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Gene Technology
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Outline: Gene Technology
• Gene Cutting & Splicing• Transferring, Cloning & Storing DNA• Polymerase Chain Reaction (PCR) • Gel Electrophoresis & RFLP Analysis• Biotechnology & Applications• Mutation• Cancer
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CCGGT
ATA
AT
AT
CCGGT
ATA
AT
AT
CG
A A T T
CG
A A T T
CG
T T A A
CG
T T A ACG
A A T TDNA ligasejoins the strands.
DNA
GATT
CG
A A T T
Sticky ends
Restriction sitesEcoRI
Recombinant DNA molecule
Restriction endonuclease cleaves the DNAEcoRI
EcoRI
EcoRI
DNA from another source cut with thesame restriction endonuclease is added.
Restriction endonuclease cleaves the DNA
Sticky ends
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
C A
Cutting and Splicing DNA Recombinant DNA Multiplying, Storing &Transferring DNAVectors - Transfer DNA
Plasmids Phages
Hosts – Store/replicate DNABacteria, yeast cells, mammalian cultured cells, plant cells
Bacterialchromosome
Plasmid DNA
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Vectors
Plasmid ampicillin gene only bacteria with plasmid will survive in medium with ampicillin.
Plasmid lacZ gene only bacteria with inactive lacZ will have foreign DNA inserted.
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DNA Libraries Store Genomes
Constructing a cDNA Library from Eukaryotes
Fig. 17.5-2
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DNA Libraries
Molecular hybridization is a technique used to identify specific DNAs in complex mixtures -A known single-stranded DNA or RNA is labeled
-It is then used as a probe to identify its complement via specific base-pairing
-Also termed annealing
Target sequence
2 copies
Cycle1
3
2
1
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
PolymeraseChainReaction (PCR)
1 copy1. Heat Denature DNACool & add primers
2. Add DNA polymerase & Nucleotides
3. New DNA synthesized
8 copies Cycle3
Repeat 1, 2 & 3
4 copiesCycle
2
Repeat 1, 2 & 3
After 20 cycles, a single fragment produces over one million (220) copies!
Longer fragments
Shorter fragmentsMixture of DNA fragmentsat top of gel Electric current applied
Powersource
Completed gel
Gel
Anode+
Cathode–
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Gel ElectrophoresisDNA andrestrictionendonuclease
DNA Fingerprinting
Original Sequence
Point Mutation
W
X
Y Y
Z
CutCut
Cut
DNA from chromosomes
CCGG
GGCC
ACGG
TGCC
CCGG
GGCC
CCGG
GGCC
Y X W
Y Z
Single base pair change
RFLP = Rrestriction Fragment Length Polymorphisms
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Different DNA duplexes
Cut DNA Gel electrophoresis ofrestriction fragments
Original Sequence
of Restriction Sites
(no mutations)
Point MutationsChange the Sequence of
Restriction Sites
Largerfragments
Smallerfragments
restriction enzymecutting sites
+ +
+–
–
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Restriction Fragments Reflect DNA Differences
Single basePair change
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
STR site 1
Crime scene DNA
STR site 2
Suspect’s DNA
Number of short tandemrepeats match
Number of short tandemrepeats do not match
Short tandem repeats (STRs) are genetic markersSTRs are short DNA sequences repeated many times in a row at thesame location. Number of STR units differs between individuals.
