Cloning and Expression of Genes

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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 dont 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
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Ligase Joins 5' phosphate to 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
Electrode
Samples
Slower
Gel
Buffer
Electrode
Faster
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RESTRICTION ENDONUCLEASES
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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
Add oligo with Biotin label

B
GTGGTACACTG
Anneal
Heat and cool
Add Paramagnetic-Streptavidin Particles

B
GTGGTACACTG
Bind
Isolate with Magnet
GTGGTACACTG
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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