Southern Blotting

1. Southern Blotting FascioScapuloHumeral Muscular Dystrophy (FSHD) Laura Yarram & Kate Poulter Bristol Genetics

2. FSHD: Background • Incidence • 1 in 20,000 (3rd most common of the muscular dystrophies) • Inheritance • Autosomal dominant • Clinical Symptoms • Muscle weakness and atrophy • Affected muscles; face, scapula, upper arm, hip girdle, lower leg • Variable age of onset • Variable severity • Method of testing: Southern Blotting

3. Bristol Genetics Laboratory Service • Managing the case load • High throughput blotting! • 2005/06 – 317 cases • ~2 blots/week • Aim: To achieve full capacity by fitting a maximum number of patients onto every gel • Maximum of 8 patients per blot

4. Steps in Southern Blotting DNA extraction Disease gene Fragments of DNA appear as a smear DNA digestion Gel electrophoresis Paper towels Denaturation of patient’s DNA in gel Chromatography paper support Gel in NaOH Nylon filter Southern blot Gel 10x SSC Blot dismantled Autoradiography X ray film Hybridisation: Stringency washes Radioactive probe added to filter cassette filter filter

5. E E B A A A A A A A A A A A P13-E11 Chrs. 4q35 3.3kb 3kb E E B A B B B B B B B B B B P13-E11 5kb Southern Blotting for FSHD testing <35kb Chrs. 10q26 E = EcoR1 B = Bln1 A = Apo1

6. E = EcoR1 EB = EcoR1/Bln1 A = Apo1 E | A E | E/B E | E/B Assay: Probe p13E-11; RE: EcoR1, EcoR1/Bln1, Apo1Linear Gel Electrophoresis >48kb assay detection limit 32kb 27kb 21kb 18 kb 8.9kb male specific fragment Bln1 reduces 4q fragment by 3kb and completely digests 10q fragment. Apo1 reduces 10q fragment by 5kb and completely digests 4q fragment.

7. Troubleshooting • How do we work out what’s gone wrong? • 8 day process • Multiple steps • Important to understand each step

8. Steps in Southern Blotting DNA Extraction Disease gene Restriction Enzyme Digestion Fragments of DNA appear as a smear Agarose Gel Electrophoresis Depurination (HCL), Denaturation (NaOH), Neutralisation Paper towels Southern Blot Whatman paper Nylon filter Gel 10x SSC

9. Prehybridisation Probe Labelling Hybridisation Stringency Washes Autoradiography Steps in Southern Blotting Hybridisation buffer + Salmon spermat 65°C dNTPs + Reaction buffer + enzyme + disease specific probe (p13-E11) + 32-P dCTP Labelled probe + salmon sperm + hyb. buffer at 65°C 2xSSC at specific temperature e.g. 65°C X ray film cassette filter

10. DNA Extraction Restriction Enzyme Digestion Gel Electrophoresis Depurination, Denaturation, Neutralisation Southern Blot Too much DNA

12. Prehybridisation Probe Labelling Hybridisation Stringency Washes Autoradiography Probe Labelling Kit Degradation Clue: Markers were pre-labelled

13. Prehybridisation Probe Labelling Hybridisation Stringency Washes Autoradiography Partially Dissolved Salmon Sperm

14. Prehybridisation Probe Labelling Hybridisation Stringency Washes Autoradiography Inadequate Stringency Washes – High Background

15. Prehybridisation Probe Labelling Hybridisation Stringency Washes Autoradiography Incomplete Fixing

16. Prehybridisation Probe Labelling Hybridisation Stringency Washes Autoradiography Exposed Film, Damaged Membrane

17. Marker transfer? Why?

18. Advantages and Disadvantages of Southern Blotting • Advantages • It is the only way to diagnose FSHD! • The exact location of a disorder does not need to be known • It detects large fragments of DNA • Disadvantages • Process takes 7-14 days • Needs high quality/quantity DNA (Phenol chloroform extraction may be required) • Radioactive • Not usually suitable for detecting mutations at base pair level • Accurate sizing is difficult • Difficult to troubleshoot