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
Suspect’sDNA
Crime sceneDNA
Suspect’s DNACrime scene DNA
Short tandem repeats (STRs) are genetic markers
DNA fragments separated by
Gel Electrophoresis
DNA Fingerprinting
RFLP & Genetic Disorder Markers
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
Viruses
1892 Russian Dmitri Ivanovski Could not filter disease agents causing Tobacco mosaic disease.
1898 Dutch botanist, Martinus Biejerinck“kills’ tobacco mosaic disease agent at 90C.
1935 - Wendell Stanley isolated and crystallized the Tobacco Mosaic Virus
Nature of Viruses
1. Agents of Diseasefound in virtually all organismshost range usually specific
2. No cell organization3. No Metabolism4. No organelles5. Cannot reproduce independent of cells6. Set of genetic instructions7. Renegade nucleic acid coated with protein
Virus Structure
2. Protein Capsid
Phospholipid bilayerProteinsGlycoproteins
3. Envelope
1. Nucleic Acid Core
Bacteriophage HIV
SHAPE1. Helical ≈ rods2. Isometric ≈ icosahedron
Viral Genome Structure
1. DNA Viruses have Double-Stranded DNA2. RNA Viruses have Single Stranded RNA
(+) Stranded RNARNA = mRNA
(-) Stranded RNARNA bases complimentary to mRNA
Virus Genome1. – strand RNA Virus2. 29,751 nucleotides3. Six encoding genes make
2 ReplicasesSpike proteinsEnvelope glycoproteinsMembrane glycoproteinNucleocapsid protein
SARS virusSevere acute respiratory syndrome
Viral genome of E. coli bacteriophage48,502 bases23 proteins development & maturationOther enzymes integrate virus DNA into host genome
Viruses cause disease in animals and plants
Both DNA viruses and RNA viruses cause disease in animals & plants
Reproductive cycle of an RNA virus
– Entry
– Glycoprotein spikes contact host cell receptors
– Viral envelope fuses with host plasma membrane
– Uncoating of viral particle to release the RNA genome
– mRNA synthesis using a viral enzyme
– Protein synthesis
– RNA synthesis of new viral genome
– Assembly of viral particles
Copyright © 2009 Pearson Education, Inc. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• HIV, the AIDS virus– A retrovirus
Envelope
Glycoprotein
Protein coat
RNA (two identical strands)
Reverse transcriptase
Human Immunodeficiency Virus
Plasma membraneof host cell
VIRUS
Entry
Viral RNA(genome)
Viral RNA(genome)
2
Membranousenvelope
Protein coatGlycoprotein spike
Uncoating
RNA synthesisby viral enzyme
3
1
Human Immunodeficiency Virus
Double-strandedDNA
ViralRNAandproteins
DNAstrand
Viral RNA
NUCLEUS
CYTOPLASM
ChromosomalDNA
ProvirusDNA
RNA
2
1
5
3
4
6
Human Immunodeficiency Virus
HIV replication animation
Genetically modified organisms
Genetically modified (GM) organisms contain one or more genes introduced by artificial means
Transgenic organisms contain at least one gene from another species GM plants
– Resistance to herbicides– Resistance to pests– Improved nutritional profile
GM animals– Improved qualities– Production of proteins or therapeutics
Copyright © 2009 Pearson Education, Inc. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
GM Plants: Recombinant DNA technology in Plants
Agrobacterium tumefaciens
DNA containinggene for desired trait
Tiplasmid
1
Insert plant geneinto plasmid usingrestriction enzymeand DNA ligase
RecombinantTi plasmid
2
IntroductionInto
plant cells
3
Regenerationof plant
Plant with new trait
T DNA carrying newgene within plant chromosome
Plant cell
Restriction site
Agrobacterium tumefaciens tumor on a plant
Glyphosate Resistance1. Cotton2. Corn3. Soybeans4. Canola5. Wheat
Bt Crops• Cotton• Corn
Genetic Engineering & genetically modified (GM) crops
Enhancement of Longevity1. “Flavr Savr” Tomato
Other engineered crops1. Papaya virus resistance2. Carnation longevity3. Flax herbicide resistance4. Lentil herbicide resistance5. Potato insect resistance6. Squash virus resistance7. Sugar beet herbicide resistance8. Cucumber virus resistance9. Watermelon virus resistance
Enhancement of Nutritional Value1. Golden Rice
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
Golden Rice
Vitamin A Deficiency
Vitamin A Roles
•Vision
•Immune defense
•Reducing morbidity of measles
•Reducing respiratory infections
•Cell differentiation and morphogenesis
Agricultural Applications of DNA Technology
Daffodil
Ferritin geneis transferredinto rice frombeans.
Phytase gene istransferred intorice from afungus.
Metallothioningene istransferred intorice from wildrice.
β-carotene enzymeSynthesis genes aretransferred intorice from daffodils.
Fe Pt SRicechromosome A1
Ferritin proteinincreases ironcontent of rice.
