copyright © 2005 pearson education, inc. publishing as benjamin cummings figure 20.1 biotechnology...

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

Figure 20.1

Biotechnology

Chapter 20


The DNA Toolbox

• Sequencing of the genomes of more than 7,000 species was under way in 2010

• Recombinant DNA nucleotide sequences from two different sources combined in vitro into the same DNA molecule

© 2011 Pearson Education, Inc.


• Genetic engineering direct manipulation of genes for practical purposes

• Biotechnology manipulation of organisms or their genetic components to make useful products



• Foreign DNA inserted into plasmid* plasmid inserted into bacterial cell

• Reproduction in bacterial cell cloning of plasmid with foreign DNA

multiple copies of a single gene

*Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome


DNA Cloning


Figure 20.2Bacterium

Bacterialchromosome

Plasmid

2

1

3

4

Gene inserted intoplasmid

Cell containing geneof interest

RecombinantDNA (plasmid)

Gene of interest

Plasmid put intobacterial cell

DNA ofchromosome(“foreign” DNA)

Recombinantbacterium

Host cell grown in culture toform a clone of cells containingthe “cloned” gene of interest

Gene of interest

Protein expressed fromgene of interest

Protein harvestedCopies of gene

Basic researchand variousapplications

Basicresearchon protein

Basic research on gene

Gene for pestresistance insertedinto plants

Gene used to alterbacteria for cleaningup toxic waste

Protein dissolvesblood clots in heartattack therapy

Human growthhormone treatsstunted growth


Using Restriction Enzymes to Make Recombinant DNA

• Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites

• Yields restriction fragments

• Most useful restriction enzymes give staggered cut “sticky ends.”


Animation: Restriction Enzymes


• Sticky ends bond with complementary sticky ends of other fragments

• DNA ligase seals bonds between fragments



Figure 20.3-1

Restriction enzymecuts sugar-phosphatebackbones.

Restriction site

DNA5

5

5

5

5

5

3

3

3

3

3

3

1

Sticky end

GAATTCCTTAAG

CTTAAG AATTC

G


Figure 20.3-2

One possible combination

DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs.


Restriction site

DNA5

5

5

5

5

5

5

5

55

5

5

55

3

3

3

3

3

3

3

3

3

3

3

3

3

3

2

1

Sticky end

GAATTCCTTAAG

CTTAAG AATTC

G

GGAATTC

CTTAA

GG

GG

AATT CAATT CC TTAA C TTAA


Figure 20.3-3

Recombinant DNA molecule

One possible combinationDNA ligaseseals strands

DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs.


Restriction site

DNA5

5

5

5

5

5

5

5

55

5

5

55

5

5

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

2

3

1

Sticky end

GAATTCCTTAAG

CTTAAG AATTC

G

GGAATTC

CTTAA

GG

GG

AATT CAATT CC TTAA C TTAA


Cloning a Eukaryotic Gene in a Bacterial Plasmid

• A cloning vector (original plasmid) DNA molecule that carries foreign DNA into a host cell



Figure 20.4

Bacterial plasmidTECHNIQUE

RESULTS

ampR gene lacZ gene

Restrictionsite

Hummingbird cell

Sticky ends Gene of

interest

Humming-bird DNAfragments

Recombinant plasmids Nonrecombinant plasmid

Bacteria carryingplasmids

Colony carrying non-recombinant plasmidwith intact lacZ gene

Colony carrying recombinantplasmidwith disruptedlacZ gene

One of manybacterialclones


Figure 20.4a-1


ampR gene lacZ gene

Restrictionsite

Hummingbird cell

Sticky ends Gene of

interest



Figure 20.4a-2


ampR gene lacZ gene

Restrictionsite

Hummingbird cell

Sticky ends Gene of

interest




Figure 20.4a-3


ampR gene lacZ gene

Restrictionsite

Hummingbird cell

Sticky ends Gene of

interest





Figure 20.4b

RESULTS


Colony carrying non-recombinant plasmidwith intact lacZ gene

Colony carrying recombinantplasmidwith disruptedlacZ gene

One of manybacterialclones


Storing Cloned Genes in DNA Libraries

• A genomic library is made using plasmids or bacteriophages



Figure 20.5

Foreign genome

Cut with restriction enzymes into eithersmallfragments

largefragments

or

Recombinantplasmids

Plasmidclone

(a) Plasmid library

(b) BAC clone

Bacterial artificialchromosome (BAC)

