif-lips) are differences in DNA
sequences on homologous chromosomes that
result in restriction fragments of different lengths
• A RFLP can serve as a genetic marker for a particular location (locus) in the genome
• RFLPs are detected by Southern blotting
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Concept 20.3: Entire genomes can be mapped at the DNA level
• The most ambitious mapping project to date has
been the sequencing of the human genome
• Officially begun as the Human Genome Project in
1990, the sequencing was largely completed by
2003
• Scientists have also sequenced genomes of other
organisms, providing insights of general biological
significance
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Genetic (Linkage) Mapping: Relative Ordering of Markers
• The first stage in mapping a large genome is
constructing a linkage map of several thousand
genetic markers throughout each chromosome
• The order of markers and relative distances
between them are based on recombination
frequencies
LE 20-11
Cytogenetic map
Genes located
by FISH
Chromosome
bands
Genetic
markers
Genetic (linkage)
mapping
Physical mapping
Overlapping
fragments
DNA sequencing
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Physical Mapping: Ordering DNA Fragments
• A physical map is constructed by cutting a DNA
molecule into many short fragments and arranging
them in order by identifying overlaps
• Physical mapping gives the actual distance in
base pairs between markers
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DNA Sequencing
• Relatively short DNA fragments can be sequenced
by the dideoxy chain-termination method
• Inclusion of special dideoxyribonucleotides in the
reaction mix ensures that fragments of various
lengths will be synthesized
LE 20-12
DNA(template strand)
5
3
Primer3
5
DNApolymerase
Deoxyribonucleotides Dideoxyribonucleotides(fluorescently tagged)
3
5DNA (templatestrand)
Labeled strands3
Directionof movementof strands
Laser Detector
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• Linkage mapping, physical mapping, and DNA
sequencing represent the overarching strategy of
the Human Genome Project
• An alternative approach to sequencing genomes
starts with sequencing random DNA fragments
• Computer programs then assemble overlapping
short sequences into one continuous sequence
LE 20-13
Cut the DNA from
many copies of an
entire chromosome
into overlapping frag-
ments short enough
for sequencing
Clone the fragments
in plasmid or phage
vectors
Sequence each fragment
Order the
sequences into one
overall sequence
with computer
software
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Concept 20.4: Genome sequences provide clues to important biological questions
• In genomics, scientists study whole sets of genes
and their interactions
• Genomics is yielding new insights into genome
organization, regulation of gene expression,
growth and development, and evolution
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Identifying Protein-Coding Genes in DNA Sequences
• Computer analysis of genome sequences helps
identify sequences likely to encode proteins
• The human genome contains about 25,000 genes,
but the number of human proteins is much larger
• Comparison of sequences of “new” genes with
those of known genes in other species may help
identify new genes
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Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Determining Gene Function
• One way to determine function is to disable the gene and observe the consequences
• Using in vitro mutagenesis, mutations are introduced into a cloned gene, altering or destroying its function
• When the mutated gene is returned to the cell, the normal gene’s function might be determined by examining the mutant’s phenotype
• In nonmammalian organisms, a simpler and faster method, RNA interference (RNAi), has been used to silence expression of selected genes
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Studying Expression of Interacting Groups of Genes
• Automation has allowed scientists to measure expression of thousands of genes at one time using DNA microarray assays
• DNA microarray assays compare patterns of gene
expression in different tissues, at different times,
or under different conditions
LE 20-14
Make cDNA by reverse
transcription, using
fluorescently labeled
nucleotides.
Apply the cDNA mixture to a
microarray, a microscope slide
on which copies of single-
stranded DNA fragments from
the organism’s genes are fixed,
a different gene in each spot.
The cDNA hybridizes with any
complementary DNA on the
microarray.
Rinse off excess cDNA; scan
microarray for fluorescent.
Each fluorescent spot
(yellow) represents a gene
expressed in the tissue
sample.
