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Recombinant DNA TechnologyRecombinant DNA Technology
Methods for Isolating, Methods for Isolating, Amplifying and Studying Amplifying and Studying Specific DNA SequencesSpecific DNA Sequences
Cutting and Cutting and Joining DNA Joining DNA Fragments Fragments
Restriction Enzyme:
cleaves DNA at specific sequences
DNA Ligase: joins DNA
fragments by sealing nicks in backbone
Use of the Restriction Enzyme Bam H1Use of the Restriction Enzyme Bam H1
5’— G G A T C C — 3’5’— G G A T C C — 3’ 3’— C C T A G G — 5’3’— C C T A G G — 5’
5’— G G A T C C — 3’5’— G G A T C C — 3’3’— C C T A G G — 5’3’— C C T A G G — 5’
sticky endsticky end
sticky endsticky end
Results inResults in
DNA fragments with complementary sticky ends DNA fragments with complementary sticky ends can associate to form new DNA combinations. can associate to form new DNA combinations.
Viewing DNA Fragments:Viewing DNA Fragments: Agarose Gel Electrophoresis Agarose Gel Electrophoresis
Locating DNA Fragments by Locating DNA Fragments by Southern BlottingSouthern Blotting
DNA Fragments are transferred to membrane
A subset of fragments
binds to the probe
Probe is complementary to
DNA of interest
Cloning GenesCloning Genes• Gene cloning = isolation of a specific Gene cloning = isolation of a specific
segment of DNA in a form that can be segment of DNA in a form that can be duplicated in another cellduplicated in another cell
• Requires a Requires a – Vector = carrier of DNA segmentVector = carrier of DNA segment– Host = cell in which foreign DNA can be Host = cell in which foreign DNA can be
duplicated and its gene product may be duplicated and its gene product may be synthesizedsynthesized
• Recombinant DNA = DNA produced by Recombinant DNA = DNA produced by joining fragments from two or more joining fragments from two or more different sourcesdifferent sources
Cloning VectorsCloning Vectors
• For Bacterial Hosts For Bacterial Hosts – PlasmidPlasmid
ampR
tetR
Bam H1Bam H1
ampR
tettetRR gene disrupted, no longer functional gene disrupted, no longer functional
Cloning VectorsCloning Vectors
• For Bacterial Hosts For Bacterial Hosts – BacteriophageBacteriophage– CosmidCosmid– Expression VectorExpression Vector
Cloning VectorsCloning Vectors
• For Eukaryotic HostsFor Eukaryotic Hosts– Shuttle VectorShuttle Vector– YAC = yeast artificial chromosomeYAC = yeast artificial chromosome– Ti plasmidTi plasmid
Tools for Finding GenesTools for Finding Genes
• DNA library = collection of clones from DNA library = collection of clones from one DNA donorone DNA donor– Categorized by: vector, source of Categorized by: vector, source of
DNADNA– Genomic library = all of the Genomic library = all of the
sequences from the genome of a sequences from the genome of a single organismsingle organism
– cDNA library= complementary DNA, cDNA library= complementary DNA, made using mRNA as a templatemade using mRNA as a template
Producing a Genomic LibraryProducing a Genomic Library
Many recombinant molecules are formed, each with a different
DNA insert
Source of DNA for Library
Producing cDNAProducing cDNAPrimer binds to
poly-A tail
DNA produced from RNA template
Partial digestion of RNA template
Second DNA strand produced using remaining RNA
segments as primers
Gaps in backbone sealed
Tools for Finding GenesTools for Finding Genes
Probe: sequence of DNA or RNA that Probe: sequence of DNA or RNA that is complementary to the gene of is complementary to the gene of interest; Used to locate a copy of the interest; Used to locate a copy of the gene in a library by hybridizationgene in a library by hybridization
