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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

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Page 1: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

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

Page 2: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

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

Page 3: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

• 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

Page 4: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Page 5: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

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

Page 6: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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

Page 7: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

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

Page 8: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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

Page 9: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

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

Page 10: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Animation: Restriction Enzymes

Page 11: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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

Page 12: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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

Page 13: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

• 5. Se siembran las bacterias sobre agar que

contiene ampilcilina y X – gal. se incuban hasta

que proliferen las colonias

Page 14: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

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

Page 15: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

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

Page 16: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

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

Page 17: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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

Page 18: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

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.

Page 19: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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

Page 20: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

LE 20-6

Bacterial

clonesRecombinant

plasmidsRecombinant

phage DNA

or

Foreign genome

cut up with

restriction

enzyme

Phage

clones

Plasmid library Phage library

Page 21: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

• 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

Page 22: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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

Page 23: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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

Page 24: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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

Page 25: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

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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

Page 26: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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

Page 27: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

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

Page 28: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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

Page 29: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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

Page 30: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

LE 20-8

Cathode

Powersource

Anode

Mixtureof DNAmoleculesof differ-ent sizes

Gel

Glassplates

Longermolecules

Shortermolecules

Page 31: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

• 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

Page 32: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

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

Page 33: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

• 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

Page 34: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

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

Page 35: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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

Page 36: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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

Page 37: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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

Page 38: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

LE 20-11

Cytogenetic map

Genes located

by FISH

Chromosome

bands

Genetic

markers

Genetic (linkage)

mapping

Physical mapping

Overlapping

fragments

DNA sequencing

Page 39: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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

Page 40: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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

Page 41: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

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

Page 42: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

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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

Page 43: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

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

Page 44: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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

Page 45: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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

Page 46: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

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Page 47: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

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

Page 48: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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

Page 49: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

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)

Page 50: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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

Page 51: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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

Page 52: DNA Technology and Genomics · ... Inc. publishing as Benjamin Cummings ... Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings ... Biotechnology Lab. LE 20-8

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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

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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

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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

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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

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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

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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