restriction enzymes , molecular biology, rDNA

1. Restriction Enzymes: Molecular Scissors
• Restriction enzymes (endonuleases) are
specific endonucleases which cut
dsDNA (both backbones)
• Recognize specific short sequences
of DNA called recognition sequence
and cleave the DNA at or near the
recognition sequence
• What kinds of bonds are broken when
restriction enzymes cut?
– Covalent phosphodiester bonds
between nucleotides (within a single
strand)
– Hydrogen bonds (between strands)
as a result of the strands coming
apart
• Originally found in bacteria (natural
defense)
• Now cloned and commercially available
• Some leave blunt ends
• If do staggered cuts: leave single-
stranded 5’ overhangs or 3’overhangs
called sticky ends.
• Important for molecular biologists
because restriction enzymes create
unpaired "sticky ends" which anneal
with any complementary sequence
Hydrogen
bond
Covalent bond
More than 3000 are known

2. Restriction Enzymes
 called "restriction enzymes“ because restrict host range for certaincalled "restriction enzymes“ because restrict host range for certain
bacteriophagebacteriophage
 bacterial" immune system": destroy any "non-self" DNAbacterial" immune system": destroy any "non-self" DNA
 methylase recognizes same sequence in host DNA and protects it by
methylating it; restriction enzyme destroys unprotected = non-
self DNA (restriction/modification systems)
There are many examples of enzymes isolated from different sources which
recognize the same target sequence. These are known as isoschizomers.Ex-
MboI and Sau3AI

3. AGCCAG GATCCGGGCTGCAAGCGGTTAAG AATTCGTCGACGTCGACGAATTCTTAACCGCTTCCAGCCCGGATCCTGGCT
GATCCGGGCTGCAAGCGGTTAAGAATTCTTAACCGCTTCCAGCCCG
GATCCTGGCTAGCCAG AATTCGTCGACGTCGACG+
“sticky ends”
AGCCAGAT ATCGGGCTGCAAGCGGTTAACAG CTGGTCGACGTCGACCAGCTGTTAACCGCTTCCAGCCCGATATCTGGCT
ATCGGGCTGCAAGCGGTTAACAGCTGTTAACCGCTTCCAGCCCGAT
AGCCAGATATCTGGCT
+ CTGGTCGACGTCGACCAG
•“Sticky ends”: 5’ or 3’ over-hangs that allow the DNA to
anneal even though it is not covalently bound
•Help with the next step: ligation
Most common
restriction enzymes
Blunt-end restriction
enzymes No sticky ends
Blunt vs Sticky Ends
Digestion
Digestion

4. Restriction endonuclease
recognition sequences
• Will occur randomly in any dsDNA
• Statistically more often for e.g. 4-base cutters than 6-base cutters
• If sites are known, we can use restriction enzyme cutting patterns to
verify the identity of our DNA - called restriction mapping
• Recognition sequences: usually 4 or 6 bases but there are some
that are 5, 8, or longer
• Recognition sequences are palindromes
• Palindrome: sequence of DNA that is the same when one strand is
read from left to right or the other strand is read from right to left–
consists of adjacent inverted repeats
• Recognition sequence may be interrupted or ambiguous
– Acc I: GT(at/gc)AC
– Bgl I: GCCNNNNNGGC
– Afl III: ACPuPyGT

5. E.g. the recognition sequence for enzyme EcoRI (E. coli strain
R1st enzyme found happens to be found) in this piece of DNA:
5’ -----G-A-A-T-T-C----- 3’
3’ -----C-T-T-A-A-G----- 5’
Cutting the backbones between the red and blue nucleotides
gives 2 nicks:
5’ -----G A-A-T-T-C----- 3’
3’ -----C-T-T-A-A G----- 5’
The velcro bonds between the A’s and Ts break easily, giving 2
fragments with 5’ overhangs (sticky ends):
5’-----G 3’ + 5’ A-A-T-T-C----- 3’
3’-----C-T-T-A-A 5’ 3’ G----- 5’

6. Note that whenever you have antiparallel, complementary sticky
ends, they could potentially H-bond back together again:
5’-----G 3’ + 5’ A-A-T-T-C----- 3’
3’-----C-T-T-A-A 5’ 3’ G----- 5’
back to: 5’ -----G A-A-T-T-C----- 3’
3’ -----C-T-T-A-A G----- 5’
Only now there will be nicks between the red and blue bases
(unless repaired by DNA ligase)

