1

DNACloningandBiotechnologyBIOL100

Ch.6

Fig.16-7

(c) Space-filling model

Hydrogen bond 3ʹ end

5ʹ end

3.4 nm

0.34 nm 3ʹ end

5ʹ end

(b) Partial chemical structure (a) Key features of DNA structure

1 nm

DNAStructure

DNACloningandItsApplica?ons

•  DNAcloning

•  preparesgene-sizedpiecesofDNAin

iden>calcopies

•  cloningvectors

•  SmallDNAmoleculesintowhichforeignDNA

maybeinserted

•  UsedforcloningofDNA

•  Plasmids,bacteriophages,BAC’s,YAC’s

2

•  Genecloning

•  usingbacteria(mostcommon)

tomakemul>plecopiesofa

gene

•  ForeignDNAisinsertedintoa

plasmid

•  recombinantplasmidis

insertedintoabacterialcell

•  Bacteriareproduce

•  resultsinmul>plecopiesofa

singlegene

DNACloningandItsApplica?ons

DNA of chromosome

Cell containing gene of interest

Gene inserted into plasmid

Plasmid put into bacterial cell

Recombinant DNA (plasmid)

Recombinant bacterium

Bacterial chromosome

Bacterium

Gene of interest

Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest

Plasmid

Gene of Interest

Protein expressed by gene of interest

Basic research and various applications

Copies of gene Protein harvested

Basic research on gene

Basic research on protein

Gene for pest resistance inserted into plants

Gene used to alter bacteria for cleaning up toxic waste

Protein dissolves blood clots in heart attack therapy

Human growth hor- mone treats stunted growth

2

4

1

3

UsingRestric?onEnzymestoMakeRecombinantDNA•  Bacterialrestric?onenzymescutDNA

molecules

•  atspecificDNAsequencescalledrestric?onsites

•  usuallymakesmanycuts

•  yieldingrestric?onfragments

•  mostR.E.’scutDNAinastaggeredway

•  producingfragmentswith“s?ckyends”

•  bondwithcomplementarys>ckyendsofotherfragments

Fig.20-3-3

Restriction site

DNA

Sticky end

Restriction enzyme cuts sugar-phosphate backbones.

5ʹ 3ʹ

3ʹ 5ʹ

1

One possible combination

Recombinant DNA molecule

DNA ligase seals strands.

3

DNA fragment added from another molecule cut by same enzyme. Base pairing occurs.

2

Libraryscreening

•  nucleicacidhybridiza?on

•  Usesnucleicacidprobe

•  DNAsequencecomplementarytothetargetgene

•  Fluorescentorradioac>velylabeled

•  Caniden>fyaclonecarryingthegeneofinterest

•  Bacterialcolonycontainingclonecanthenbecultured

G 5ʹ 3ʹ … … G G C C C T T T A A A

C 3ʹ 5ʹ C C G G G A A A T T T

3

Fig.20-7

Probe DNA

Radioactively labeled probe

molecules

Film

Nylon membrane

Multiwell plates holding library clones

Location of DNA with the complementary sequence

Gene of interest

Single-stranded DNA from cell

Nylon membrane

TECHNIQUE

•

ThePolymeraseChainReac?on(PCR)

•  polymerasechainreac?on,PCR

•  producemanycopiesofaspecific

targetsegmentofDNA

•  Athree-stepcycle

•  hea>ng,cooling,andreplica>on

•  chainreac>onproduces

exponen>allygrowingpopula>onof

iden>calDNAmolecules

5ʹ

Genomic DNA

TECHNIQUE

Cycle 1 yields

2 molecules

Denaturation

Annealing

Extension

Cycle 2 yields

4 molecules

Cycle 3 yields 8

molecules; 2 molecules

(in white boxes)

