1. Molecular Biology
Restriction enzymes

2. Restriction enzymes Endonuclease
Cleaves internal phosphodiester linkages
Recognize specific double stranded DNA sequences
Different endonucleases recognize different sequences
Recognize palindrome sequences

3. 5’-G G A T C C-3’ 3’-C C T A G G-5’ Palindromes
The same sequence is read in the 5’ » 3’ direction on both strands
5’-G G A T C
C-3’
3’-C C T A G
G-5’

4. 5’-G 3’-C C T A G G A T C C-3’ G-5’
The same phosphodiester linkages are cleaved on both strands!
5’-G
3’-C C T A G G A T C
C-3’
G-5’

5. Different ends are generated
3’-C C T G A A G T C
C-3’
G-5’
Blunt ends

6. Different ends are generated
3’-C C T A G G A T C
C-3’
G-5’
5’ overhangs

7. Different ends are generated
3’-C
5’-G G A T C
C-3’ C T A G
G-5’
3’ overhangs

8. Compatibility of ends
Blunt ends HO P OH O P
Compatible

9. Compatibility of ends
Overhangs HO P OH HO P O
Incompatible

10. Compatibility of ends Annealing Compatible Overhangs P-CTAG HO GATC-POH
Annealing
GATC-P O
P-CTAG O
Compatible

11. Compatibility of ends Annealing Incompatible Overhangs GATC-P HO OH
P-TCCA HO
GATC-P OH
Annealing
GATC-P OH
P-TCCA HO
Incompatible

12. Restriction Maps

13. Restriction maps Determining the positions of restriction sites
Linear DNA maps
Circular DNA maps (plasmids)
Maps of inserts within vectors
Mapping genomes
Southern analysis

14. Approach Determine whether the DNA has digested
Is the digestion complete or partial?
How many cuts?
Determine the relative positions

15. Is the DNA digested? Compare to the undigested control Ladder Control
Which samples were not digested?
1 and 4
Which samples were digested?
2 and 3 1 2 3 4

16. Is the digestion complete?
Complete digestion
All the DNA molecules are cleaved at all the possible sites
Partial digestion
A fraction of the molecules are not digested
Partial undigested
A fraction of the molecules were digested, but not at all the possible sites

17. Complete digestion
Digestion

18. Partial digestion: Partial undigested
Non digested
Digestion

19. Partial digestion
partial
Digestion

20. Is the digestion complete or partial?
Ladder
Control 1 2 3 4
Compare to control
Verify the intensity of the bands
Verify the sizes

21. Determine the relative positions
How many cuts?
Number of sites
Circular DNA = number of bands
Linear DNA = Number of bands – 1
Determine the relative positions
The fragment sizes represent the distances between the sites

22. Linear DNA maps HindIII HindIII + SalI Enzyme Fragments (Kb) HindIII
3 and 4
SalI
2 and 5
HindIII + SalI
2 and 3
7.0
3.0
4.0
HindIII
HindIII + SalI
2.0
3.0

23. Circular DNA maps (plasmids)
Enzyme
Fragments (Kb)
BamHI
2, 3 and 5
HindIII
1 and 9
BamHI + HindIII
1, 1.5, 2, 2.5 and 3
3.0
2.0
1.0
1.5
2.5
7.0
10.0
1.0
9.0
10.0

24. Recombinant plasmids
MCS X
Digested with X
Vector

25. Recombinant plasmid +
Insert X
Recombinant
Vector + insert

26. Determining restriction map of a DNA insert
Step 1: Determining Insertion Site +
Insert X
Cut with X

27. Insertion maps Total size Insert size Insertion site 7.7Kb
Generates 2 fragments one of which is the size of the vector
XbaI
Enzyme
Fragments
BamHI
7.7Kb
EcoRI
1.0, 3.0, 3.7Kb
PstI
2.0 and 5.7
XbaI
2.7 and 5.0

28. Determining Relative Orientation
Insertion site delimits Right & Left
Ex. If Pst is the insertion site: Bam is to the left.
If Xba is the insertion site, then Bam is to the right

29. Determining Relative OrientationX A X
Orientation 2 A X A X

30. Relative mapping Sites to map Enzyme Fragments Total cuts
Sites in vector
Sites in insert
BamHI
7.7Kb 1
EcoRI
1.0, 3.0, 3.7Kb 3 2
PstI
2.0 and 5.7
XbaI
2.7 and 5.0
Insertion site

31. Map of PstI : 2 and 5.7Kb
5.7 Kb
2.0 Kb
5.0

32. Map of EcoRI: 1, 3 and 3.7Kb P
3.7
1.0
3.0
1.0
1.0
3.0

33. Restriction analysis of large DNA
Purpose
Find the position of a sequence in a large DNA fragment
Determine the number of copies of a gene
Find similar sequences in other organisms
Analyze unsequenced DNA from an unknown organism

34. Digestion of Large DNA
Number of fragments makes analysis impossible

35. Southern Analysis Separate DNA fragments according to size Denature
Hybridize with single stranded probe representing region of interest
Probe allows visualization of only the region of interest

36. Southern
Probe
Agarose gel
Hybridize to probe