Restriction Enzymes
Lecture 15: 1
11/20/2006
1. Definition: enzymes that recognize specific double-stranded sequences and
hydrolyze the phosphodiester bonds on both strands.
2. How many REs have been known? ~2500
3. How is the activity of a RE defined?
The mount of the enzyme required to digest one microgram of lamda DNA in
one hour at the optimum conditions for the enzyme.
4. How many types of RES? 3.
Type I and III REs have both endonuclease and methylase activities, cutting a
random sequence away from the recognition sequence.
Type II REs carry endonuclease activities. They cleave a specific sequence site
within the recognition site.
5. Palindromes are common in the recognition sites of REs.
EcoRI
5’-GAATTC-3’
3’-CTTAAG-5’
6. The rules for naming REs
EcoRI is the first (I)restriction enzyme isoltated from Escherichia coli RY 13 strain.
Restriction Enzymes
7. Frequencies of Restriction Sites in a given sequence
4 nucleotides: G, A, T, C. If the recognition sequence is 1
nucleotide long, then a sequence of 4 nucleotides should contain
one site of the RE. 41=4. G, A, T, and C.
How about 2: 42=16
G
A
T
C
G
GG GA
GT
GC
A
AG AA
TT
AC
T
TG TA
TT
TC
C
CC CA
CT CC
Let’s try 3: 43=64 (triplet codons)
GG
GA
GT
GC
AG
AA TT
AC
G GGG GGA GGT GGC GAG GAA GTT GAC
A AGG AGA AGT AGC AGA AAA ATT AAC
T TGG TGA TGT TGC TAG TAA TTT TAC
C CGG CGA CGT CGC CAG CAA CTT CAC
TG
TA
TT
TC
CC
CA
CT
CC
G GTG GTA GTT GTC GCC GCA GCT GCC
A ATG ATA ATT ATC ACC ACA ACT ACC
T TTG TTA TTT TTC TCC TCA TCT TCC
C CTG CTA CTT CTC CCC CCA CCT CCC
Lecture 15: 2
11/20/2006
Restriction Enzymes
Let’s try 3: 43=64 (triplet codons)
GG
GA
GT
GC
AG
AA TT
AC
G GGG GGA GGT GGC GAG GAA GTT GAC 1
A AGG AGA AGT AGC AGA AAA ATT AAC
2
T TGG TGA TGT TGC TAG TAA TTT TAC
3
C CGG CGA CGT CGC CAG CAA CTT CAC 4
TG
TA
TT
TC
CC
CA
CT
CC
G GTG GTA GTT GTC GCC GCA GCT GCC 5
A ATG ATA ATT ATC ACC ACA ACT ACC
6
T TTG TTA TTT TTC TCC TCA TCT TCC
7
C CTG CTA CTT CTC CCC CCA CCT CCC
8
For the 4-base cutters: 44=256 (=32X8=256)
The smallest sequence recognized by any REs is at least 4
nucleotides long.
GGG GGA GGT GGC GAG GAA GTT GAC 1
G GGGG GGGA GGGT GGGC GGTG GGAA GGTT GGAC
A AGGG AGGA AGGT AGGC AGAG AGAA AGTT AGAC
T TGGG TGGA TGGT TGGC TGAG TGAA TGTT TGAC
C CGGG CGGA CGGT CGGC GAG CGAA CGTT CGAC
………, For a sequence of 256 nts, there is going to be at least
one site for a 4 base recognition RE.
Lecture 15: 3
11/20/2006
Restriction Enzymes
Lecture 15: 4
11/20/2006
Useful 4 cutters:
1. Sau3A: 5’…..GATC…...3’
3’…..CTAG…...5’
Isolated from Staphylococcus aureus 3A
2. MboI
MboI: 5’…..GATC…...3’
3’….CTAG…...5’
Isolated from Moraxella bovis
Isoschizomers: REs that recognize the same sequence are isochizomers.
Sau3A and MboI are used to produce a partial digestion of a complex DNA in creating genomic DNA
libraries.
3. Dpn I
5’…GmA TC…3
3’…C TmAG…3
Dpn I is often used in site-direted mutagenesis.
