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. Ligase Joins 5' phosphate
DNA LIGATION   ’ ’   ’ ’
SK: ’ ’
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
SK:
Faster

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

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
SK:

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?

42. 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

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