match target sequence

Target sequence

Primers

New nucleo- tides

3ʹ

3ʹ

3ʹ

3ʹ

5ʹ

5ʹ

5ʹ 1

2

3

GelElectrophoresisandSouthernBloTng•  gelelectrophoresis

•  Oneindirectmethodofrapidlyanalyzingandcomparinggenomes

•  usesagelasamolecularsieve

•  toseparatenucleicacidsorproteinsbysize

•  Electricalcurrentisapplied

•  causeschargedmoleculestomovethroughgel

•  Moleculessortedinto“bands”bysize

Mixture of DNA mol- ecules of different sizes

Power source

Power source

Longer molecules

Shorter molecules

Gel

Anode Cathode

TECHNIQUE

RESULTS

1

2

+

+

–

–

4

•  restric>onfragmentanalysis

•  DNAfragmentsproducedbyrestric>onenzymes

•  sortedbygelelectrophoresis

GelElectrophoresisandSouthernBloTng

Normal allele

Sickle-cell allele

Large fragment

(b) Electrophoresis of restriction fragments from normal and sickle-cell alleles

201 bp 175 bp

376 bp

(a) DdeI restriction sites in normal and sickle-cell alleles of β-globin gene

Normal β-globin allele

Sickle-cell mutant β-globin allele

DdeI

Large fragment

Large fragment

376 bp

201 bp 175 bp

DdeIDdeI

DdeI DdeI DdeI DdeI

DNASequencing•  Sangerchaintermina>onmethod

•  DNAfragmentsequencing

•  ~1-5000nt

•  Modifiednucleo>des

•  aWachtosynthesizedDNAstrandsof

differentlengths

•  Eachistaggedwithadis>nct

fluorescentlabel

•  iden>fiesthenucleo>deatendof

DNAfragment

•  sequencecanbereadfrom

theresul>ngchromatogram

DNA (template strand)

TECHNIQUE

RESULTS

DNA (template strand)

DNA polymerase

Primer Deoxyribonucleotides

Shortest

Dideoxyribonucleotides (fluorescently tagged)

Labeled strands

Longest

Shortest labeled strand

Longest labeled strand

Laser

Direction of movement of strands

Detector

Last base of longest

labeled strand

Last base of shortest

labeled strand

dATP dCTP dTTP dGTP

ddATP ddCTP ddTTP ddGTP

•  Organismalcloning

•  producesoneormore

organisms

•  gene>callyiden>caltothe

“parent”thatdonatedthe

singlecell

•  to?potentcell

•  cangenerateacompletenew

organism

Cloningorganisms

5

Fig.20-16

EXPERIMENT

Transverse section of carrot root

2-mg fragments

Fragments were cultured in nu- trient medium; stirring caused single cells to shear off into the liquid.

Single cells free in suspension began to divide.

Embryonic plant developed from a cultured single cell.

Plantlet was cultured on agar medium. Later it was planted in soil.

A single somatic carrot cell developed into a mature carrot plant.

RESULTS

CloningAnimals:NuclearTransplanta?on•  Soma>cCellNuclearTransfer(SCNT)

•  nucleusofanunfer>lizedeggcellorzygote

•  replacedwiththenucleusofadifferen>atedcell

•  Experimentswithfrogembryos

•  showthatatransplantednucleus

•  cano[ensupportnormaldevelopmentoftheegg

•  However,theolderthedonornucleus

•  lowerthepercentageofnormallydevelopingtadpoles

Fig.20-17

EXPERIMENT

Less differ- entiated cell

RESULTS

Frog embryo Frog egg cell UV

Donor nucleus trans- planted

Frog tadpole

Enucleated egg cell

Egg with donor nucleus activated to begin

development

Fully differ- entiated (intestinal) cell

Donor nucleus trans- planted

Most develop into tadpoles

Most stop developing before tadpole stage

6

Fig.20-18

TECHNIQUE

Mammary cell donor

RESULTS

Surrogate mother

Nucleus from mammary cell

Cultured mammary cells

Implanted in uterus of a third sheep

Early embryo

Nucleus removed

Egg cell donor

Embryonic development Lamb (“Dolly”)

genetically identical to mammary cell donor

Egg cell from ovary Cells fused

Grown in culture

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3 3

4

5

6

2

ProblemsAssociatedwithAnimalCloning

•  Inmostnucleartransplanta>onstudies

•  onlyasmallpercentageofclonedembryoshavedevelopednormallytobirth

•  Manyepigene&cchangesmustbereversedinthenucleusfromadonoranimal

•  inorderforgenestobeexpressedorrepressedappropriatelyforearlystagesofdevelopment