For 6-cutter REs, 46 = 4096 nts
These REs are frequently used to clone the genes into plasmids.
For 8-cutter RE, 48=65,536 nts
Used at the flanking sites of cloning.
Not I: 5’…..GCGGCCGC……3’
3’…..CGCCGGCG……5’
Restriction Enzymes
For 8-cutter RE, 48=65,536 nts
Used at the flanking sites of cloning.
Not I: 5’…..GCGGCCGC……3’
3’…..CGCCGGCG……5’
From Nocardia otitidis-cavarun
Asc I: 5’….GGCGCGCC……3’
3’…..CCGCGCGG……5’
8. Three types of ends are produced by RE digest:
1)Blunt ends:
5’-CCCGGG-3’
3’-GGGCCC-5’
Sma I, Serratia marcescens
5’-GATATC-3’
3’-CTATAG-5’
EcoR V, Escherichia coli
2) Sticky ends (5’-protruding or overhang)
5’-GGATCC-3’
3’-CCTAGG-5’
BamH I, Bacillus amyolquefaciens H
3) Sticky ends (3’-protruding or overhang)
5’-GGTACC-3’
3’-CCTTGG-5’
Kpn I: Klebsiella pneumoniae
Lecture 15: 5
11/20/2006
Restriction Enzymes
Lecture 15: 6
11/20/2006
9. Sticky ends can be converted into blunt ends or partially filled in:
5’-GGATCC-3’
3’-CCTAGG-5’
BamH I
5’-G-3’
Fill-in buffer w/dGTP, dATP, dTTP, and dCTP, Klenow
3’-CCTAG-5’
5’-GGATC-3’
3’-CCTAG-5
5’-AAGCTT-3’
3’-TTCGAA-5’ Hind III
5’-A-3’
Fill-in buffer w/dGTP, dATP, and [α-32P]dCTP, Klenow
3’-TTCGA-5’
5’-AAG*C-3’
3’-TTCGA-5’
Restriction Enzymes
Lecture 15: 7
11/20/2006
T7 DNA Polymerase can convert 5’-overahng or 3’ overhang into
blunt ends due to its DNA-dependent DNA polymerase and 3’exonuclease (stronger than that of Klenow).
5’-GGTACC-3’
3’-CCTTGG-5’
5’-GGTAC-3’
3’-C-5’
Kpn I
T7 DNA polymerase
5’-GGTAC-3’
3’-C-5’
5’-G-3’
3’-C-5’
The DNA polymerase of T7 bacteriophage has DNA polymerase and 3' -> 5'
exonuclease activities, but lacks a 5' -> 3' exonuclease domain. It is thus very similar in
activity to Klenow fragment and T4 DNA polymerase.
The claim to fame for T7 DNA polymerase is it's processivity. That is to say, the
average length of DNA synthesized before the enzyme dissociates from the template is
considerably greater than for other enzymes. Due to this talent, the principle use of T7
DNA polymerase is in DNA sequencing by the chain termination technique.
T7 DNA polymerase can be chemically-treated or genetically engineered to abolish it's
3' -> 5' exonuclease activity. These forms of the enzyme are marketed under the name
Sequenase and Sequenase 2.0, and are widely used for DNA sequencing reactions.
Restriction Enzymes
Lecture 15: 8
11/20/2006
10. Activities of some restriction enzymes are methylationdependent.
CH3
5’……GATC……...3’
5’……CTAG……...3’
CH3
This property is exploited to develop a site-directed mutagenesis
(Quickchange Site-Directed Mutagenesis kit from Stratagene).
(See transparency)
11. RFLP and RAPD
Restriction Fragment Length Polymorphism (RFLP)
Restriction Fragment Length Polymorphism (RFLP) is a technique
in which organisms may be differentiated by analysis of patterns
derived from cleavage of their DNA. If two organisms differ in the
distance between sites of cleavage of a particular restriction
endonuclease, the length of the fragments produced will differ when
the DNA is digested with a restriction enzyme. The similarity of the
patterns generated can be used to differentiate species (and even
strains) from one another.
cystic fibrosis (CF)