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
description
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. Isolating the Gene. - PowerPoint PPT Presentation
Transcript of Cloning and Expression of Genes
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
Isolating the Gene
• Isolate mRNA• Copy to DNA with reverse transcriptase• cDNA (copy or complementary DNA)
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
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
Isolating the Insulin
Analysis of DNA
• Gel Electrophoresis• DNA Sequencing• Hybridization• Polymerase Chain Reaction• DNA Chips• Human Genome Project
DNA LIGATION
’ ’
’ ’
Ligase Joins 5' phosphateto 3' hydroxyl
’ ’
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
GEL ELECTROPHORESIS - SIZE SORTING
BufferGel
Electrode
Electrode
Samples
Faster
Slower
RESTRICTION ENDONUCLEASES
EcoRI
HindIII
AluI
HaeIII
- OH 3’
5’ P -
- P 5’
3’ OH -
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
DNA Sequencing
Hybridization
• Binding of complementary DNA strands to each other
• Find insulin sequence? Need prior information
• Hybridize with part of the insulin sequence
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
Polymerase Chain Reaction
• Amplify a single piece of DNA • Makes a large number of copies• Target specific primers allow specific
sequences to be amplified
DNA Polymerase
POLYMERASE CHAIN
REACTION
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?
http://www.affymetrix.com
http://cmgm.stanford.edu/pbrown/
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
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
