Chapter 20Chapter 20DNA Technology and DNA Technology and

GenomicsGenomics

DNA CloningDNA Cloning

Tools and techniques:Tools and techniques:– Making Recombinant DNA using Making Recombinant DNA using

restriction enzymesrestriction enzymes– Cloning vector – the bacterial plasmidCloning vector – the bacterial plasmid– Nucleic Acid Probe Hybridization – Nucleic Acid Probe Hybridization –

identifying clonesidentifying clones Genomic librariesGenomic libraries PCRPCR

LE 20-2Bacterium

Bacterialchromosome

Plasmid

Gene inserted intoplasmid

Cell containing geneof interest

Gene ofinterest DNA of

chromosome

RecombinantDNA (plasmid)

Plasmid put intobacterial cell

Recombinantbacterium

Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest

Protein expressedby gene of interest

Protein harvested

Gene ofinterest

Copies of gene

Basicresearchon gene

Basicresearchon protein

Basic research andvarious applications

Gene for pestresistance insertedinto plants

Gene used to alterbacteria for cleaningup toxic waste

Protein dissolvesblood clots in heartattack therapy

Human growth hor-mone treats stuntedgrowth

LE 20-2a

Bacterium

Bacterialchromosome

Plasmid

Gene inserted intoplasmid

Cell containing geneof interest

Gene ofinterest DNA of

chromosome

RecombinantDNA (plasmid)

Plasmid put intobacterial cell

Recombinantbacterium

LE 20-2b

Recombinantbacterium

Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest

Protein expressedby gene of interest

Protein harvested

Gene ofinterest

Copies of gene

Basicresearchon gene

Basicresearchon protein

Basic research andvarious applications

Gene for pestresistance insertedinto plants

Gene used to alterbacteria for cleaningup toxic waste

Protein dissolvesblood clots in heartattack therapy

Human growth hor-mone treats stuntedgrowth

LE 20-3Restriction site

DNA 53

35

Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow.

One possible combination

DNA fragment from anothersource is added. Base pairingof sticky ends producesvarious combinations.

Fragment from differentDNA molecule cut by thesame restriction enzyme

DNA ligaseseals the strands.

Recombinant DNA molecule

Sticky end

LE 20-4_1

Isolate plasmid DNAand human DNA.

Cut both DNA samples withthe same restriction enzyme.

Mix the DNAs; they join by base pairing.The products are recombinant plasmidsand many nonrecombinant plasmids.

Bacterial cell lacZ gene(lactosebreakdown)

Humancell

Restrictionsite

ampR gene(ampicillinresistance)

Bacterialplasmid Gene of

interest

Stickyends

Human DNAfragments

Recombinant DNA plasmids

LE 20-4_2

Isolate plasmid DNAand human DNA.

Cut both DNA samples withthe same restriction enzyme.

Mix the DNAs; they join by base pairing.The products are recombinant plasmidsand many nonrecombinant plasmids.

Bacterial cell lacZ gene(lactosebreakdown)

Humancell

Restrictionsite

ampR gene(ampicillinresistance)

Bacterialplasmid Gene of

interest

Stickyends

Human DNAfragments

Recombinant DNA plasmids

Introduce the DNA into bacterial cellsthat have a mutation in their own lacZgene.

Recombinantbacteria

LE 20-4_3

Isolate plasmid DNAand human DNA.

Cut both DNA samples withthe same restriction enzyme.

Mix the DNAs; they join by base pairing.The products are recombinant plasmidsand many nonrecombinant plasmids.

Bacterial cell lacZ gene(lactosebreakdown)

Humancell

Restrictionsite

ampR gene(ampicillinresistance)

Bacterialplasmid Gene of

interest

Stickyends

Human DNAfragments

Recombinant DNA plasmids

Introduce the DNA into bacterial cellsthat have a mutation in their own lacZgene.

Recombinantbacteria

Plate the bacteria on agarcontaining ampicillin and X-gal.Incubate until colonies grow.

Colony carrying non-recombinant plasmidwith intact lacZ gene

Colony carryingrecombinantplasmid withdisrupted lacZ gene

Bacterialclone

LE 20-5

Master plate

Filter

Solutioncontainingprobe

Filter liftedand flipped over

Radioactivesingle-strandedDNA

ProbeDNA

Gene ofinterest

Single-strandedDNA from cell

Film

Hybridizationon filter

Master plate

Coloniescontaininggene ofinterest

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.