Largeinsertwithmanygenes

(c) Storing genome libraries


• Made by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a particular cell

• A cDNA library represents only the subset of genes transcribed into mRNA in the original cells


Complementary DNA (cDNA) library


Figure 20.6-1

DNA innucleus

mRNAs incytoplasm


Figure 20.6-2

DNA innucleus

mRNAs incytoplasm

mRNA

Reversetranscriptase Poly-A tail

DNAstrand

Primer

55

33

A A A A A AT T T T T


Figure 20.6-3

DNA innucleus

mRNAs incytoplasm

mRNA


DNAstrand

Primer

55

55

33

33

A A A A A A

A A A A A A

T T T T T

T T T T T


Figure 20.6-4

DNA innucleus

mRNAs incytoplasm

mRNA


DNAstrand

Primer

DNA polymerase

55

55

55

33

33

33

A A A A A A

A A A A A A

T T T T T

T T T T T


Figure 20.6-5

DNA innucleus

mRNAs incytoplasm

mRNA


DNAstrand

Primer

DNA polymerase

cDNA

55

55

55

55

33

33

33

33

A A A A A A

A A A A A A

T T T T T

T T T T T


Screening a Library for Clones Carrying a Gene of Interest

• Identified with a nucleic acid probe having a sequence complementary to the gene

• Process is called nucleic acid hybridization



• A probe can be synthesized that is complementary to the gene of interest

• For example, if the desired gene is

– Then we would synthesize this probe


5 3 CTCAT CACCGGC

53G A G T A G T G G C C G


Figure 20.7

Radioactivelylabeled probemolecules Gene of

interestProbeDNA

Single-strandedDNA fromcell

Film

Location ofDNA with thecomplementarysequence

Nylonmembrane

Nylon membrane

Multiwell platesholding libraryclones

TECHNIQUE 5

53

3

GAGTAGTGGCCG CTCATCACCGGC


Eukaryotic Cloning and Expression Systems

• Avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells, such as yeasts or cultured mammal cells, as hosts for cloning and expressing genes



• Electroporation: applying a brief electrical pulse to create temporary holes in plasma membranes introduces recombinant DNA into eukaryotic cells

• Alternatively, scientists can inject DNA into cells using microscopically thin needles



Cross-Species Gene Expression and Evolutionary Ancestry

• The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species

• e.g., Pax-6 (gene that directs formation of vertebrate eye); also directs the formation of insect eye



Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR)

• Polymerase chain reaction, PCR produce many copies of a specific target segment of DNA

• Key to PCR is an unusual, heat-stable DNA polymerase called Taq polymerase.



Figure 20.8

Genomic DNA

Targetsequence

Denaturation

Annealing

Extension

Primers

Newnucleotides

Cycle 1yields

2molecules

Cycle 2yields

4molecules

Cycle 3yields 8

molecules;2 molecules

(in white boxes)match target

sequence

5

5

5

5

3

3

3

3

2

3

1

TECHNIQUE


Figure 20.8a

Genomic DNA

Targetsequence

5

5

3

3

TECHNIQUE


Denaturation

Annealing

Extension

Primers

Newnucleo-tides

Cycle 1yields

2molecules

5

5

3

3

2

3

1

Figure 20.8b


Figure 20.8c

Cycle 2yields

4molecules


Figure 20.8d

Cycle 3yields 8

molecules;2 molecules

(in white boxes)match target

sequence


Gel Electrophoresis and Southern Blotting

• Gel electrophoresis method of rapidly analyzing and comparing genomes

• Uses a gel as a molecular sieve to separate nucleic acids or proteins by size, electrical charge, and other properties


