Comment for any topic requirement regarding lifesciences [or] for any suggestions
APPGCET - 2022 all about 2nd councelling Click here
Restriction endonucleases
[or]
restriction enzymes
[or]
molecular scissors
---Restriction endonucleases these name ends with a suffix ASE which means this an enzyme.
---Endonucleases are proteinitious enzymes
---enzymes are worked as catalysts. We all know about catalysts that they enhance the rate of reaction and reduce time taken for a successive reaction
--- for example all the living organisms are blessed with a life. Right?
But most us ( humans) lead life in routine manner just like a normal chemical reaction.
--- when we add some goals, aims, aspirations to our life, just like a catalyst to reaction this will enhance the efficiency of our life.
Enzymes with somewhat similar activity of restriction endonucleases
Enzymes those perform degradation of DNA similar to endonucleases are as follows
1. S1 nucleases
2 . Exonucleases
S1 nucleases act upon only single standard genetic materials without out any recognition and accuracy.
Exonucleases also removes nucleotides from the single standard DNA. Exonuclease is a co enzymes of DNA POLYMERASE 1. which removes only 1 to few nucleotides, starts its activity either from 5' or from 3' end, not from both ends.
RestrictionEndonucleases
History
---once upon a time, before the discovery of R.Eases a technique named SONICATION is used to agitate the DNA.
Restriction enzymes, a Revolutionary discovery in molecular biology.
Why they are called so??
A name RESTRICTION is given because of their ability of restricting (host restriction and modification phenomenon)
restriction enzymes are found in bacterium. This enzymes acts as defense system, just like our immune system
--- we are infected by bacteria where as bacteria are infected by bacteriophage.
( virus ) when a bacteriophage invade on bacteria and injects its genetic material into the bacterium these restrication enzymes acts as defensive barriers. R.E cuts the viral dna and meanwhile protects own genetic material by methylating ( adding methyle group) specific or recognition sequence.
This idea of restriction enzymes started as a hypothesis by Werner Arber who noticed that certain bacterial strains fought off bacteriophage infection by chopping off its DNA. But bacterial DNA remains unchanged why?
Arber and meselson hypothesized that bacterial cells produce two types of enzymes: one called a “restriction” enzyme that can identify and cut foreign DNA, and a “modification” enzyme that recognizes the host DNA and protects it from cleavage. This hypothesis was proven in an experiment in which two enzymes were isolated from E. coli. The modification enzyme, methylase, protected DNA of the bacterium, while the restriction enzyme chopped off phage (non-methylated) DNA.
Chronological order of discovery:
1969 Werner Arber and Stewart Linn discovered that most
bacteria contained a class of enzymes which modify the bases in
DNA termed methylases.
The phenomena of restriction and modification were illustrated by experimenting with bacteriophage λ on two E. coli host strains. C and K
In 1970, Hamilton O. Smith, Thomas Kelly and Kent Wilcox isolated
and characterized the first type II restriction enzyme, HindII, from
the bacterium Haemophilus influenzae.
In 1972 Daniel Nathans and Kathleen Danna showed that cleavage of SV40
DNA by restriction enzymes yielded specific fragments which can
be separated using polyacrylamide gel electrophoresis, thus
showing that restriction enzymes cut at specific sites and can be
used for mapping of the DNA.
Arber discovered restriction enzymes.
Smith verified Arber's hypothesis.
Nathans pioneered the applications of restriction enzymes
1n 1978 three of these collectively awarded with noble prize.
Although primarily found in bacterial genomes
and plasmids, restriction endonucleases also exist
in archaea, viruses, and eukaryotes.
As manimum as 7 and maximum of 14 genes are found in bacteria, but some of them are inactive.
Including all types, there are 3500 restric-
tion enzymes found in 10000 speices that recognize 259 different DNA
sequences are now known.
Data of enzymes discovered are updated every month in follow website
www.neb.com
Host restriction modification phenomenon:
Restriction endonucleases were originally named for their ability to restrict the growth of phage in a host bacterial cell by cleavage of the invading DNA. The endonucleases recognize specific sites for action called Restriction sites.
In this manner, they may be acting as bacterial protection systems. The DNA of the host is protected from restriction by the activity of a methylase(s),which recognizes the same sequence as the restriction enzyme and methylates a specific nucleotide (4-methylcytosine, 5-methylcytosine, 5-hydroxymethylcytosine, or 6-methyladenine) on each strandwithin this sequence. Once methylated, the host DNA is no longer a substrate for the endonuclease.