Phytate, whichinhibits ironreabsorption,is destroyed by thephytase enzyme.
Metallothioninprotein suppliesextra sulfur toincrease ironuptake.
β-carotene, aprecursor tovitamin A, issynthesized.
Beans Aspergillus fungus Wild rice
A2 A3 A4
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Genetically Engineered Golden RiceApplications of Plant
Genetic Engineering
European Corn Borer
Bt Corn Bt Corn Non-Bt Corn
Genetically Engineered Corn=Bt corn
Bacillus thuringensis1. bacterium2. produces Bt toxin3. kills larvae
Table 16.1
Medical Applications – Subunit Vaccines
Human immuneresponse
Gene specifying herpessimplex surface protein
Harmless vaccinia(cowpox) virus
Herpes simplex virus
2. Herpes simplexgene is isolated.
3. Vaccinia DNA is extracted and cleaved.
4. Fragment containingsurface gene combines with cleaved vaccinia DNA.
5. Harmless engineered virus (the vaccine) with surface like herpes simplex is injected into the human body.
6. Antibodies directedagainst herpes simplex viral coat are made.
1. DNA is extracted.
Gene Therapy & ADA Deficiency
ENDGENE
TECHNOLOGY
Mutation
Mutation1. Definition: Change in DNA2. Frequency: 1 in 50 million base pairs
1 in a million gametes
White grapes
Seedless navel orange
Albino rainbow trout
Blue Trout
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
Mutation = change in the nucleotide sequence of DNA
Why mutation?1. Spontaneous
errors in DNA replication errors in DNA recombination
2. Induced to form by mutagensHigh-energy radiationChemicals
Mutation
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
Point mutations Changes in 1-few nucleotides
Normal gene mRNA
Base substitution
Base deletionMissing
Met Lys Phe Gly Ala
Met Lys Phe Ser Ala
Met Lys Leu Ala His
A U G A A G U U U G G C G C A
A U G A A G U U U A G C G C A
A U G A A G U U G G C G C A U
U
Protein
Mutation – gene alteration
mRNAProtein
mRNAProtein
Base insertion
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Point mutations alter one or a few DNA bases.What happens when a point mutation occurs?Silent no change in mRNA codonNonsense create stop codonFrameshift shifts reading of mRNA codons
Mutation: Altered Genes
DNA
mRNA
Amino acid
ATG
UAC
Tyrosine
Normal Silent Nonsense FrameshiftMutation
ATA
UAU
Tyrosine
ATT
UAA
Stop
TTAGGCC
TTAGCGCC
UCG
Serine
Normal hemoglobin DNA Mutant hemoglobin DNA
Sickle-cell hemoglobinNormal hemoglobin
mRNAmRNA
ValGlu
Examples of Mutation – Sickle Cell Anemia
250,000 base pairs
27 Exons + Introns
61,000 base pair mRNA
1,480 Amino Acid Sequence of CTFR protein
Examples of Mutation – Cystic Fibrosis
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Mutation: Altered GenesChromosomal mutations change chromosome structure.
deletion part of chromosome is lostduplication part of chromosome is copiedinversion part of chromosome in reverse ordertranslocation/transposition part of chromosome
moves to a new location
Deletion
Duplication
Inversion
Translocation Transposition
Chromosomal mutations
part of chromosome is lost
part of chromosome is copied
part of chromosome is reversed in order
chromosome segments move/swap places
Chromosomal Mutations Transposition = Jumping genes
Chromosomal mutations – Transposons
Transposon
Chromosome A
Chromosome B
Consequences of transposition (48% Human genome = transposons)1. Cause mutations 2. May disable functional genes3. May cause cancer by insertion of transposon promoter near
cancer-causing gene4. Transposon diseases: Hemophilia, SCID, Muscular Distrophy5. Viruses like HIV behave like transposons
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
Alterations of chromosome structure - Deletion
Deletion
Cri du chatChromosome 5 deletion1 in 25,000-50,000Detected by amniocentesisMental retardationMay live normal life span
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
Reciprocal translocation associated with chronic myelogenous leukemia (CML)
“Philadelphia chromosome”
Chromosome 9
Chromosome 22 Reciprocaltranslocation
Activated cancer-causing gene
DNA Damage and Repair
DNA Damage and Repair DNA Damage and Repair
Xeroderma pigmentosa
THE GENETIC BASIS OF CANCER
Copyright © 2009 Pearson Education, Inc.