7. Restriction Endonucleases
Type I- multisubunit, endonuclease and methylase activities, cleave at random
nonspecific site > than 1,000 bp away
Type II- cleave DNA within recognition sequence, require no ATP, most
monomers
Type III- Recognize specific 5-7 bp sequences multisubunit, endonuclease and
methylase about 24-27 25 bp from recognition sequence
TypeII b-

8. Origins of Restriction Enzymes
• Naturally found in different types of bacteria
• Bacteria use restriction enzymes to protect themselves from foreign DNA
• Bacteria have mechanisms to protect themselves from the actions of their own
restriction enzymes
• Have been isolated and sold for use in lab work
• Restriction enzymes are isolated from bacteria
• Derive names from the bacteria
• Genus- first letter capitalized
• Species- second and third letters (small case)
• Additional letters from “strains”
• Roman numeral designates different enzymes from the same
bacterial strain, in numerical order of discovery
• Example:
PstI
Providencia stuartii
1st enzyme isolated
EcoRI
E Escherichia
Co coli
R R strain
I first enzyme
discovered from
Escherichia coli
R
HindIII
Haemophil
us
influenzae
strain RD
3rd
enzyme
TaqI
Thermus aqua
1st enzyme iso

9. Theoretical Basis
Using Restriction Enzymes
 The activity of restriction enzymes is dependent upon
precise environmental condtions:
PH
Temperature
Salt Concentration
Ions
 An Enzymatic Unit (u) is defined as the amount of enzyme
required to digest 1 ug of DNA under optimal conditions:
3-5 u/ug of genomic DNA
1 u/ug of plasmid DNA
Stocks typically at 10 u/ul

10. Conditions for activity
Restriction Buffer provides optimal conditions
• NaCI provides the correct ionic strength
• Tris-HCI provides the proper pH
• Mg2+
is an enzyme co-factor
Why incubate at 37°C?
• Body temperature is optimal for these and most other enzymes
What happens if the temperature is too hot or cool?
• Too hot = enzyme may be denatured (killed)
• Too cool = enzyme activity lowered, requiring
longer digestion time

11. •Endonucleases (or restriction enzymes) are
enzymes which cut DNA at specific internal
recognition sequences
•Compare to exonucleases, which cut from one end
•You must choose restriction sites that are available
in the plasmid you are cloning into
•They must not appear in your gene (silent mutation
can remove unwanted sites in your designed gene)
Choice of Restriction Sites/Enzymes
Once you have your gene, you need to design
a way to get it into your plasmid

12. •Restriction sites must exist only once in your
plasmid
•They must be in the correct position relative to
the purification tag
•Restrictions sites usually add extra residues to
your gene product; make sure they are compatible
with your peptide/protein
•Some restriction sites are sub-optimal for cloning
•Blunt end sites
•dam and dcm methylation-affected enzymes
Really Important Factors to Remember
When Choosing Restriction Enzymes

13. dam Methylation
N
O
N
N
O
N
N
HO
P
P
O
O O
O
O
O
MeN
O
N
N
O
N
NH2
O
P
P
O
O O
O
O
O
Dam methylase
•Dam methylase puts a methyl group on the nitrogen of 6th
position of adenosine at the site: GATC
•All of the E. Coli that we use generate DNA with dam
methylation
•Some enzymes only cut dam methylated DNA: eg DpnI
•Some enzymes do not cut dam methylated DNA: eg XbaI
http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/dam_dcm_methylases_of_ecoli.asp

14. dcm Methylation
http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/dam_dcm_methylases_of_ecoli.asp
Dcm methylase
O
P
O
O O
O N
N
NH2
O
O
P
O
O O
Me
O
P
O
O O
O N
N
NH2
O
O
P
O
O O
•Dcm methylase puts a methyl group on the carbon of 5th
position of cytidine at the site: CCAGG and CCTGG
•The enzyme we use most that can be affected by dcm
methylation is SfiI
•XL1-Blues and BL21s are both Dcm
+

15. Application 1: Restriction Analysis
• Using restriction enzymes to find out
information about a piece of DNA
• We can use restriction enzymes to find out
– The size of a plasmid
– If there are any restriction sites for a particular
enzyme on a piece of DNA (ex. EcoRI)
– How many restriction sites for a particular enzyme
– Where the restriction sites are located