Isolate mRNA.Tissue sample
mRNA molecules
Labeled cDNA molecules
(single strands)
DNA
microarray
Size of an actual
DNA microarray
with all the genes
of yeast (6,400 spots)
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Comparing Genomes of Different Species
• Comparative studies of genomes from related and
widely divergent species provide information in
many fields of biology
• The more similar the nucleotide sequences between two species, the more closely related these species are in their evolutionary history
• Comparative genome studies confirm the relevance of research on simpler organisms to understanding human biology
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Future Directions in Genomics
• Genomics is the study of entire genomes
• Proteomics is the systematic study of all proteins
encoded by a genome
• Single nucleotide polymorphisms (SNPs) provide
markers for studying human genetic variation
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Concept 20.5: The practical applications of DNA technology affect our lives in many ways
• Many fields benefit from DNA technology and
genetic engineering
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Medical Applications
• One benefit of DNA technology is identification of
human genes in which mutation plays a role in
genetic diseases
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Diagnosis of Diseases
• Scientists can diagnose many human genetic
disorders by using PCR and primers
corresponding to cloned disease genes, then
sequencing the amplified product to look for the
disease-causing mutation
• Even when a disease gene has not been cloned,
presence of an abnormal allele can be diagnosed
if a closely linked RFLP marker has been found
LE 20-15
DNA
RFLP marker
Disease-causing
allele
Normal allele
Restriction
sites
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Human Gene Therapy
• Gene therapy is the alteration of an afflicted
individual’s genes
• Gene therapy holds great potential for treating
disorders traceable to a single defective gene
• Vectors are used for delivery of genes into cells
• Gene therapy raises ethical questions, such as whether human germ-line cells should be treated to correct the defect in future generations
LE 20-16
Cloned gene
Retrovirus
capsid
Bone
marrow
cell from
patient
Inject engineered
cells into patient.
Insert RNA version of normal allele
into retrovirus.
Viral RNA
Let retrovirus infect bone marrow cells
that have been removed from the
patient and cultured.
Viral DNA carrying the normal
allele inserts into chromosome.
Bone
marrow
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Pharmaceutical Products
• Some pharmaceutical applications of DNA
technology:
– Large-scale production of human hormones
and other proteins with therapeutic uses
– Production of safer vaccines
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Forensic Evidence
• DNA “fingerprints” obtained by analysis of tissue
or body fluids can provide evidence in criminal and
paternity cases
• A DNA fingerprint is a specific pattern of bands of RFLP markers on a gel
• The probability that two people who are not identical twins have the same DNA fingerprint is very small
• Exact probability depends on the number of markers and their frequency in the population
LE 20-17Defendant’s
blood (D)
Blood from defendant’s
clothes
Victim’s
blood (V)
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Environmental Cleanup
• Genetic engineering can be used to modify the
metabolism of microorganisms
• Some modified microorganisms can be used to
extract minerals from the environment or degrade
potentially toxic waste materials
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Agricultural Applications
• DNA technology is being used to improve
agricultural productivity and food quality
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Animal Husbandry and “Pharm” Animals
• Transgenic organisms are made by introducing genes from one species into the genome of another organism
• Transgenic animals may be created to exploit the attributes of new genes (such as genes for faster growth or larger muscles)
• Other transgenic organisms are pharmaceutical “factories,” producers of large amounts of otherwise rare substances for medical use
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Genetic Engineering in Plants
• Agricultural scientists have endowed a number of
crop plants with genes for desirable traits
• The Ti plasmid is the most commonly used vector
for introducing new genes into plant cells
LE 20-19Agrobacterium tumefaciens
Ti
plasmid
Site where
restriction
enzyme cuts
DNA with
the gene
of interest
T DNA
Recombinant
Ti plasmid
Plant with
new trait
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Safety and Ethical Questions Raised by DNA Technology
• Potential benefits of genetic engineering must be
weighed against potential hazards of creating
harmful products or procedures
• Most public concern about possible hazards
centers on genetically modified (GM) organisms
used as food