1. Genomic Southern Blotting and DNA Fingerprinting ABE Workshop 2007 Jamie Lum

2. Southern Blotting is a method to check for the presence of a gene or DNA in a sample.

3. Group 1 Group 2 Group 3 Group 4 PDI-2PDI-2PDI-3PDI-3 Bam HI Hind III Bam HI Hind III 1kB WT HM HZ LD 1kb WT HM HZ LD 1kB WT HM HZ LD 1kB WT HM HZ LD Mark U C U C U C Mark C U C U C U Mark C U C U C U Mark U C U C U C

4. Quantitate DNA with serial dilutions Quantitate DNA with serial dilutions In the presence of NBT/BCIP, alkaline phosphatase (AP) precipitates into purple spots. 10 -1 10 -2 10 -3 10 -4 10 -1 10 -2 10 -3 10 -4Control Group 1 Group 2 Group 3 Group 4

5. Capillary transfer of the DNA from the gel to a nylon membrane Capillary transfer of the DNA from the gel to a nylon membrane  Cut off top right corner of the gel.  Immerse gel in 0.125M HCl and shake for 10 minutes.  Soak the gel in Denaturation Buffer for 30 minutes on shaker.  Rinse the gel with water.  Soak in Neutralization Buffer for 30 minutes. Cut nylon membrane and two 3M filter papers to the size of the gel. Cut off top right corner of membrane. Cut nylon membrane and two 3M filter papers to the size of the gel. Cut off top right corner of membrane. Add transfer buffer to a glass tub. Add transfer buffer to a glass tub. LAYER, LAYER, AND LAYER!! LAYER, LAYER, AND LAYER!!

6. Tray with transfer buffer Tray with transfer buffer Wick (3M paper) Wick (3M paper) Gel Gel Nitrocellulose Nitrocellulose Parafilm cut as a picture frame Parafilm cut as a picture frame Filter paper Filter paper Paper towels Paper towels Glass dish Glass dish Weight Weight Allow to sit overnight Allow to sit overnight

7. Mark gel wells on the membrane with a pencil. Rinse the membrane in 6X SSC for 10 minutes. Lay the membrane, nucleic acid side up, on a piece of foil and expose to 120,000 microjoules/cm 2.

8. Prehybridization and Hybridization

9. Add 10 mL of 42 o C DIG Easy Hyb solution to a roller tube with the membrane. Add 10 mL of 42 o C DIG Easy Hyb solution to a roller tube with the membrane. Prehybridize for 20-30 minutes in a 42 o C hybridization oven. Prehybridize for 20-30 minutes in a 42 o C hybridization oven. Denature probe in boiling water for 5 minutes and quickly place in ice water. Denature probe in boiling water for 5 minutes and quickly place in ice water. Remove prehybridization solution and add probe/hybridization mixture into roller tube. Remove prehybridization solution and add probe/hybridization mixture into roller tube. Hybridize for at least 2 hours in the hybridization oven. Hybridize for at least 2 hours in the hybridization oven. Pour off the probe. Pour off the probe.

10. Wash membrane twice in 2X Wash solution for 5 minutes per wash. Wash membrane twice in 2X Wash solution for 5 minutes per wash. Wash membrane twice in 0.1X Wash solution for 10 minutes per wash. Wash membrane twice in 0.1X Wash solution for 10 minutes per wash. Equilibrate membrane in Washing Buffer for 2 minutes. Equilibrate membrane in Washing Buffer for 2 minutes. Incubate in Blocking solution for 30 minutes. Incubate in Blocking solution for 30 minutes. Remove blocking solution and add Antibody solution. Incubate for 30 minutes. Remove blocking solution and add Antibody solution. Incubate for 30 minutes. Wash membrane twice in Washing Buffer for 15 minutes per wash. Wash membrane twice in Washing Buffer for 15 minutes per wash. Equilibrate membrane in Detection Buffer for 2 minutes. Equilibrate membrane in Detection Buffer for 2 minutes.

11. Place membrane on a piece of transparency film and spot with CSPD/Detection Buffer over membrane. Place membrane on a piece of transparency film and spot with CSPD/Detection Buffer over membrane. Wrap up edges, make sure no air bubbles. Wrap up edges, make sure no air bubbles. Tape membrane in pre-warmed X-ray cassette and incubate for 10 minutes. Tape membrane in pre-warmed X-ray cassette and incubate for 10 minutes. In dark room, place cut X-ray film over membrane and close cassette. In dark room, place cut X-ray film over membrane and close cassette. Expose the film for 30 minutes. Expose the film for 30 minutes. Remove film from cassette and place in developer for 1 minute. Remove film from cassette and place in developer for 1 minute. Transfer film to fixer for 5 minutes. Transfer film to fixer for 5 minutes. Rinse with tap water and hang to dry. Rinse with tap water and hang to dry.

12. FINALLY!!FINALLY!!FINALLY!!FINALLY!!

13. Group 1 MW U C U C U C LamHindIII WT HMPDI2 HZPDI2

14. Group 2 MW C U C U C U LamHindIII WT HMPDI2 HZPDI2

15. Group 3 MW C U C U C U LamHindIII WT HMPDI3 HZPDI3

16. Group 4 MW U C U C U C LamHindIII WT HMPDI3 HZPDI3

17. So how is this information useful? Restriction Fragment Length Polymorphisms or RFLPs differentiates by patterns derived from cleavage of their DNA. The similarity of the patterns generated by RFLPs can be used to differentiate species or individuals from one another. Restriction Fragment Length Polymorphisms or RFLPs differentiates by patterns derived from cleavage of their DNA. The similarity of the patterns generated by RFLPs can be used to differentiate species or individuals from one another. Isolation of DNA for analysis is both time consuming and labor intensive. Isolation of DNA for analysis is both time consuming and labor intensive. Polymerase Chain Reaction or PCR is a technique for amplifying a specific region of DNA, defined by a set of two "primers" at which DNA synthesis is initiated by a thermostable DNA polymerase. Polymerase Chain Reaction or PCR is a technique for amplifying a specific region of DNA, defined by a set of two "primers" at which DNA synthesis is initiated by a thermostable DNA polymerase. Used to amplify small amounts of DNA in a few hours so more samples can be analyzed in a shorter time. Used to amplify small amounts of DNA in a few hours so more samples can be analyzed in a shorter time. Paternity testing Paternity testing Positive identification of crime suspect Positive identification of crime suspect

18. Someone ate the last cookie. A small DNA sample was taken from the plate and DNA was taken from seven of the workshop facilitators. Based on the RFLP, who did it? 1-Dr. Christopher 2-Dong Ping 3-Prof. Kabi CS-Crime Scene DNA 4-Eun Ju 5-Dr. White 6-Dr. Unabia 7-Dr. Berestecky 1 2 3 CS 4 5 6 7

19. A woman has accused a man of fathering her twins. Of course, he denies it, so a paternity test was done. 1-Mother 2-Child 1 3-Child 2 4-Potential father 1 2 3 4 1 2 3 4