1. Southern Blotting DNA Fingerprinting

2. Southern Blot A Southern Blot identifies specific sequences of DNA A Southern Blot may be used to determine a DNA fingerprint A Southern Blot may be used in forsenic medicine

3. Southern Blot Restriction Digest of DNA Electrophoresis Denaturation/Depurination Blotting Step Probing done To do 2 wks

4. Today’s laboratory: DNA fingerprinting in a hypothetical paternity determination.

5. In this hypothetical case, DNA was extracted from samples obtained from two possible fathers, a mother and child. The DNA was were cleaved with a restriction enzyme. A Paternity Case

6. Loading Gels Each group should load their gel Run gel at approximately 125 volts for 45-60 minutes Lecture will be given while samples electrophorese.

7. How to handle your gel after electrophoresis…

8. Load Gels and then we will discuss recombinant DNA technology

9. Sample Volume Add 40 ul per well MUST WARM 5 min. at 65C Samples –A. Standard DNA fragments –B. Mother DNA cut with restriction enzyme –C. Child DNA cut with restriction enzyme –D. Possible Father #1 DNA with restriction enzyme –E. Possible Father #2 DNA cut with restriction enzyme

10. Depurination Step 8 minute (maximum) incubation in 100 ml.25N HCl Rinse gel several times with 100 ml distilled water depurination

11. Denaturation Step After rinsing gel with water Add 100 ml DNA denaturation solution 10-15 min. Replace with fresh denaturation solution for 10-15 minutes

12. General Schematic for Southern Blot: DNA

13. Setting up the Southern Blot: pg. 3-82 Line a tray with plastic wrap Place “denatured” gel upside down on wrap Pre-wet nylon membrane in denaturation solution for 2-5 minutes Place nylon on top of inverted gel

14. Setting up Southern Blot: pg. 3-82 Place filter paper on top of nylon membrane Remove air bubbles Place stack of paper towels on top of filter paper Place empty 400 ml beaker on towel Incubate overnight

15. Setting up the Southern Blot transfer Plastic wrap Inverted Gel Nylon Membrane Filter paper Paper towels Weight Overnight incubation at room temperature

16. Overnight the ssDNA will diffuse by capillary transfer from gel onto nylon membrane!

17. Next week we will stain this membrane for similarities between child and parents.

19. Southern Blotting DNA Fingerprinting

20. Analysis of Southern Blot A non-isotopic method of detection

21. Remember the Southern Blot requires… that genomic DNA be first “digested” into smaller fragments that the DNA be separated on a gel that the DNA be denatured into single stranded DNA

22. Southern Blot requires that… you “probe” the fragments with a complementary sequence of DNA or RNA you have a means to “visualize” the binding between the probe and the DNA

23. You will probe the blot today Be sure and refer to your manual during these steps!

24. You will probe the blot today First you must prepare the nytran membrane –Use warm blocking buffer 45 minutes –Remove blocking buffer –Rinse container with water –Add probe and mix well –Incubate 10 minutes

25. Next: many rinsing steps Rinse with 400 mls detection buffer for 10 min Rinse again with 200 mls for 15 min Be sure and agitate the solution with the buffer for complete rinsing

26. Color Development I will prepare the substrate Add 8 mls of substrate Place DNA face down Place in 37C water bath Color should develop within 15-20 min

27. http://vector.cshl.org/resources/dnadetective.html Let’s look at some animations and examples of Southern Blots used in actual cases: http://vector.cshl.org/resources/BiologyAnimationLibrary.htm

28. Agarose gel electrophoresis Biotinylated DNA fragments labelled probes Avidin labelled enzyme Avidin has 4 binding sites for biotin

29. Biotin-DNA binds to avidin labelled alkaline phosphatase.

30. Substrate: 5-Bromo-4-chloro-3 indolyl phosphpate (BCIP) Product: Nitro Blue Tetrazolium to be reduced to an insoluble visible product.

32. Southern Blot Analysis