Restriction Mapping & Southern Blotting Made Simple

1. Restriction Mapping & Southern Blotting Made Simple Class instructions

2. In this lesson • Importance of complementary base pairing • Importance of 5’ and 3’ ends of DNA • Electrophoresis separates by size • Blotting keeps the DNA in the same position • The bands visible on the blot are the labeled ones but other DNA bands are still present • Southern blotting can be used to find exact matches or similar matches • Dye labels are used instead of radioisotopes due to safety considerations • How to interpret restriction maps and Southern blots • How restriction enzymes produce sticky ends

3. The devil is in the detail! • The 5’ prime and 3’ prime ends of the bases must be round the right way! IMPORTANT: Do not take bases apart!!!

4. Correct base pairing is critical! • Green (Guanine) pairs with yellow (Cytosine) • Blue (Adenine) pairs with orange (Thymine)

5. DNA Black sugars = sense strand Red sugars = antisense strand

6. Probe • Single strand of DNA • Labeled with radioactivity • Used to visualise DNA • Sticks ONLY to sense strand (black sugars)

7. The activity • Divide class into three groups - each group gets a double stranded section of DNA • Group 1 digest DNA with Taq1 • Group 2 digest DNA with Sma1 • Group 3 digest DNA with Taq1 and Sma1

8. The activity overview • Cut the template with enzyme and separate the DNA at these sites leaving sticky ends.   • How many fragments do you get and how big are they (measure the black sense strand only)?  • Separate the fragments using DNA gel electrophoresis. Put fragments in size order (measure the black strand) from largest to smallest as in a DNA gel. • Blot the DNA bands onto a membrane. Denature the DNA so it is single stranded by removing the red antisense strands from the bands leaving the black sense strands. • DNA is blotted onto a nitrocellulose or nylon membrane so you have a replica of the positions of the DNA in the gel preserved on the membrane. • Hybridise the DNA probe. In our example, the probe binds to one region. The probe has white sugars. Remember to keep the 5’ to 3’ prime orientation the correct way round. • Visualise the band. Probe is labeled so it can be visualised after hybridisation. Which band does the probe bind to?

9. Restriction Digest Sites Taq1 Sma1 5’ - C C C G G G - 3' 3’ - G G G C C C - 5’ 5’ - T C G A - 3' 3’ - A G C T - 5’

10. Starting DNA Black sugars = sense strand Red sugars = antisense strand

11. Taq1 digest Result after electrophoresis and denaturation Blot probe is visible Probe is visible

12. Sma1 digest Result after electrophoresis and denaturation Blot probe is visible Probe is visible

13. Taq1& Sma1 double digest Result after electrophoresis and denaturation probe is visible Probe is visible

14. TaqI & SmaI TaqI & SmaI Ladder Ladder Uncut TaqI SmaI Uncut TaqI SmaI Gel results DNA gel electrophoresis Southern Blotting

15. Restriction mapping • Find restriction enzyme sites in DNA • Combine single & double digest results • Use to localize where the region of interest is on a piece of DNA (such as a genome) Hint – start with SmaI, then double digest and use TaqI result to put them in the correct order

16. TaqI & SmaI TaqI & SmaI Ladder Ladder Uncut TaqI SmaI Uncut TaqI SmaI Gel results DNA gel electrophoresis Southern Blotting

17. Restriction Mapping Uncut TaqI digest SmaI digest TaqI and SmaI digest

18. uncut TaqI SmaI TaqI SmaI & TaqI TaqI TaqI TaqI SmaI SmaI Restriction Map 17 2 6 4 5 5 2 10 8 9