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Cloning and Expression of Genes
• Insulin: Harmone regulating blood sugar (glucose)
• Obtain Insulin Gene from Human DNA
• Insert gene in bacterial cells
• Select cells that have desired gene
• Induce bacteria to express foreign gene to produce insulin
• Collect and purify insulin
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Isolating the Gene
• Isolate mRNA
• Copy to DNA with reverse transcriptase
• cDNA (copy or complementary DNA)
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Inserting the Gene
• Carrier DNA is a vector• Plasmids: Circular DNA found in
microorganisms• Cloning Vector• Easily transferred, incorporated, and isolated• Inserted with restriction enzymes• Cut up foreign DNA
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Selecting Cells with Desired Gene
• Most cells don’t take up DNA
• Ampicillin resistance gene in plasmid vectors
• Only cells with plasmids grow
• Blue-White Screening for gene or not
• Intact bacteria gene or not, Blue or White
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Isolating the Insulin
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Analysis of DNA
• Gel Electrophoresis
• DNA Sequencing
• Hybridization
• Polymerase Chain Reaction
• DNA Chips
• Human Genome Project
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DNA LIGATION
’ ’
’ ’
Ligase Joins 5' phosphateto 3' hydroxyl
’ ’
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Gel Electrophoresis
• Sort DNA by Size
• Electrophoresis is separation of particles in an electric field based on charge
• Thin Agarose Gels: Matrix of interlocking fibers
• DNA has negative charge
• Repates through matrix
• Ethidium Intercalator for visualization
• Entanglement and speed related to charge and size
• Restriction Map: Different restriction enzymes, different size maps, ID restriction sites
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GEL ELECTROPHORESIS - SIZE SORTING
BufferGel
Electrode
Electrode
Samples
Faster
Slower
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RESTRICTION ENDONUCLEASES
EcoRI
HindIII
AluI
HaeIII
- OH 3’
5’ P -
- P 5’
3’ OH -
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Sequencing
• Isolate fragment to be sequenced as SS
• 4 Tubes, 4 Bases
• Add mix of nucleotides, DNA polymerase, and primers with radioactive labels
• In each tube as single base is modified (dideoxynucleotide)
• Once added no other nucleotides added
• Chain Termination Sequencing
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DNA Sequencing
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Hybridization
• Binding of complementary DNA strands to each other
• Find insulin sequence? Need prior information
• Hybridize with part of the insulin sequence
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Gene Expression
• Study production of mRNA in the cell
• Clone genes of interest
• Make cDNA probes
• Sample mRNA populations of cell
• Detect by hybridization to radioactive probe
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Polymerase Chain Reaction
• Amplify a single piece of DNA
• Makes a large number of copies
• Target specific primers allow specific sequences to be amplified
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DNA Polymerase
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POLYMERASE CHAIN
REACTION
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DNA Arrays
• DNA Sequences Affixed to Solid Support
• Genotyping and Gene Expression
• Entire Genomes on Chips
• Look at Relative Intensity Levels
• Massively Parallel
• Loads of Data
• Interpretation, Storage, Analysis?
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http://www.affymetrix.com
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http://cmgm.stanford.edu/pbrown/
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ANTIBODY AFFINITY
CACCATGTGAC
GTGGTACACTG B
PMP
+
Anneal
CACCATGTGAC
GTGGTACACTG B+
CACCATGTGAC
GTGGTACACTG B PMP
Bind
Add oligo withBiotin label
Heat and cool
Add Paramagnetic-Streptavidin
Particles
Isolate with MagnetN
S
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Mapping and Sequencing the Human Genome
• Find all genes in Human Genome
• Develop Tools for Analysis in Biology and Medicine
• Tools:– Restriction Enzymes– PCR– DNA Arrays