2/24/17

Molecular Biology
BIOL2164

Restriction
Endonucleases

GCB2017 http://www.planet-science.com/categories/over-11s/natural-world/2012/08/molecular-scissors.aspx

Lecture Objectives:
Restriction Endonucleases

Students should be able to:

1. Describe Restriction Endonucleases

2. Understand their biological role
3. Understand their nomenclature and classification
4. Describe their general mode of action/operation
5. Understand the importance of REs to R-DNA Technology

GCB2017

1
 2/24/17

Restriction Endonucleases

Also called restriction enzymes

1962: molecular scissors discovered in bacteria
E. coli bacteria have an enzymatic immune system that
recognizes and destroys foreign DNA

.~ 3,000 enzymes identified

~ 200 with unique properties
~ 100s purified and available
commercially

GCB2017

DNA Degradation via Nucleases

Degradation:
DNA nucleases (DNAse)
RNA nucleases (RNAse)

Nucleases
endonuclease (within ss or
ds fragments)
exonuclease (ends of ss
fragments; direction specific)

GCB2017

2
 2/24/17

Restriction
Endonucleases
E. coli bacteria have an enzymatic immune
system that recognizes and destroys foreign DNA

Why dont bacteria

destroy their own DNA
with their restriction
enzymes?

GCB2017

Bacterial Defense from RE

Restriction Modification System

restriction enzymes paired with methylases (methyltransferases).

DNA Methyltransferases add methyl groups to specific

nucleotides within the recognition sequence (on each strand).

The methylation prevents recognition by the restriction enzyme.

Host DNA is protected, but foreign (invading DNA) is not, and is

degraded.

GCB2017

3
 2/24/17

The host DNA is uncut

Methylation protects target sites

Enzyme Site Sequence

EcoRV 5-'GA*TATC-3

EcoRI 5-GAA*TTC*-3'

HindIII 5'-A*AGC*TT-3'

XbaI 5'-TC*TAGA*-3'

An enzymatic immune system that recognizes and destroys foreign DNA

GCB2017

Restriction Endonucleases -
three types : I, II and III
Restriction endonucleases are categorized into
three general groups (Types I, II and III) based on:

Composition
Enzyme co-factor requirements
Nature of target sequence
Position of DNA cleavage site relative to target
sequence

4
 2/24/17

Restriction Endonucleases - three

types I, II and III

1. Restriction activity and modifying activity are in separate enzymes

2. Restriction endonucleases cleave within the recognition site or adjacent to it.
3. Do not need ATP for activity. Needs only Mg++
4. Cuts in a precise manner, very predictable
5. Short palindromic recognition sequence

Restriction Endonucleases - three types

type I, II and III

Question: Which of these endonucleases is best for use in

Molecular Biology?

GCB2017

5
 2/24/17

Type II. Best for R-DNA

Most useful for gene analysis and cloning

More than 3500 REs
Recognize 4-8 bp sequences
Need Mg 2+ as cofactor
Cut in close proximity of the recognition site
Homodimers
ATP hydrolysis is not required

Restriction Endonucleases:
Nomenclature
eg. BamH1
Bacillus amyloliquefaciens Strain H
First enzyme characterised

eg. HindIII
Haemophilus influenzae Strain D - 3rd enzyme
Named for bacterial genus, species, strain, and type

eg. EcoRI Example: EcoR1

Genus: Escherichia
Escherichia coli Strain R 1st enzyme Species: coli
discovered Strain: R
Order discovered: 1
GCB2017

6
 2/24/17

Classification of Restriction
Endonucleases
1. Sticky - end cutter vs blunt-end cutter

2. Frequent cutter vs rare cutter

Recognition sequence Cleavage frequency

How4frequently
bp (Alu1, HpaII,
does eachHhaI) 44 = 256
of the above restriction enzymes cut DNA?
-There are 4 different bases, so the probability of finding a particular base at one
5 bpon(Hga1)
location a DNA strand = . 45 = 1024
-The6probability
bp (BamH1, HinD111)
of finding the required base4at
6= 4096
each of n locations = ()n (n =
length of recognition sequence).
7 bp (PpuM1) 47 = 16,384
-frequency of sites = 4n

-44 =8256,
bp 4(Not1) 9 bp
6 = 4,096, 48 = (AlwN1)
65,536. 10 bp

Sticky End Cutters

Most restriction enzymes make staggered cuts

Staggered cuts produce single stranded
sticky-ends
DNA from different sources can be spliced
easily because of sticky-end overhangs.
5 P
- OH -
3
HindIII
5
-P
H-
3 O

EcoRI

7
 2/24/17

Blunt End Cutters

Some restriction enzymes cut DNA at the opposite base

They leave blunt ended DNA fragments

These are called blunt end cutters

AluI

HaeIII

GCB2017

8
 2/24/17

Classification of
Restriction
Endonucleases
3. Ambiguous vs precise cutters

eg. BsiE1

CGPuPyCG
GCPyPuGC

Classification of Restriction
Endonucleases
4. Isoshizomers

Some enzymes have the same target site (recognition site)

Two subtypes:

Same target site different cleavage site

eg. Sma1 and Xma1 Different REs can produce compatible ends

Note:
Same target site - same cleavage site
There are some Restriction endonucleases that have different
eg. Aat1 and Stu1 target sites, produce compatible cohesive ends.