StemCellsofAnimals•  stemcell

•  unspecializedcellthatcanreproduceitselfindefinitely

•  anddifferen>ateintospecializedcellsofoneormoretypes

•  embryonicstemcells

•  Stemcellsisolatedfromearlyembryosattheblastocyststage

•  abletodifferen>ateintoallcelltypes

•  Theadultbodyalsohasstemcells,whichreplacenonreproducingspecializedcells

•  IPSC’s

•  Inducedpluripotentstemcells

•  Adultcellsrevertedtostemcell

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Fig.20-20

Cultured stem cells

Early human embryo at blastocyst stage (mammalian equiva-

lent of blastula)

Different culture conditions

Different types of differentiated cells

Blood cells Nerve cells Liver cells

Cells generating all embryonic cell types

Adult stem cells

Cells generating some cell types

Embryonic stem cells

From bone marrow in this example

•  Singlenucleo?depolymorphisms(SNPs)

•  usefulgene>cmarkers

•  singlebase-pairsitesthatvaryinapopula>on

•  restric?onfragmentlengthpolymorphism(RFLP)

•  arestric>onenzymeisadded

•  SNPsresultinDNAfragmentswithdifferentlengths

DiagnosisofDiseases

Fig.20-21

Disease-causing allele

DNA

SNP Normal allele

T

C

8

HumanGeneTherapy•  Genetherapy

•  altera>onofanafflictedindividual’sgenes

•  holdsgreatpoten>alfortrea>ngdisorders

•  traceabletoasingledefec>vegene

•  Vectorsareusedfordeliveryofgenesintospecifictypesofcells

•  Iebonemarrow

•  Virusvectorsturnedouttobeproblema>c

Bone marrow

Cloned gene

Bone marrow cell from patient

Insert RNA version of normal allele into retrovirus.

Retrovirus capsid

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.

Inject engineered cells into patient.

1

2

3

4

•  Transgenicanimals

•  madebyintroducinggenesfromonespecies

•  intothegenomeofanotheranimal

•  pharmaceu>cal“factories”

•  “Pharm”plantsarealsobeingdeveloped

•  tomakehumanproteinsformedicaluse

ProteinProduc?onby“Pharm”AnimalsandPlants

ForensicEvidenceandGene?cProfiles

•  gene?cprofile

•  Anindividual’suniqueDNAsequence

•  canbeobtainedbyanalysisof>ssueorbodyfluids

•  canbeusedtoprovideevidenceincriminalandpaternitycases

•  andtoiden>fyhumanremains

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•  shorttandemrepeats(STRs)

•  gene>cmarkers

•  varia>onsinthenumberofrepeatsofspecificDNAsequences

•  PCRandgelelectrophoresisareusedtoamplify

•  andtheniden>fySTRsofdifferentlengths

•  Theprobabilitythattwopeoplewhoarenotiden>caltwinshavethesameSTRmarkersisexcep>onallysmall

ForensicEvidenceandGene?cProfiles

This photo shows Earl Washington just before his release in 2001, after 17 years in prison.

These and other STR data exonerated Washington and led Tinsley to plead guilty to the murder.

(a)

Semen on victim

Earl Washington

Source of sample

Kenneth Tinsley

STR marker 1

STR marker 2

STR marker 3

(b)

17, 19

16, 18

17, 19

13, 16

12, 12

14, 15

11, 12

13, 16

12, 12