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Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.

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Presentation on theme: "Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa."— Presentation transcript:

1 Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa

2 Outline History/Background Info Goals of Southern hybridization Example Other applications

3 History/Background ‘Southern’ hybridization named after Sir Edwin Southern Developed in 1975 One of the most highly cited scientific publications Earned Sir Southern a Lasker Award in 2005

4 History/Background Spawned naming of related techniques: Southern blot (DNA) Northern blot (RNA) Western blot (Protein) Eastern blot (???)

5 Goals of Southern Hybridization Immobilize DNA onto a permanent substrate Identify DNA sequence (gene) of interest

6 Example – Looking for Gene X Arabidopsis thaliana 2 copies of gene X

7 Capsella rubella ? copies of gene X extract DNA Example – Looking for Gene X

8 Step 1. Restriction Enzyme Digestion EcoR I

9 Step 1. Restriction Enzyme Digestion

10 Step 2. Gel Electrophoresis _ +

11 _ +

12

13 Goals of Southern Hybridization Immobilize DNA onto a permanent substrate ‘Membrane’ –paper-like matrix –nylon or nitrocellulose –usually has a slight positive charge

14 TGAATC ACATTG Step 3. DNA Denaturation Eliminate hydrogen bonds with sodium hydroxide (NaOH)

15 Step 4. Transfer DNA to Membrane Two methods for transferring DNA to a membrane –capillary –electrophoretic

16 Step 4. Transfer DNA to Membrane

17 Goals of Southern Hybridization Immobilize DNA onto a permanent substrate Identify DNA sequence (gene) of interest

18 Step 5. Making a Probe A probe is a small (25-2000 bp) length of DNA or RNA –Complementary to the sequence (gene) of interest –Labeled for subsequent detection procedures

19 Step 5. Making a Probe Arabidopsis thaliana 2 copies of gene X

20 Step 5. Making a Probe Gene X from Arabidopsis Partial or full-length probes by PCR

21 Step 5. Making a Probe Gene X from Arabidopsis Partial probes by random-priming

22 Step 5. Making a Probe Denature template with heat

23 Step 5. Making a Probe Add random primers

24 Step 5. Making a Probe Extend random primers with polymerase

25 Step 5. Making a Probe A probe complementary to the sequence (Gene X) of interest!

26 Step 5. Making a Probe How do we detect the probe? –Radioactivity ( 32 P)

27 Step 5. Making a Probe How do we detect the probe? –Digoxigenin (DIG) U

28 Step 4. Transfer DNA to Membrane

29 Step 6. Pre-hybridization Prehybridization buffers contain ‘blocking reagents’ that occupy available binding sites on the membrane

30 Step 7. Hybridization

31

32 Step 8. Washes

33 Step 9. Anti-DIG

34

35 Step 10. Washes

36 Step 11. CSPD

37 Step 12. Detection DIG-labeled probes emitting minute amounts of light (chemiluminescence) 32 P-labeled probes emitting ß-particles

38 Step 12. Detection DIG-labeled probes emitting minute amounts of light (chemiluminescence) 32 P-labeled probes emitting ß-particles Autoradiography film can detect this radiation

39

40 Conclusion How many copies of ‘Gene X’ does Capsella rubella possess? Capsella rubella 3

41 Other Applications DNA fingerprinting –RFLP of VNTRs Dot or slot blot Colony or plaque lifts Microarray analysis

42 Other Applications DNA fingerprinting –RFLP of VNTRs Dot or slot blot Colony or plaque lifts Microarray analysis

43 Other Applications DNA fingerprinting –RFLP of VNTRs Dot or slot blot Colony or plaque lifts Microarray analysis

44 Other Applications DNA fingerprinting –RFLP of VNTRs Dot or slot blot Colony or plaque lifts Gene Expression

45 Other Applications Microarray technology


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