BamH1 and Bst1 eg. BamH1, Bgl111, Mbo1, Sau3A1, DpnII, BstY1

9
 2/24/17

Restriction enzyme
animation

http://www.dnai.org/b/index.html

GCB2017

Restriction Endonucleases:
Mode of Action
Scanning
GGACGCTAGCTGATGAATTCGCATCGGATCCGAATCCGCTCTTTCAA
CCTGCGATCGACTACTTAAGCGTAGCCTAGGCTTAGGCGAGAAAGTT

Recognition Sequence
GGACGCTAGCTGATGAATTCGCATCGGATCCGAATCCGCTCTTTCAA
CCTGCGATCGACTACTTAAGCGTAGCCTAGGCTTAGGCGAGAAAGTT

Cleavage
GGACGCTAGCTGATG AATTCGCATCGGATCCGAATCCGCTCTTTCAA
CCTGCGATCGACTACTTAA GCGTAGCCTAGGCTTAGGCGAGAAAGTT

GCB2017

10
 2/24/17

RE: Mode of action

Enzymes recognize specific 4-8 bp sequences

Some enzymes cut in a staggered fashion - sticky ends

EcoRI 5GAATTC3 Look at the recognition

Sequences..
3CTTAAG5

Recognize anything??
Some enzymes cut in a direct fashion blunt ends

PvuII 5CAGCTG3

3GTCGAC5
GCB2017

Restriction Endonucleases

Recognition sites have symmetry (palindromic)

ABLE WAS I, ERE, I SAW ELBA

5-GGATCC-3
Bam H1 site:
3-CCTAGG-5

GCB2017

11
 2/24/17

Restriction Endonucleases Uses

Discovery of enzymes that cut and paste DNA make

genetic engineering possible.

Restriction enzyme cuts DNA and generates

fragments

Ligase joins different DNA fragments

DNA fragments from different species can be

ligated (joined) to create Recombinant DNA

handout

Molecular Cloning
Basic process of cloning a gene (from a chromosome)

1)Isolate gene of interest PCR/ Restriction digest

GCB2017

12
 2/24/17

Importance to Cloning
Strategy

Cohesive end cutter or sticky end cutter

GCB2017

Restriction Enzymes for R-DNA

Human DNA cleaved with EcoRI Corn DNA cleaved with EcoRI
5-C-G-G-T-A-C-T-A-G-OH
3-G-C-C-A-T-G-A-T-C-T-T-A-A-PO4
+ PO4-A-A-T-T-C-A-G-C-T-A-C-G-3
HO-G-T-C-G-A-T-G-C-5

Complementary base
pairing
5-A-C-G-G-T-A-C-T-A-G A-A-T-T-C-A-G-C-T-A-C-G-3
3-T-G-C-C-A-T-G-A-T-C-T-T-A-A G-T-C-G-A-T-G-C-5

+ DNA Ligase, + rATP

5-A-C-G-G-T-A-C-T-A-G-A-A-T-T-C-A-G-C-T-A-C-G-3
3-T-G-C-C-A-T-G-A-T-C-T-T-A-A-G-T-C-G-A-T-G-C-5

recombinant DNA (R-DNA)molecule

GCB2017

13
 2/24/17

Step 1: RE Digestion

EcoR I EcoR EcoR

I I EcoR I

Problem: Genomic DNA too large (quantity)

Solution: Smaller fragments

Restriction Enzymes and Gel

Electrophoresis
_
DNA is negatively
charged from the
phosphate backbone
+

Visualize DNA with ethidium

bromide fluoresces ONLY
when bound to DNA

GCB2017

14
 2/24/17

Step 2. Gel Electrophoresis

_ +

Step 2. Gel Electrophoresis

digested

undigested

_ +

15
 2/24/17

After Restriction Digestion of

Genomic DNA Digest Plasmid DNA Digest

Restriction digestion:
Commercial restriction enzymes

1. Restriction
Restriction digestion:
enzymes are commercially available at 5 units per
L Commercial
concentration.restriction enzymes

2.1. unit = 1enzymes

IRestriction unit (U)
areis the amount
commercially if enzyme
available at 5 units that
per catalyses the
reaction of 1 nmol of substrate per minute under standard
L concentration.

conditions.
2. I unit = 1 unit (U) is the amount if enzyme that catalyses the
reaction of 1 nmol of substrate per minute under standard
conditions.
3. Enzymes come in storage buffer containing EDTA, DTT, BSA
3. and Glycerol
Enzymes come in storage buffer containing EDTA, DTT, BSA
and Glycerol

4.4. Too much enzyme in the reaction mix can cause star activity
Too much enzyme in the reaction mix can cause star activity

16
 2/24/17

Star activity

Loss of specificity of the restriction enzyme (typeII) under

extreme non-standard conditions is called star activity.