Animation: Biotechnology Lab


Figure 20.9

Mixture ofDNA mol-ecules ofdifferentsizes

Powersource

Powersource

Longermolecules

Cathode Anode

Wells

Gel

Shortermolecules

TECHNIQUE

RESULTS

1

2


Figure 20.9a

Mixture ofDNA mol-ecules ofdifferentsizes

Powersource

Powersource

Longermolecules

Cathode Anode

Wells

Gel

Shortermolecules

TECHNIQUE

2

1


Figure 20.9b

RESULTS


• Restriction fragment analysis DNA fragments produced by restriction enzyme digestion sorted by gel electrophoresis



• Variations in DNA sequence are called polymorphisms

• Sequence changes that alter restriction sites are called RFLPs (restriction fragment length polymorphisms)



Figure 20.10

Normal -globin allele

Sickle-cell mutant -globin allele

Largefragment

Normalallele

Sickle-cellallele

201 bp175 bp

376 bp

(a) DdeI restriction sites in normal andsickle-cell alleles of the -globin gene

(b) Electrophoresis of restrictionfragments from normal andsickle-cell alleles

201 bp175 bp

376 bp

Large fragment

Large fragment

DdeI DdeI DdeI DdeI

DdeI DdeI DdeI


Figure 20.10a

Normal -globin allele

Sickle-cell mutant -globin allele

(a) DdeI restriction sites in normal andsickle-cell alleles of the -globin gene

201 bp175 bp

376 bp

Large fragment

Large fragment

DdeI DdeI DdeI DdeI

DdeI DdeI DdeI


Figure 20.10b

Largefragment

Normalallele

Sickle-cellallele

201 bp175 bp

376 bp

(b) Electrophoresis of restrictionfragments from normal andsickle-cell alleles


• Southern blotting combines gel electrophoresis of DNA fragments with nucleic acid hybridization



Figure 20.11

DNA restriction enzyme

321

4

TECHNIQUE

I Normal-globinallele

II Sickle-cellallele

III Heterozygote

Restrictionfragments

Nitrocellulosemembrane (blot)

Heavyweight

Gel

Sponge

Alkalinesolution Paper

towels

III III

III III III III

Preparation ofrestriction fragments

Gel electrophoresis DNA transfer (blotting)

Radioactively labeledprobe for -globingene

Nitrocellulose blot

Probe base-pairswith fragments

Fragment from sickle-cell -globin allele

Fragment from normal - globin allele

Filmoverblot

Hybridization with labeled probe Probe detection5


DNA Sequencing

• Relatively short DNA fragments can be sequenced by the dideoxy chain termination method, the first automated method to be employed

• Modified nucleotides called dideoxyribonucleotides (ddNTP) attach to synthesized DNA strands of different lengths

• Each type of ddNTP is tagged with a distinct fluorescent label that identifies the nucleotide at the end of each DNA fragment

• The DNA sequence can be read from the resulting spectrogram



Figure 20.12

DNA(template strand)

TECHNIQUE

5

3

C

C

C

C

T

TT

G

G

A

A

AA

GTT

T

DNApolymerase

Primer

5

3

P P P

OH

G

dATP

dCTP

dTTP

dGTP

Deoxyribonucleotides Dideoxyribonucleotides(fluorescently tagged)

P P P

H

G

ddATP

ddCTP

ddTTP

ddGTP

5

3

C

C

C

C

T

TT

G

G

A

A

AA

DNA (templatestrand)

Labeled strands

Shortest Longest5

3

ddCddG

ddAddA

ddA

ddG

ddG

ddTddC

GTT

TGTT

TC

GTT

TC

T T

G

GTT

TCT

GA

GTT

TCT

GAA

GTT

TCT

GAAG

GTT

TCT

GAAGT

GTT

TCT

GAAGTC

GTT

TCT

GAAGTCA

Directionof movementof strands

Longest labeled strand

Detector

LaserShortest labeled strand

RESULTS

Last nucleotideof longestlabeled strand

Last nucleotideof shortestlabeled strand

G

G

G

A

AA

C

C

T


Figure 20.12a

DNA(template strand)

TECHNIQUEPrimer Deoxyribonucleotides Dideoxyribonucleotides

(fluorescently tagged)