LE 20-6

Bacterialclones

Recombinantplasmids

Recombinantphage DNA

or

Foreign genomecut up withrestrictionenzyme

Phageclones

Plasmid library Phage library

LE 20-7

Genomic DNA

Targetsequence

5

3

3

5

5

3

3

5

Primers

Denaturation:Heat brieflyto separate DNAstrands

Annealing:Cool to allowprimers to formhydrogen bondswith ends oftarget sequence

Extension:DNA polymeraseadds nucleotides tothe 3 end of eachprimer

Cycle 1yields

2molecules

Newnucleo-

tides

Cycle 2yields

4molecules

Cycle 3yields 8

molecules;2 molecules

(in white boxes)match target

sequence

Restriction Fragment AnalysisRestriction Fragment Analysis

Gel electrophoresisGel electrophoresis Southern blottingSouthern blotting Restriction Fragment Length Restriction Fragment Length

Differences - RFLPsDifferences - RFLPs

LE 20-8

Cathode

Powersource

Anode

Mixtureof DNAmoleculesof differ-ent sizes

Gel

Glassplates

Longermolecules

Shortermolecules

LE 20-9Normal -globin allele

175 bp 201 bp Large fragment

Sickle-cell mutant -globin allele

376 bp Large fragment

Ddel Ddel Ddel Ddel

Ddel Ddel Ddel

Ddel restriction sites in normal and sickle-cell alleles of-globin gene

Normalallele

Sickle-cellallele

Largefragment

376 bp201 bp175 bp

Electrophoresis of restriction fragments from normaland sickle-cell alleles

LE 20-9a

Normal -globin allele

175 bp 201 bp Large fragment

Sickle-cell mutant -globin allele

376 bp Large fragment

Ddel

Ddel restriction sites in normal and sickle-cell alleles of-globin gene

Ddel Ddel Ddel

Ddel Ddel Ddel

LE 20-9b

Normalallele

Sickle-cellallele

Largefragment

376 bp201 bp175 bp

Electrophoresis of restriction fragments from normaland sickle-cell alleles

LE 20-10

DNA + restriction enzyme Restrictionfragments

Normal-globinallele

Sickle-cellallele

Heterozygote

Preparation of restriction fragments. Gel electrophoresis. Blotting.

Nitrocellulosepaper (blot)

Gel

Sponge

Alkalinesolution

Papertowels

Heavyweight

Hybridization with radioactive probe.

Radioactivelylabeled probefor -globingene is addedto solution ina plastic bag

Paper blot

Probe hydrogen-bonds to fragmentscontaining normalor mutant -globin

Fragment fromsickle-cell-globin allele

Fragment fromnormal -globinallele

Autoradiography.

Film overpaper blot

Mapping GenesMapping Genes

Human Genome ProjectHuman Genome Project Steps of mapping the genomeSteps of mapping the genome

– Genetic mappingGenetic mapping– Physical Mapping Physical Mapping – DNA sequencingDNA sequencing

Sanger (Fig. 20.12)Sanger (Fig. 20.12)Venter (Fig. 20.13)Venter (Fig. 20.13)

LE 20-11

Cytogenetic map

Genes locatedby FISH

Chromosomebands

Geneticmarkers

Genetic (linkage)mapping

Physical mapping

Overlappingfragments

DNA sequencing

LE 20-12DNA(template strand)

5

3

Primer3

5

DNApolymerase

Deoxyribonucleotides Dideoxyribonucleotides(fluorescently tagged)

3

5DNA (templatestrand)

Labeled strands3

Directionof movementof strands

Laser Detector

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 phagevectors

Sequence each fragment

Order the sequences into one overall sequence with computer software

Genomics - ApplicationsGenomics - Applications

Determining Gene functionDetermining Gene function– In vitro mutagenesisIn vitro mutagenesis– RNA interferenceRNA interference

Gene expression studiesGene expression studies– DNA microarray assaysDNA microarray assays

Comparing genomesComparing genomes ProteonomicsProteonomics

– SNPsSNPs

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)

DNAmicroarray

Size of an actualDNA microarraywith all the genesof yeast (6,400 spots)

Practical ApplicationsPractical Applications

MedicalMedical Diagnosis of diseaseDiagnosis of disease

– RT-PCRRT-PCR Gene therapyGene therapy PharmaceuticalsPharmaceuticals ForensicsForensics AgricultureAgriculture EnvironmentalEnvironmental

LE 20-15

DNA

RFLP marker

Disease-causingallele

Normal allele

Restrictionsites

LE 20-16

Cloned gene

Retroviruscapsid

Bonemarrowcell frompatient

Inject engineeredcells into patient.

Insert RNA version of normal alleleinto retrovirus.

Viral RNA

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

Viral DNA carrying the normalallele inserts into chromosome.

Bonemarrow

LE 20-19

Agrobacterium tumefaciens

Tiplasmid

Site whererestrictionenzyme cuts

DNA withthe geneof interest

T DNA

RecombinantTi plasmid

Plant withnew trait