Enzymolysis of DNA
Exp. 3: Enzymolysis of DNA
【Objective】
1. To learn and understand the concepts, principles and the characteristics of
restriction enzymes.
2. To become familiar with the experimental procedures of DNA enzymolysis.
3. To become able to cleave DNA chains with restriction enzymes and analyze
the DNA fragments.
【Principle】
A restriction enzyme is an enzyme that catalyzes the cleavage of
double-stranded DNA at specific sites to produce discrete fragments. Restriction
enzyme is also called as restriction endonuclease. The DNA fragments cleaved by
restriction enzymes can be rejoined by enzymes known as ligases, provided the
base sequences of their ends are complementary. Therefore, many techniques in
molecular biology and genetic engineering rely on restriction enzymes.
The term restriction comes from the fact that these enzymes were discovered in
E.coli strains that appears to restrict the infection by certain bacteriophages.
Restriction enzymes, therefore, are believed to be involved in bacteria to resist viral
attack and to help in the removal of viral sequence.
Rather than cutting DNA indiscriminately, restriction enzymes cleave DNA
double-helical strands that contain particular nucleotide sequences, meaning that
the restriction enzymes will make incisions only within that sequences known as
“recognition sequences”.
Recognition sequences are usually four to eight nucleotides long. Many
recognition sequences are palindromic, that is, the sequence on one strand reads the
same in the opposite direction on the complementary strand. The meaning of
“palindromic” in this context is different from what one might expect from its
linguistic usage: GTAATG is not a palindromic DNA sequence, but GTATAC is.
Restriction enzymes are named based on the bacteria in which they are isolated
in the following manner. For example: EcoR I
E:
Escherichia (genus)
co:
coli (species)
R:
RY13 (strain)
I:
the first restriction enzyme identified in bacterium
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Enzymolysis of DNA
Examples
Enzyme
Source
Restriction Sequence
Cut
Hind III
Haemophilus influenzae
5’ AAGCTT 3’
3’ TTCGAA 5’
5’ A
3’ TTCGA
AGCTT 3’
A 5’
Pst I
Providencia stuartii
5’ CTGCAG 3’
3’GACGTC 5’
5’ CTGCA
3’ G
G 3’
ACGTC 5’
Alu I
Arthrobacter luteus
5’ AGCT 3’
3’ TCGA 5’
5’ AG
3’ TC
CT 3’
GA 5’
Restriction enzymes are classified biochemically into three types, designated
Type I, Type II and Type III. Type I and III systems, in general, are a single large
enzyme complex of methylase and restriction cutting functions. Although these
enzymes recognize specific DNA sequences, the sites of actual cleavage are from
these recognition sites with variable distances, and can be hundreds of bases away.
In type II systems, the restriction enzyme is independent of its methylase, and
cleavages occur at very specific sites that are within or close to the recognition
sequence. The majority of known restriction enzymes are type II, which
demonstrates the most versatile uses as laboratory tools.
The restriction enzymes most used in molecular biology labs cut within their
recognition sites and generate one of three different types of ends. In the diagrams
below, the recognition site is boxed.

5’overhangs:
5’-A-T-G-G-A-T-C-C-A-A-3’
3’-T-A-C-C-T-A-G-G-T-T-5’

5’-ATG
3’-TACCTAG’
GATCCAA-3’
GTT-5’
3’overhangs:
5’-G-A-G-G-T-A-C-C-C-T-3’
3’-C-T-C-C-A-T-G-G-G-A-5’

Bam H1
Kpn Ⅰ
5’-GAGGTAC
3’-CTC
CCT-3’
CATGGGA- 5’
Bluts
5’-T-A-C-C-C-G-G-G-T-C-3’
3’-A-T-G-G-G-C-C-C-A-G-5’
Sma Ⅰ
5’-TACCC
3’-ATGGG
GGGTC-3’
CCCAG-5’
The 5’or 3’ overhangs generated asymmetrically by enzymes are called sticky
ends or cohesive ends, because they readily stick or anneal with their partner by
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Enzymolysis of DNA
base pairing.
【Experimental Materials】
1. λDNA (Lamda DNA) (0.38μg/μl)
2. Hind III: 16u/μl, XHo Ⅰ
3. Enzymolysis buffer (Tris-HCl, NaCl, MgCl2, DTT)
4. Stopping solution (Bromophenol blue 1%, SDS 1%, EDTA 2%, glycerol 50%)
【Procedure】
1. Prepare 4 eppendorf tubes. Add reagents listed in the table below into each
tube.
Volume: μl
λDNA
Hind III
XHo I
ddH2O
2.
3.
4.
5.
Tube 1
Tube 2
Tube 3
Tube 4
10
/
/
10
10
5
/
5
10
/
5
5
10
5
5
/
Close the cap of each tube tightly. Mix well using vertex and centrifuge the
tube.
【Note: The centrifugation is very important. The total amount of
sample is very small, vertex can cause sample to stick on the inter wall of
the tube.】
Rest these eppendorf tubes onto a tube rack. Incubate the rack into a water
bath at 37℃ for 2 hours.
Take out the tubes from the water bath. Add 5μl of stopping solution in each
tube.
Run electrophoresis to analyze the DNA fragments (See next).
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