WHODUNNIT?

1. WHODUNNIT?

2. DNA Technology- What is it? • Making recombinant DNA (DNA in which genes from 2 different sources are combined) • Biotechnology-manipulation of genes of organisms for our benefit • Genetic engineering-directly changing genes of an organism • Examples: • Cloning • Gel electrophoresis • Gene therapy • Test-tube babies • Sequence DNA/genes • Stem cell research /

3. What can we do with it?The Big Picture • Diagnose diseases • Cure diseases • Produce better medicines/vaccines more efficiently • Solve crimes • Paternity tests • Produce more nutritious foods • Produce food in countries with little or no arable land • More efficiently Dispose of waste

4. Important Techniques/Tools Used in DNA Technology • Restriction Enzymes • DNA libraries • Gel electrophoresis • Bacterial transformation, plasmids • cDNA • Polymerase chain reaction (PCR) • probing/DNA hybridization • DNA sequencing • Bioinformatics • Southern blotting • Microarray analysis • RFLPs • vectors

6. A few words about bacterial genetics…. • Prokaryotes • Nucleoid region • Reproduce by binary fission-108 in 12 hrs!! • Genome • 1 DS circular chromosome • Plasmids-smaller circles of DNA • Self replicating • Confer new characteristics • R plasmids, F plasmids • Can be genetically engineered • Genetic recombination in bacteria • Transformation-uptake of naked foreign DNA • Transduction-viruses • Conjugation using pilli

8. Restriction Enzymes • Used in almost areas of recombinant DNA technology • Create DNA “libraries”- collections of bacteria each having a random fragment of DNA

9. Restriction Enzymes/Endonucleases • Enzymes able to cleave DNA at specific sequences 4-8 bps in length • Originally found in bacteria, used as a defense against viral infection • Presently over 200 known REs, each recognizing a different DNA sequence

10. May make blunt (linear) cuts or staggered (zig zag) cuts • Staggered cuts leave “sticky” complementary ends-this allows DNA from 2 sources to join together (recombinant DNA)

11. Not every sequence is found in every genome/DNA fragment • Enzymes recognizing smaller sequences usually cut more often than enzymes recognizing larger sequences • Since every organisms DNA is different, cutting 2 or more samples w/ the same RE will yield different sizes & numbers of fragments • Cutting one DNA sample w/ the SAME RE will yield fragments which can be used to create a restriction map

12. Gel Electrophoresis • Uses • Paternity testing • Solving crimes • Diagnosing diseases • Finding a particular gene or DNA sequence in a DNA library • Taxonomical studies

13. The basics…….http://gslc.genetics.utah.edu/units/biotech/gel

14. A few more points….. • Can be used to separate proteins, lipids, nucleic acids • Gel is porous agarose (starch or cellulose) and consistency can vary • Rate of movement is a function of fragment length/agarose consistency/time/ and voltage • Gels/DNA have dyes added to increase visibility of fragments both during & after electrophoresis

15. Whodunnit?Crime Scenes

16. Who’s my Baby Daddy?Aka paternity cases

17. Size DOES Matter!Determining the size (in bps) of DNA fragments of unknown lengths • Generate a STANDARD CURVE using known sample • Compare distance migrated of unknown fragments to standard curve • Estimate unknown fragment sizes