Cloning and Expression of Genes

1. 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

2. Isolating the Gene • Isolate mRNA • Copy to DNA with reverse transcriptase • cDNA (copy or complementary DNA)

4. 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

8. 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

12. Isolating the Insulin

15. Analysis of DNA • Gel Electrophoresis • DNA Sequencing • Hybridization • Polymerase Chain Reaction • DNA Chips • Human Genome Project

16. DNA LIGATION   ’ ’   ’ ’ ’ ’ Ligase Joins 5' phosphate to 3' hydroxyl

17. 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

18. GEL ELECTROPHORESIS - SIZE SORTING Electrode Samples Slower Gel Buffer Electrode Faster

20. 5’ P - - OH 3’ EcoRI - P 5’ 3’ OH - HindIII AluI HaeIII RESTRICTION ENDONUCLEASES

23. 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

25. DNA Sequencing

27. Hybridization • Binding of complementary DNA strands to each other • Find insulin sequence? Need prior information • Hybridize with part of the insulin sequence

29. 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

31. Polymerase Chain Reaction • Amplify a single piece of DNA • Makes a large number of copies • Target specific primers allow specific sequences to be amplified

32. DNA Polymerase

34. POLYMERASE CHAIN REACTION

36. 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?

38. http://www.affymetrix.com

39. http://cmgm.stanford.edu/pbrown/

42. CACCATGTGAC CACCATGTGAC PMP CACCATGTGAC N B PMP S ANTIBODY AFFINITY Add oligo with Biotin label + B GTGGTACACTG Anneal Heat and cool Add Paramagnetic-Streptavidin Particles + B GTGGTACACTG Bind Isolate with Magnet GTGGTACACTG

43. 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