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Chapter 20. DNA Technology and Genomics. DNA Cloning. Tools and techniques: Making Recombinant DNA using restriction enzymes Cloning vector – the bacterial plasmid Nucleic Acid Probe Hybridization – identifying clones Genomic libraries PCR. Cell containing gene of interest. Bacterium. - PowerPoint PPT Presentation
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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