DNApolymerase

5

5

3

3

OH H

GG

dATP

dCTP

dTTP

dGTP

P P P P P P

ddATP

ddCTP

ddTTP

ddGTP

T

TT

G

G

G

C

C

C

CT

TT

A

A

AA


Figure 20.12b

DNA (templatestrand)

Labeled strands

Shortest Longest



Detector


TECHNIQUE (continued)

5

3

G

G

C

C

C

CT

TT

A

A

AA

T

TT

G

ddC

ddC

ddG

ddG

ddG

ddA ddA

ddA

ddT

3

5

T

TT

G

CT

TT

G

CG

T

TT

G

CGA

T

TT

G

CGAA

T

TT

G

CGAAG

T

TT

CGAAGT

T

TT

CGAAGTC

A

T

TT

CGAAGTC

G G G


Figure 20.12c

RESULTS

Last nucleotideof longestlabeled strand

Last nucleotideof shortestlabeled strand

G

G

G

A

AA

C

C

T



Detector



• Reverse transcriptase-polymerase chain reaction (RT-PCR)

• Reverse transcriptase + mRNA cDNA, which serves as a template for PCR amplification of the gene of interest



Figure 20.13

cDNA synthesis

PCR amplification

Gel electrophoresis

mRNAs

cDNAs

Primers

-globingene

Embryonic stages1 2 3 4 5 6

2

3

1

RESULTS

TECHNIQUE


Expression of Interacting Groups of Genes

• DNA microarray assays compare patterns of gene expression in different tissues, at different times, or under different conditions



Isolate mRNA.

2

1

3

4

TECHNIQUE

Make cDNA by reversetranscription, usingfluorescently labelednucleotides.

Apply the cDNA mixture to a microarray, a different genein each spot. The cDNA hybridizeswith any complementary DNA onthe microarray.

Rinse off excess cDNA; scan microarrayfor fluorescence. Each fluorescent spot(yellow) represents a gene expressedin the tissue sample.

Tissue sample

mRNA molecules

Labeled cDNA molecules(single strands)

DNA fragmentsrepresenting aspecific gene

DNA microarray

DNA microarraywith 2,400human genes

Figure 20.15


Figure 20.15a

DNA microarraywith 2,400human genes


Determining Gene Function

• in vitro mutagenesis mutations are introduced into a cloned gene, altering or destroying its function

• Mutated gene returned to the cell normal gene function determined by examining the mutant’s phenotype

• Gene expression can also be silenced using RNA interference (RNAi)



• Genetic markers called SNPs (single nucleotide polymorphisms) occur on average every 100–300 base pairs

• SNPs can be detected by PCR, and any SNP shared by people affected with a disorder but not among unaffected people may pinpoint the location of the disease-causing gene



Figure 20.16

DNA

SNPNormal allele

Disease-causingallele

T

C


• Organismal cloning produces one or more organisms genetically identical to the “parent” that donated the single cell

Cloning



Cloning Plants: Single-Cell Cultures

• Totipotent cell can generate a complete new organism

• Plant cloning is used extensively in agriculture



Figure 20.17

Crosssection ofcarrot root

2-mgfragments

Fragments werecultured in nu-trient medium;stirring causedsingle cells toshear off intothe liquid.

Single cellsfree insuspensionbegan todivide.

Embryonicplant developedfrom a culturedsingle cell.

Plantlet wascultured onagar medium.Later it wasplanted in soil.

Adultplant


Cloning Animals: Nuclear Transplantation

• Nucleus of an unfertilized egg cell is replaced with the nucleus of a differentiated cell



Frog embryo Frog egg cell Frog tadpole

UV

Less differ-entiated cell

Donornucleustrans-planted

Enucleatedegg cell

Fully differ-entiated(intestinal) cell

Donornucleustrans-plantedEgg with donor nucleus

activated to begindevelopment

Most developinto tadpoles.

Most stop developingbefore tadpole stage.

