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

Ligase Joins 5' phosphate DNA LIGATION   ’ ’   ’ ’ SK: ’ ’ Ligase Joins 5' phosphate to 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 Electrode Samples Slower Gel Buffer Electrode SK: Faster

RESTRICTION ENDONUCLEASES EcoRI HindIII AluI HaeIII - OH 3’ 5’ P - - P 5’ 3’ OH - SK:

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 SK:

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/

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

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