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Recombinant DNA Recombinant DNA Technology Technology Methods for Isolating, Methods for Isolating, Amplifying and Studying Amplifying and Studying Specific DNA Sequences Specific DNA Sequences

Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

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Page 1: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

Recombinant DNA TechnologyRecombinant DNA Technology

Methods for Isolating, Methods for Isolating, Amplifying and Studying Amplifying and Studying Specific DNA SequencesSpecific DNA Sequences

Page 2: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific 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

Page 3: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

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.

Page 4: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

Viewing DNA Fragments:Viewing DNA Fragments: Agarose Gel Electrophoresis Agarose Gel Electrophoresis

Page 5: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

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

Page 6: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

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

Page 7: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

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

Page 8: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

Cloning VectorsCloning Vectors

• For Bacterial Hosts For Bacterial Hosts – BacteriophageBacteriophage– CosmidCosmid– Expression VectorExpression Vector

Page 9: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

Cloning VectorsCloning Vectors

• For Eukaryotic HostsFor Eukaryotic Hosts– Shuttle VectorShuttle Vector– YAC = yeast artificial chromosomeYAC = yeast artificial chromosome– Ti plasmidTi plasmid

Page 10: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

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

Page 11: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

Producing a Genomic LibraryProducing a Genomic Library

Many recombinant molecules are formed, each with a different

DNA insert

Source of DNA for Library

Page 12: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

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

Page 13: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

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

Page 14: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

Screening a Library with a ProbeScreening a Library with a Probe

Page 15: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

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)

Page 16: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

Polymerase Polymerase

Chain ReactionChain Reaction

Page 17: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

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

Page 18: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

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

Page 19: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

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

Page 20: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

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

Page 21: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences
Page 22: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences
Page 23: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

Applications of Recombinant Applications of Recombinant DNA TechnologyDNA Technology

Genetic Testing and Gene Therapies Genetic Testing and Gene Therapies Products of BiotechnologyProducts of Biotechnology

Page 24: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

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

Page 25: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

RFLP Analysis in Paternity Testing RFLP Analysis in Paternity Testing

Page 26: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

CC  SSRR  CCII  EE

MM  NNEE  EE

RFLP Analysis in ForensicsRFLP Analysis in Forensics

11 22 33 44 55 66 77

SuspectsSuspects SuspectsSuspects

Page 27: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

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

Page 28: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

Genetic TestingGenetic Testing

Page 29: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

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.

Page 30: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

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

Page 31: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

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

Page 32: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

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

Page 33: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

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

Page 34: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

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

Page 35: Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

What Are Some Concerns?What Are Some Concerns?