16. •Once you have your restriction enzymes chosen, it is time
to design the final complete gene
•The multiple cloning site (or whatever plasmid you are
cloning into) should already have the 5’ portion of the gene
intact (i.e. RBS, spacer, Met)
• Sequences must be in frame
NcoI
BtgI
51 CTTTAATAAG GAGATATACC ATGGGCAGCA GCCATCACCA TCATCACCAC
M G S S H H H H H H
SacI AscI SbfI SalI NotI
BamHI EcoRI EcoICRI BssHII PstI AccI HindIII
101AGCCAGGATC CGAATTCGAG CTCGGCGCGC CTGCAGGTCG ACAAGCTTGC
S Q D P N S S S A R L Q V D K L A
Application 2: Design of insert

17. Design of the Insert
71 ATGGGCAGCAGCCATCACCATCATCACCAC
M G S S H H H H H H
SacI AscI SbfI SalI
BamHI EcoRI EcoICRI PstI AccI HindIII
101AGCCAGGATCCGAATTCGAGCTCGGCGCGCCTGCAGGTCGACAAGCTTGC
S Q D P N S S S A R L Q V D K L A
The gene we want:
ggctgcgacagggcgagcccgtactgcggttaa
G C D R A S P Y C G *
BamHI PstI
AGCCAGGATCCGAATTCGAGCTCGGCGCGCCTGCAGGTCGACAAGCTTGC
S Q D P N S S S A R L Q V D K L A
G C D R A S P Y C G *
ggctgcgacagggcgagcccgtactgcggttaa
AGCCAGGATCCGggctgcgacagggcgagcccgtactgcggttaaCTGCAGGTCGACAA
Be aware of the amber
stop codon: TAG
Multiple cloning site

18. Design of the Insert
Always check and re-check your sequence!
ATGGGCAGCA GCCATCACCA TCATCACCAC
AGCCAGGATCCGggctgcgacagggcgagcccgtactgcggttaaCTGCAGGTCGACAA
atgggcagcagccatcaccatcatcaccacagccaggatccgggctgcgacagggcgagc
M G S S H H H H H H S Q D P G C D R A S
ccgtactgcggttaactgcaggtcgacaa
P Y C G - L Q V D
Everything looks good: in frame the whole way!
Translate the whole gene

19. The wrong way to do it:
AGCCAGGATCC ggctgcgacagggcgagcccgtactgcggttaaCTGCAGGTCGACAAGCTT
atgggcagcagccatcaccatcatcaccacagccaggatccggctgcgacagggcgagcc
M G S S H H H H H H S Q D P A A T G R A
cgtactgcggttaactgcaggtcgacaagctt
R T A V N C R S T S
Frame shifted = garbage!
Design of the Insert
The gene is just inserted after the restriction site, which is
out of frame with the plasmid-encoded start-codon/His-
tag
**Some plasmids, for whatever reason, have restriction
sites out of frame with the translated gene**

20. Application 3: Restriction mapping
• A restriction map is a map of known restriction
sites within a sequence of DNA. Restriction
mapping requires the use of restriction enzymes.
In molecular biology, restriction maps are used
as a reference to engineer plasmids or other
relatively short pieces of DNA, and sometimes
for longer genomic DNA.
There are other ways of mapping features on DNA
for longer length DNA molecules, such as
mapping by Transduction (Bitner, Kuempel
1981).

21. Restriction mapping involves first cutting dsDNA
with restriction enzymes (A, B, or both A and B) to
produce predictably sized fragments:
A
B
A
A only: B only: A and B:

22. Constructing a restriction map for EcoRI and BamHI in a DNA
fragment

24. Application: RFLP Analysis:
•RF stands for Restriction Fragments. Those are the fragments that were
cut by restriction enzymes.
•L stands for Length, and refers to the length of the restriction fragment.
•P stands for Polymorphisms, a Greek term for “many shapes”. The
lengths of some of the restriction fragments differ greatly
between individuals.
RFLP = Restriction Fragment Length
Polymorphism
Molecular biologists have identified regions of the human genome where
restriction fragment lengths are highly variable between individuals.
Electrophoresis of these RFLP’s produce different patterns of DNA
bands. With 3 billion base pairs in the human genome, however, RFLP
analysis would
produce a ‘smear’ of many similar sized fragments.