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 nucleotides 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’ - T C G A - 3' 3’ - A G C T - 5’
5’ - C C C G G G - 3' 3’ - G G G C C C - 5’

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

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

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

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

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

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

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

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