EXPERIMENT

RESULTS

Figure 20.18


Reproductive Cloning of Mammals

• 1997, Scottish researchers announced the birth of Dolly, a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell



Figure 20.19

Mammarycell donor

21

3

4

5

6

TECHNIQUE

RESULTS

Culturedmammarycells

Eggcell fromovary

Egg cell donor

NucleusremovedCells fused

Grown in culture

Implanted in uterusof a third sheep

Embryonicdevelopment

Nucleus frommammary cell

Early embryo

Surrogatemother

Lamb (“Dolly”) geneticallyidentical to mammary cell donor


Figure 20.19a

Mammarycell donor

21

3

TECHNIQUE

Culturedmammarycells

Eggcell fromovary

Egg cell donor

Nucleusremoved

Cells fused



4

5

6

RESULTS

Grown in culture

Implanted in uterusof a third sheep

Embryonicdevelopment


Early embryo

Surrogatemother

Lamb (“Dolly”) geneticallyidentical to mammary cell donor

Figure 20.19b


• Since 1997, cloning has been demonstrated in many mammals, including mice, cats, cows, horses, mules, pigs, and dogs

• CC (for Carbon Copy) was the first cat cloned; however, CC differed somewhat from her female “parent”

• Cloned animals do not always look or behave exactly the same



Figure 20.20


Stem Cells of Animals

• Relatively unspecialized cell that can reproduce itself indefinitely and differentiate into specialized cells of one or more types

• Stem cells isolated from early embryos at the blastocyst stage are called embryonic stem (ES) cells; these are able to differentiate into all cell types

• The adult body also has stem cells, which replace nonreproducing specialized cells



Figure 20.21

Culturedstem cells

Differentcultureconditions

Differenttypes ofdifferentiatedcells

Embryonicstem cells

Adultstem cells

Cells generatingall embryoniccell types

Cells generatingsome cell types

Livercells

Nervecells

Bloodcells


Practical applications of DNA technology



Medical Applications

• Identification of human genes in which mutation plays a role in genetic diseases



Diagnosis and Treatment of Diseases

• Can diagnose PCR and sequence-specific primers, then sequencing the amplified product to look for the disease-causing mutation

• SNPs may be associated with a disease-causing mutation



Human Gene Therapy

• The alteration of an afflicted individual’s genes



Figure 20.23Cloned gene

2

1

3

4

Retroviruscapsid

Bonemarrowcell frompatient

Viral RNA

Bonemarrow

Insert RNA version of normal alleleinto retrovirus.

Let retrovirus infect bone marrow cellsthat have been removed from thepatient and cultured.

Viral DNA carrying the normalallele inserts into chromosome.

Inject engineeredcells into patient.


Pharmaceutical Products

• Advances in DNA technology and genetic research are important to the development of new drugs to treat diseases



• Transgenic animals are made by introducing genes from one species into the genome of another animal

pharmaceutical “factories”

Protein Production by “Pharm” Animals



Figure 20.24


Forensic Evidence and Genetic Profiles

• An individual’s unique DNA sequence, or genetic profile, can be obtained by analysis of tissue or body fluids

• Can use PCR and/ or Southern Blotting/ RFLP



Figure 20.25This photo showsWashington just beforehis release in 2001,after 17 years in prison.

(a)

(b)These and other STR data exonerated Washingtonand led Tinsley to plead guilty to the murder.

Semen on victim

Earl Washington

Kenneth Tinsley

17,19

16,18

17,19

13,16

14,15

13,16

12,12

11,12

12,12

Source ofsample

STRmarker 1

STRmarker 2

STRmarker 3


Environmental Cleanup

• Modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials



Agricultural Applications

• DNA technology is being used to improve agricultural productivity and food quality

• Genetic engineering of transgenic animals speeds up the selective breeding process

• Beneficial genes can be transferred between varieties of species



• The Ti plasmid is the most commonly used vector for introducing new genes into plant cells

• Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance, increased resistance to pests, increased resistance to salinity, and improved nutritional value of crops



Figure 20.26

Plant with new trait

RESULTS

TECHNIQUE

Tiplasmid

Site whererestrictionenzyme cuts

DNA withthe geneof interest

RecombinantTi plasmid

T DNA

Agrobacterium tumefaciens


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

• Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology



• Genetically modified (GM) organisms


copyright © 2005 pearson education, inc. publishing as benjamin cummings figure 20.1 biotechnology...

Documents

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