2
Application of Restriction Enzymes
1
2.1
Detection of DNA Fragments
Separation of DNA fragments occurs in
Agarose gels (0.3 – 2%): 300 bp – >1 Mbp
Polyacrylamide gels: 1 – 1000 bp
2
Agarose
Solubilization at 92°C
Solidifying at 42°C
3
Low-melting point agarose:
Solubilization at 62°C
Solidifying at 25°C
Allows incubation of melted agarose with
most enzymes
Low-melting point agarose is purified from
agarose
4
Preparation of Polyacrylamide
5
Electrophoresis Through a Gel
Separates DNA and RNA Molecular
According To Size
 Pores in the gel matrix sieve
the DNA molecules according
to their volume
 The large molecules move
slower
 After a given time, molecule
of different size are separated
because they have moved
different distances
6
 Circular<linear<supercoiled
Electrophoretic
Separation of Linear
DNA Fragments
How to visualize the DNA
fragments ?
Molecular weight marker
7
Ethidium Bromide
Intrinsic fluorescence: weak
Fluorescence after intercalation: strong
Detection limit: ~ 5 ng DNA per band
Irradiation at 254 nm or 320 nm (1/10)
Energy is re-emitted at 590 nm
Works also with ssDNA and RNA (1/10)
8
Electrophoretic Migration Pattern of
Double-Stranded DNA
9
2.2
Pulse-Field Gel Electrophoresis
(PFGE)
Separation of large DNA fragments: 20 kb - >1
Mbp
Principle:
Alternating electric fields with different pulse
lengths
10
Electrode Configuration of FIGE (Field
Inversion)
11
Electrode Configuration of PFGE (PulsedField Gel Electrophoresis)
12
Electrode Configuration of CHEF (ContourClamped Homogeneous Electric Field)
13
Electrode Configuration of OFAGE
(Orthogenal-Field Alternation)
14
Separation of the 16 Yeast
Chromosomes
15
2.3
Establishment of Restriction
Maps
16
2.3.1
The Smith-Birnstiel Method
17
The Different Steps of the SmithBirnstiel Procedure:
1. Identification of a unique restriction site
within the recombinant plasmid (e.g., for a homing
endonuclease)
2. Linearization of the recombinant plasmid
3. Radioactive labeling at one or both ends
4. If labeled at both ends, cleavage with a second
enzyme, separation of the two fragments
5. Partial digestion, separation of the fragments,
18
autoradiography
The Smith-Birnstiel Mapping
Procedure
partial digestion
autoradiography
19
2.3.2
Restriction Maps of
Recombinant λ Phages and
Cosmids
20
Principle of Restriction Fragment Mapping
within Recombinant λ D NA Molecules
cos
21
2.4
Cloning of Restriction Fragments
22
Principle of DNA Cloning
23
2.5
Cloning Without Restriction
Enzymes and DNA Ligase
24
Seamless Cloning and Gene Fusion
Definition:
Processes that allow two or more DNA
fragments to be joined precisely so that no
unwanted nucleotides are added at the
junctions between DNA fragments
Seamless cloning does not require neither
restriction enzymes nor DNA ligase
Q Lu (2005) Trends Biotechnol. 23: 199
25
The In-Fusion Cloning Protocol
http://www.clontech.com/products/detail.a
26
sp?product_id=162275&tabno=2
The Gateway Cloning System
Cloning is independent of restriction enzymes
and DNA ligase
att: core recombination
site is 21 bp
ccdB: toxin; inactivates
DNA gyrase
ccdA: antidot
27
D Esposito (2009) Methods Mol. Biol. 498: 31
Cloning via Direct Transformation of a
PCR Product
40 – 50 bp
overlap
termini
C You (2012) Appl Environ Micobiol 28: 272
2.6
Cleavage of DNA at Any
Sequence
Objective:
To cleave DNA at any predetermined sequence
Methods:
1.ZFN DNA cleavage
2.TALEN DNA cleavage
3.crRNA-guided DNA cleavage
29
ZNF DNA Cleavage
Components:
1.Zinc finger proteins (ZFPs): recognize target
DNA sequences; 3-nt motif
2.FokI nuclease domain
30
TALEN DNA Cleavage
31
crRNA-Guided DNA Cleavage
32