AATGCCATTTACGGTA
Denature DNA by heating AATGCCAT
TTACGGTA
TTACGGTAAdd Probe
Probe Binds to gene
Screening a Library with a ProbeScreening a Library with a Probe
Amplifying DNA with the Amplifying DNA with the Polymerase Chain Reaction (PCR)Polymerase Chain Reaction (PCR)
• Copies a specific region in the DNA Copies a specific region in the DNA • Can be used with small amounts of Can be used with small amounts of
samplesample• Uses repeated cycles of heating to Uses repeated cycles of heating to
denature DNA and cooling to synthesize denature DNA and cooling to synthesize new DNAnew DNA
• Requires Requires – primers to begin synthesisprimers to begin synthesis– heat-stable polymerase (Taq polymerase)heat-stable polymerase (Taq polymerase)
Polymerase Polymerase
Chain ReactionChain Reaction
DNA Sequencing by DNA Sequencing by the Sanger Methodthe Sanger Method
Relies on Chain Termination duringRelies on Chain Termination during replication due to incorporation of replication due to incorporation of Dideoxynucleotides Dideoxynucleotides
OH H
deoxyribosedeoxyribose
H H
dideoxyribosedideoxyribose
Unknown SequenceUnknown Sequence 3’-A-G-C-C-C-A-G-A-T-T-5’3’-A-G-C-C-C-A-G-A-T-T-5’
5’-T-C-5’-T-C-GG-3’-3’
5’-T-C-G-G-G-T-C-5’-T-C-G-G-G-T-C-TT-3’-3’
Complementary Products Complementary Products Black= dideoxynucleotideBlack= dideoxynucleotide
5’-5’-TT-3’-3’5’-T-5’-T-CC-3’-3’
5’-T-C-G-G-G-T-C-T-5’-T-C-G-G-G-T-C-T-AA-3’-3’
5’-T-C-G-G-G-T-5’-T-C-G-G-G-T-CC-3’-3’
5’-T-C-G-5’-T-C-G-GG-3’-3’5’-T-C-G-G-G-5’-T-C-G-G-G-TT-3’-3’
5’-T-C-G-G-G-T-C-T-A-5’-T-C-G-G-G-T-C-T-A-AA-3’-3’
5’-T-C-G-G-5’-T-C-G-G-GG-3’-3’
Replicate unknown DNA using dideoxynucleotidesReplicate unknown DNA using dideoxynucleotides to terminate growing chains at various positions to terminate growing chains at various positions
A T C G A T C G Dideoxynucleotide Dideoxynucleotide
Use gel electrophoresis to separate replicated Use gel electrophoresis to separate replicated fragments that differ by one base fragments that differ by one base
Size of DNA on gel shows distance from start of replicationSize of DNA on gel shows distance from start of replicationLabeling of dideoxynucleotide shows complementary Labeling of dideoxynucleotide shows complementary sequence sequence
5’-T-C-G-G-G-T-C-T-5’-T-C-G-G-G-T-C-T-AA5’-T-C-G-G-G-T-C-5’-T-C-G-G-G-T-C-TT5’-T-C-G-G-G-T-5’-T-C-G-G-G-T-CC5’-T-C-G-G-G-5’-T-C-G-G-G-TT
5’-5’-TT5’-T-5’-T-CC5’-T-C-5’-T-C-GG5’-T-C-G-5’-T-C-G-GG5’-T-C-G-G-5’-T-C-G-G-GG
Complementary Complementary SequenceSequence
5’-T-C-G-G-G-T-C-T-A-5’-T-C-G-G-G-T-C-T-A-AA A T C G A T C G
Dideoxynucleotide Dideoxynucleotide
Complementary SequenceComplementary Sequence
Unknown SequenceUnknown Sequence
Determine unknown sequence from complementary Determine unknown sequence from complementary sequence with base-pairing rules sequence with base-pairing rules
Applications of Recombinant Applications of Recombinant DNA TechnologyDNA Technology
Genetic Testing and Gene Therapies Genetic Testing and Gene Therapies Products of BiotechnologyProducts of Biotechnology
RFLP AnalysisRFLP Analysis
• RFLP = Restriction RFLP = Restriction Fragment Length Fragment Length PolymorphismPolymorphism
• Identifies differences in Identifies differences in the length of restriction the length of restriction fragments derived from fragments derived from similar DNA sequencessimilar DNA sequences
• Analyzed by Southern Analyzed by Southern BlottingBlotting
• Used in gene mapping Used in gene mapping
RFLP Analysis in Paternity Testing RFLP Analysis in Paternity Testing
CC SSRR CCII EE
MM NNEE EE
