1. 免疫学信息网 http://www.immuneweb.com Techniques to manipulate DNA Phenol/chloroform extraction Ethanol precipitation Concentration determination Gel Electrophoresis DNA hybridization Southern Blotting Melting temperatures PCR

2. 免疫学信息网 http://www.immuneweb.com BRCA3 EcoRI NIH Example: Is the BRCA3 gene involved in Suppressing breast cancer? BRCA1 BRCA2 BRCA3

3. 免疫学信息网 http://www.immuneweb.com Paper Towels Southern Blotting 1 tissue SDSProtKPhenol Chloro ETOH ppt Spin

4. 免疫学信息网 http://www.immuneweb.com The “Hyperchromic Shift” 2

5. 免疫学信息网 http://www.immuneweb.com Paper Towels Southern Blotting 1 tissue SDSProtKPhenol Chloro ETOH ppt Spin

6. 免疫学信息网 http://www.immuneweb.com Agarose Gel Electrophoresis _ + 3

7. 免疫学信息网 http://www.immuneweb.com Sample Well 25 ng 1 kb ladder 0.8% Agarose 1 2 4 6 8 10 12 kb Agarose Gel Electrophoresis Sybergold Detection Limit: ~0.2-0.5 ng DNA EtBr Detection Limit: ~5-10 ng DNA (Not in handout)

8. 免疫学信息网 http://www.immuneweb.com Log (MW) is linearly related to mobility 4 100 bp Low % has no Resolving power at low MW 1 kb 10 kb High % has no resolving power at high MW

9. 免疫学信息网 http://www.immuneweb.com Separation Range Vs. % Agarose 5 Low Geling Agarose 4-6 0.02-0.4

10. 免疫学信息网 http://www.immuneweb.com TEMED (N,N,N,N-tetramethylenediamine) 7

11. 免疫学信息网 http://www.immuneweb.com PolyAcrylamide Gels 8

12. 免疫学信息网 http://www.immuneweb.com DNA topology affects mobility of DNA 9

13. 免疫学信息网 http://www.immuneweb.com PFGE can resolve large DNA fragments 10

14. 免疫学信息网 http://www.immuneweb.com Paper Towels Southern Blotting 1 tissue SDSProtKPhenol Chloro ETOH ppt Spin

15. 免疫学信息网 http://www.immuneweb.com Helix Formation: Nucleation and Propagation 11

16. 免疫学信息网 http://www.immuneweb.com A-T G-C Helix Melting 12

17. 免疫学信息网 http://www.immuneweb.com What parameters affect the T m ? (T m is defined as the temp at which half the molecules are single-stranded)

18. 免疫学信息网 http://www.immuneweb.com T m depends on DNA length 13 % dsDNA Sigmoidal Curve: Highly Cooperative Melting!

19. 免疫学信息网 http://www.immuneweb.com Effect of % GC Content on T m 14

20. 免疫学信息网 http://www.immuneweb.com Effect of [Salt] on T m 15

21. 免疫学信息网 http://www.immuneweb.com Predicting melting temperatures (see lecture notes)

22. 免疫学信息网 http://www.immuneweb.com Kinetics of Hybridization: Do I have time for a soda or a week-end in New York City?

23. 免疫学信息网 http://www.immuneweb.com C o t Curves: Hybridization vs. Complexity 16

24. 免疫学信息网 http://www.immuneweb.com A Bi-Phasic C o t Curve 17 5 kb Repeats (20% total) 100 kb fragment Repeat DNA = 5 kb Longest non-repeat DNA = 100kb - 20 kb = 80 kb Difference in complexity = 80/5 = 16

25. 免疫学信息网 http://www.immuneweb.com Paper Towels Southern Blotting 1 tissue SDSProtKPhenol Chloro ETOH ppt Spin

26. 免疫学信息网 http://www.immuneweb.com Results of Southern Blot Control TissueCancer Tissue BRCA3 EcoRI NIH

27. 免疫学信息网 http://www.immuneweb.com BRCA3 EcoRI NIH

28. 免疫学信息网 http://www.immuneweb.com 94  C (30 s) 40-60  C (30 s) 72  C (30-60 s/kb) Melt AnnealExtend The Polymerase Chain Reaction (PCR) 18 20-30X

29. 免疫学信息网 http://www.immuneweb.com 94  C (30 s) 40-60  C (30 s) 72  C (30-60 s/kb) Melt AnnealExtend Cycle #2 NIH 20-30X

30. 免疫学信息网 http://www.immuneweb.com Results of PCR #1 Control TissueCancer Tissue BRCA3 EcoRI NIH Primer Set #1

31. 免疫学信息网 http://www.immuneweb.com Results of PCR#5 Control TissueCancer Tissue BRCA3 EcoRI NIH Primer Set #5

32. 免疫学信息网 http://www.immuneweb.com PCR mutagenesis EcoRIHindIII Isolate products, mix Amplify digest, subclone sequence 20 PvuII

33. 免疫学信息网 http://www.immuneweb.com

34. Linear Amplification Geometric Amplification 19

35. 免疫学信息网 http://www.immuneweb.com hsp70 EcoRI NIH Example: Is heat shock factor bound to promoters before heat shock? EcoRI HSE HSF

36. 免疫学信息网 http://www.immuneweb.com Optimal separation of fragments > 2 kb requires low voltage 0.5 10 V/cm 6