Mata kuliah tentang enzim restriksi.Cari lebih banyak lagi di: http://muhammadhabibielecture.blogspot.com/2015/02/materi-kuliah-semester-4.html

1. Restriction endonucleasesest ct o e do uc eases
and their applications

2. Hi t f t i tiHistory of restriction
endonucleases and its rolee do uc eases a d ts o e
in establishing molecular
bi lbiology

3. Restriction enzymes
• Over 10,000 bacteria species have been
screened for restriction enzymes
O 2 500 t i ti h b f d• Over 2,500 restriction enzymes have been found
• Over 250 distinct specificities
• Occasionally enzymes with novel DNA sequence• Occasionally enzymes with novel DNA sequence
specificities are still found while most now prove
to be duplicates (isoschizomers) of already
di d ifi itidiscovered specificities.

4. Restriction Enzyme Function
• It is generally believed that the biological
function of restriction enzymes is to
protect cells from foreign DNA.
• Infecting DNA is cleaved (restricted) by
the restriction enzyme(s) preventing it
f f ll li ti dfrom successfully replicating and
parasitizing the cell.

5. Why the bacteria does not kill itself?
The Restriction Enzyme Modification Systems
if everything gets cleaved, how come the bacteria
does not kill itself?
• Usually, organisms that make restriction enzymes
also make a companion modification enzyme
(DNA methyltransferase) that protects their own( y ) p
DNA from cleavage.
• These enzymes recognize the same DNAy g
sequence as the restriction enzyme they
accompany, but instead of cleaving the sequence,
they disguise it by methylating one of the bases in
h DNA t deach DNA strand.

6. Classification of Restriction
enzymesenzymes
Class I Class II (93%) Class III
Restriction-methylase
on the same subunit
Homo-dimers,
methylase on a
separate subunit
Restriction-methylase
on the same subunit
p
ATP-dependent Mg++ dependent ATP-dependent
Binds to DNA
recognition site and
t DNA d l
recognize symmetric
DNA sequences and
l ithi th
Cut the DNA at the
recognition site and
th di i t fcuts DNA randomly -
any DNA as long as it
comes in contact
cleave within the
sequences
then dissociate from
the DNA

7. Type II Restriction enzymes
are endonucleases
that cut DNA at specific sites, and are most
useful for molecular biology research

8. Type II Restriction enzymes
Recognition sitesRecognition sites
are
P li d iPalindroimes:
121
IFFI ABAIFFI, ABA
AAGCTT
TTCGAA

9. How do I know what sequenceHow do I know what sequence
each enzyme cut?
• Test by cutting DNA of known sequencey g q
• Commercial sources are tested already,y,
and you find a catalog

10. Some popular Biotechnology Companies
• Life Technologies (BRL/GIBCO)
• New England Biolabs
• Amersham Pharmacia Biotech
• Qiagen
P• Promega
• Clonetech
• InvitrogenInvitrogen
• Stratagene
• ...

11. Nomenclature of restriction enzyme
• Eco R1: E coli
• Pst I: Providencia stuartii
• Hind III: Haemophilus influenza
• Not I: Norcardia otitidis-caviarum
• What do you name a restriction enzymeWhat do you name a restriction enzyme
isolated from Xanthomonas graminis?

12. How long is the recognition sequence
• 4 bp: e.g., Taq 1, HpaII, MspI
• 6 bp: e.g., EcoR1, HindIII, BamH1, PstI, salI
• 8 bp: Not I, Sfi I

13. Recognition sequence may beRecognition sequence may be
interrupted or ambiguous
Acc I: GT(at/gc)AC( g )
Bgl I: GCCNNNNNGGCg
Afl III: ACPuPyGTAfl III: ACPuPyGT

14. Three types of ends producedThree types of ends produced
by type II restriction enzymes
• 3’-overhang (protruding)
• 5’-overhang5 overhang
• Blunt end

15. 5’-overhang
EcoR I
5’-----------------------gaattc---------------------------3’5 -----------------------gaattc---------------------------3
3’-----------------------cttaag--------------------------5’
X EcoR1
5’-----------------------g
+
aattc---------------------------3’g
3’-----------------------cttaa +
aattc 3
g---------------------------5’

16. 3’-overhang
Pst I:
5’-----------------------ctgcag---------------------------3’5 -----------------------ctgcag---------------------------3
3’-----------------------gacgtc--------------------------5’
X PstI
5’-----------------------ctgca-3’
+
5’-g---------------------------3’g
3’-----------------------g-5’ +
5 g 3
3’- actgc---------------------------5’

17. Blunt end
EcoR V
5’-----------------------gatatc---------------------------3’5 -----------------------gatatc---------------------------3
3’-----------------------ctatag--------------------------5’
X EcoR V
5’-----------------------gat
+
atc---------------------------3’g
3’-----------------------cta +
atc 3
tag---------------------------5’

18. Only Compatible,
base-pairable ends
li tcan ligate

19. Odds of cutting at a segment of DNA
• 4 bp cutter: 44 = 256 bp
• 6 bp cutter: 46 = 4 kbp
• 8 bp cutter: 48 = 64 kb
• ??? How many do you predict Eco R1 to
cut catfish genome of 8 x 109 bpg p

20. What do you expect ifWhat do you expect if
you digest with youryou digest with your
plasmid DNA with 4-p
bp cutters

21. What do you expect ifWhat do you expect if
you digest with youryou digest with your
plasmid DNA with 6-p
bp cutters

22. What do you expect ifWhat do you expect if
you digest with youryou digest with your
plasmid DNA with 8-p
bp cutters

23. Restriction mapping
EcoR1
EcoR1
EcoR1
PstI
PstI
E
E
E
P
P
10 kb
0.6 kb 2.4 kb 6.0 kb 1.0 kb
1.5 kb 0.8 kb
7.7 kb
10 kb
What do you expect to see with double digest,
if reaction is complete?
0.6 kb, 0.9 kb*, 1.5 kb, 6.0 kb, 0.2 kb, 0.8 kb.

24. What do you expect ifWhat do you expect if
you digest with youryou digest with your
genomic DNA with 4-g
bp cutters

25. What do you expect ifWhat do you expect if
you digest with youryou digest with your
genomic DNA with 6-g
bp cutters

26. What do you expect ifWhat do you expect if
you digest with youryou digest with your
genomic DNA with 8-g
bp cutters

27. Selection of restrictionSelection of restriction
enzymes
Of Over 250 commercially available and
over 2,000 total, how do I know what to
use?
• Cutting frequency
• Easy to work with
• Economical