RFLP Analysis in ForensicsRFLP Analysis in Forensics
11 22 33 44 55 66 77
SuspectsSuspects SuspectsSuspects
DNA FingerprintingDNA Fingerprinting
• Detects patterns for short repeated Detects patterns for short repeated sequences that are highly variable among sequences that are highly variable among humanshumans
• Described as Described as – VNTR: Variable VNTR: Variable
Number of Tandem Number of Tandem RepeatsRepeats
– STR: Short Tandem STR: Short Tandem RepeatsRepeats
– MicrosatellitesMicrosatellites
Analysis using Microsatellite sequencesAnalysis using Microsatellite sequences
Genetic TestingGenetic Testing
Gene Gene TherapyTherapy
Andrew Gobea was born with SCID, an immune Andrew Gobea was born with SCID, an immune deficiency due to a defect in the ADA gene. deficiency due to a defect in the ADA gene. Four days after his birth, he received an injection Four days after his birth, he received an injection of his own stem cells carrying functional ADA genes. of his own stem cells carrying functional ADA genes.Andrew’s immune system began producing ADA, Andrew’s immune system began producing ADA, but the amount was not sufficient to maintain full but the amount was not sufficient to maintain full health. He needs daily injections of ADA enzyme. health. He needs daily injections of ADA enzyme.
RNA Interference as a Possible RNA Interference as a Possible Treatment for High CholesterolTreatment for High Cholesterol
• High levels of ApoB, a protein High levels of ApoB, a protein component of blood lipid carriers, are component of blood lipid carriers, are associated with high cholesterol levelsassociated with high cholesterol levels
• In monkeys, siRNA for ApoB has In monkeys, siRNA for ApoB has lowered the mRNA for ApoB in liver cells lowered the mRNA for ApoB in liver cells and also reduced serum cholesterol and also reduced serum cholesterol levelslevels
siRNA for ApoB was delivered in
lipid capsules
Recombinant DNA ProductsRecombinant DNA Products
PharmaceuticalPharmaceutical Used for Used for
Factor VIIIFactor VIII Blood ClottingBlood Clotting
Human Growth Human Growth HormoneHormone
Pituitary DwarfismPituitary Dwarfism
InsulinInsulin DiabetesDiabetes
InterferonInterferon CancerCancer
Tissue Plasminogen Tissue Plasminogen ActivatorActivator
Heart Attack Heart Attack
VaccineVaccine Hepatitis BHepatitis B
Genetically Modified Genetically Modified OrganismsOrganisms
Introducing or modifying Introducing or modifying specific genes to alter the specific genes to alter the phenotype of an organismphenotype of an organism
Transgenic: organism that Transgenic: organism that contains a gene from another contains a gene from another species in all of its cellsspecies in all of its cells
Transgenic Animals Transgenic Animals Models of Human DiseaseModels of Human Disease Produce PharmaceuticalsProduce Pharmaceuticals
Studying Gene Studying Gene Function with Function with Knockout MiceKnockout Mice
• neoneo++ gene gives gene gives resistanceresistance to G418 to G418
• tktk++ gene gives gene gives sensitivitysensitivity to to gancyclovirgancyclovir
• Insertion of neoInsertion of neo++ into target gene into target gene yields neoyields neo++ and tk and tk-- cells, resistant to cells, resistant to G418 and to G418 and to gancyclovir gancyclovir
Transgenic Transgenic PlantsPlants
Bt Corn produces Bt Corn produces its own pesticideits own pesticide
““Golden” rice with Golden” rice with beta-carotene and beta-carotene and extra ironextra iron
Round Up Ready Soybeans Round Up Ready Soybeans are resistant to herbicide are resistant to herbicide
What Are Some Concerns?What Are Some Concerns?