Under such conditions, the restriction enzyme may cleave similar

but non identical target sequences.

Non-standard conditions include:

1. high glycerol concentration >5% v/v
2. >100 units per ug of DNA
3. Low ionic strength buffer <25mM (usually is 100-150 mM)
4. high pH (> 8.0) usually pH is around 7
5. presence of organic solvents, DMSO, ethanol, PEG,
dimethyl acetamide
6. Cations other than Mg++ eg. Cu++, Co++,Zn++, Mn++

Typical Restriction Digest

Sterile, deionized water 16.3 l

RE 10X Buffer 2.0 l
Acetylated BSA, 10g/l 0.2 l
DNA, 1g/l 1.0 l
Mix by pipetting, then add:
Restriction Enzyme, 10u/l 0.5 l
Final volume 20.0 l

17
 2/24/17

Restriction digestion:
Stopping the Restriction digestion

1. Heat inactivation of restriction enzymes

Place reaction mix in a waterbath set at 65oC
Note: not all Restriction enzymes can be heat inactivated. eg.
Pvu1, Bgl II, BamH1, Acc1

2. Add 2ul of 25 mM EDTA

EDTA chelates the Mg++ and makes it unavailable to the
Restriction Endonuclease.

3. Phenol-chloroform extraction/ purification

Removes all proteins, including the enzymes.

Digestions
Fragment is 4kbp long

EcoRI (150bp) BamHI (500bp) EcoRI (1.5kbp) NcoI (3 kbp)

Single Digests:

EcoRI BamHI NcoI

Number of
3 2 2
fragments

150bp
Size of 500bp 3kb
1.35kpb
fragments 3.5kbp 1kb
2.5kbp

18
 2/24/17

Digestions

Undigested
EcoRI BamHI NcoI

BamHI
Ladder

EcoRI

NcoI
Number
of 3 2 2
fragments
150bp
Size of 500bp 3kb
1.35kpb
fragments 3.5kbp 1kb 4kbp
2.5kbp

3kbp

2kbp

1kbp

0.5kbp

0.25kbp

Digestions
Fragment is 4kbp long

EcoRI (150bp) BamHI (500bp) EcoRI (1.5kbp) NcoI (3 kbp)

Double Digests:

EcoRI/BamHI EcoRI/NcoI BamHI/NcoI

Number of
4 4 3
fragments

150bp 150bp
500bp
Size of 350bp 1.35kbp
2.5kbp
fragments 1kbp 1.5kpb
1kbp
2.5kbp 1kbp

19
 2/24/17

Digestions
EcoRI/BamHI EcoRI/NcoI BamHI/NcoI

Undigested
Number of
4 4 3

BamHI/
fragments

EcoRI/

EcoRI/
BamHI
Ladder

NcoI

NcoI
150bp 150bp
500bp
Size of 350bp 1.35kbp
2.5kbp
fragments 1kbp 1.5kpb
1kbp
2.5kbp 1kbp

4kbp

3kbp

2kbp

1kbp

0.5kbp

0.25kbp

handout

Molecular Cloning Strategies

Basic process of cloning a gene (from a chromosome)

1) Isolate gene of interest

2) Construct a recombinant DNA (R-DNA) molecule

3) Introduce R-DNA into a host bacterium (copies)

4) Identify and isolate (pure culture) cells carrying

recombinant DNA (selection)

20
 2/24/17

Elements of Recombinant-
DNA technology
METHODS
Vector
DNA fragment
n Restriction Digestion AmpR
P tetR

n Dephosphorylation
P P

n DNA ligation
DNA ligase P
n Preparation of E. coli competent cells

n Transformation Cut with restriction

endonuclease

n Selection / Screening of colonies

- Recombinant DNA
- Recombinant vector
- DNA chimera

handout

Molecular Cloning

Basic process of cloning a gene (from a chromosome)

1)Isolate gene of interest PCR/ Restriction digest

21
 2/24/17

EcoR1 EcoR1

TTCGTCGAATTCGTTATGCGAATTCTGCATAATGGTC

EcoR1
TTCGTCGAATTCGTTATGCTAATTCTGCATAATGGTC

(EcoR1 site destroyed)

GCB2017

GCB2017

22
 2/24/17

45

SNPs, RFLPs, point mutations

GAATTC GAATTC GAATTC GAATTC GAATTC GAATTC

GACTTC
GAATTC GAATTC GAGTTC GAATTC GAATTC RFLP
RFLP Pt mut
SNP SNP
Pt mut
SNP SNP

GCB2017

23