Cancer results from mutations in genes that control cell division
Mutations in two types of genes can cause cancer
– Proto-oncogenes
– Proto-oncogenes normally promote cell division
– Mutations to proto-oncogenes enhance activity
– Tumor-suppressor genes
– Normally inhibit cell division
– Mutations inactivate the genes and allow uncontrolled division to occur
Cancer & Proto-Oncogenes
Promote cancer when present in a single copy
Can be viral genes inserted into host chromosomes
Can be mutated versions of proto-oncogenes, normal genes that promote cell division and differentiation
Converting a proto-oncogene to an oncogene can occur by
– Mutation causing increased protein activity
– Increased number of gene copies causing more protein to be produced
– Transposition - Change in location putting the gene under control of new promoter for increased transcription
Copyright © 2009 Pearson Education, Inc.
Mutation withinthe gene
Hyperactivegrowth-stimulatingprotein in normalamount
Proto-oncogene DNA
Multiple copiesof the gene
Gene moved tonew DNA locus,
under new controls
Oncogene New promoter
Normal growth-stimulatingprotein in excess
Normal growth-stimulatingprotein in excess
Cancer & Proto-Oncogenes
Mutated tumor-suppressor geneTumor-suppressor gene
Defective,nonfunctioningprotein
Normalgrowth-inhibitingprotein
Cell divisionunder control
Cell division notunder control
Cancer & Tumor-suppressor genes
Promote cancer when both copies are mutated
Signaling cell
DNA
Nucleus
Transcriptionfactor(activated)
Signaling molecule
PlasmamembraneReceptor
protein
Relayproteins
TranscriptionmRNANewprotein
Translation
Target cell
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3
4
5
6
Signal Transduction Pathways &
Proto-Oncogenes
Cell Division
DNA RNA
DNA RNA
DNA RNA
DNA RNA
DNA RNA
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Rasprotein
Srckinase
Cell cyclecheckpoints
Cytoplasm
Nucleus
Rbprotein
p53protein
Growth factor receptor:more per cell in manybreast cancers.
Ras protein:activated by mutationsin 20–30% of all cancers.
Src kinase:activated by mutationsin 2–5% of all cancers.
PROTO-ONCOGENES
Rb protein:mutated in 40% of all cancers.
p53 protein:mutated in 50% of all cancers.
TUMOR-SUPPRESSOR GENES
Proteins Regulate Cell Cycle
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
DNA damage caused by heat,radiation,chemicals.
Step 1 Step 2
Damaged cells mayturn cancerous if other mutations appear.
ABNORMAL p53
Abnormalp53 protein
Cancercell
Step 3p53 protein fails to stop cell division and DNA repair.Cell division continues without repair.
Benzopyrene
Activity of Abnormal p53 gene
Cancer Carcinogens Cases in 1999
Prostate Testosterone; dietary fat 179,300
Breast Estrogen; possibly dietary fat 176,300
Lung Cigarette smoke 171,600
Colon & Rectum High dietary fat; low dietary fiber 129,400
Bladder Cigarette smoke 54,200
Skin Ultraviolet light 44,200
Kidney Cigarette smoke 30,000
Mouth and Throat Tobacco & alcohol 29,800
Pancreas Cigarette smoke 28,600
Stomach Table salt; cigarette smoke 21,900
Cervix Viruses; cigarette smoke 12,800
Cancer in the United States
Chromosomes 1 mutation
Normalcell
4mutations
3mutations
2mutations
MalignantCell &
metastasis
Mutation of Tumor
Suppressor Gene APC
Increased Cell
Division
Mutation of Proto-
OncogeneK-ras
Mutation of Tumor
Suppressor Gene DCC
Mutation of Tumor
Suppressor Gene p53
Benign polyp
Benign polyp
Multiple mutations lead to cancer
END