1. Last class REs Paper reading Data analysis Intro to Paper 1

2. Type II REs GCATGC CGTACG RE1 GC CGTA ATGC CG

3. The RE conundrum Why doesn’t RE cut up genomic DNA in bacteria? - No nuclear membrane to protect DNA - Genomic DNA has multiple RE sites Inactivate enzyme - Then no longer protective 

4. Solving the RE conundrum ____________ the DNA at RE site! Make __________ at specific times (Why?) gDNA = Protected viral DNA = Degraded! gDNA = Protected viral DNA = Degraded! NOTE: Methylation on “C” or “A” residue

5. The Restriction-Methylation system GCATGC CGTACG RE1 GC CGTA ATGC CG RE1 GCATGC CGTACG CH 3 X X

6. Lab 4 Methylation specificity

7. Analytical uses of RE DNA 1 = 10000 bp You have:Pure DNA 1 Pure DNA 2 DNA 2 = 10000 bp Unknown DNA: Maybe pure 1/2 or mixture Sequencer is broken! Need a quick answer: What is unknown?

8. RE Mapping to quickly test sequences RE 1 10 Kb RE 1 10 Kb 4 Kb 6 Kb 10 Kb 6 Kb 4 Kb

9. RE Map Locations of RE sites on DNA Relative locations

10. RE Maps  Size + Pattern Known: Circular DNA, Uncut ~ 12Kb Known: RE2 = 6Kb DNA Known: RE1 = 6Kb DNA Known: RE1 + RE2 = 3Kb DNA RE MAP?

11. RE Mapping example Known: Circular DNA, Uncut ~ 12Kb Known: RE1 + RE3 = 4Kb + 8Kb DNA Known: RE1 or RE3 = 12Kb DNA Known: RE2 = 2Kb + 8Kb DNA Known: RE2 + RE3 = 2Kb + 6Kb DNA Known: RE2 + RE1 = 2Kb + 6Kb DNA RE MAP?