Because both strands of the host DNA are methylated and even hemi-methylated DNA is protected,
freshly replicated host DNA is not digested by the endonucleases.
Digested protected
CH3
|
5' G| A A T T C 3' 5' G A A T T C 3'
|________
3' C T T A A |G 5' 3' C T T A A G 5'
|
CH3
Staggered cut methylated
EcoR1 modification
As so often in biology exceptions exists
Here in some cases instead of methyl group DNA is modified by inserting sulfur.
Recently discovered dnd system illustrate about the insertion of sulfur into the PHOSPHORUS backbone forms a PHOSPHOROTHIOATE group one of the non- bridging oxygen bonds in the phosphate backbone can be replaced by sulfur.
This type of modification are carried out by five types dnd's I.e., dnd A B C D E
or 4 ssp enzymes I.e., ssp A B C D
L- cysteine desulfarse provides sulfur atoms to dnd A, which is needed for modification.
dnd's have operons and other activities
This is a very vast Topic (dnd and ssp ) . if you want more data you may work on it by books
Nomenclature:
Restriction enzymes are named according to the proposal of Smith and Nathans. Enzymes are named with three letters in italics are derived from the
first letter of the genus and the first two letters of the microbial species from which the enzyme was
derived. An additional letter without italics may be used to designate a particular strain. This is followed by a roman numeral to signify the first, sec-
ond, and so on, enzyme discovered from the organism.
Enzyme Source Organism Recognition Sequence
HpaII Haemophilus parainfluenzae C/CGG GGC/C
MboI Moraxella bovis /GATC CTAG/
NdeII Neisseria denitrificans /GATC CTAG/
EcoRI Escherichia coli RY13 G/AATTC CTTAA/G
EcoRII Escherichia coli RY13 /CC(A or T)GGGG(T or A)CC/
EcoRV Escherichia coli J62/pGL74 GAT/ATC CTA/TAG
BamHI Bacillus amyloliquefaciens G/GATCC CCTAG/G
SauI Staphylococcus aureus CC/TNAGG GGANT/CC
BglI Bacillus globigii GCCNNNN/NGGC
CGGN/NNNNCCG
NotI Nocardia otitidis-caviarum GC/GGCCGC CGCCGG/CG
DraII Deinococcus radiophilus RG/GNCCY YCCNG/GR
/ = position where enzyme cuts; N = any base, R = any purine, Y = any pyrimidine
Isoschizomers
Different types of restriction enzymes isolated from different bacteria which
have same recognition sequence.
Ex. SphI (CGTAC/G) and BbuI (CGTAC/G) are isoschizomers of each
other
Neoschizomers
Different R. enzymes having same recognition sequence but cutting site (base) is not same.
Working of restriction enzymes in simply in 3 words
Finding కనిపెట్టుట recognition
Covering కాపాడుట methylation
Cutting కత్తిరించుట digestion
Strategies followed by restriction enzymes for recognition:
Cutting of DNA by Restriction Enzymes:
It might seem logical for the DNA to be cut at the recognition site where the restriction enzyme binds. This is often true, but not always. There are two major types of restriction enzyme that differ in where they cut the DNA, relative to the recognitionsite. How restriction enzymes find their binding sites in vast lengths of DNA is considered
When the restriction enzymes diffused to the target DNA, check the site of contact for a match to their recognition sequence, and released the DNA if it did not match. This procedure would take so long that invading viral DNA would be expressed and take over the cell before the restriction enzyme destroyed it.
Two major strategies have been proposed for proteins that must locate target sequences within long DNA molecules in a reasonable time. This applies not only to restriction and modification enzymes but to regulatory proteins that must locate promoters or other specific sites on DNA.
One way is for the protein to slide along the DNA strand without letting go.
The protein monitors the sequence rapidly, but without extreme accuracy, as it moves along the DNA strand. If a possible match is found, the enzyme stops and tests if the match is accurate. The alternative is hopping from one site on the DNA sequence to another that is usually reasonably close but may sometimes be far away . The enzyme does not usually lose contact with the DNA but relies on DNA looping to bring the sites together . After hopping, the protein will prescan the DNA by local sliding for a while before hopping again.
● ---- protein or enzyme reading DNA by sliding or by hopping
Classification of restriction enzymes:
Type 1
Type 2 (orthodox)
Type 3
Type 4
Type 1 enzyme :
It is the first identified enzyme in two strains of E. coli (K-12 and B).
Type I enzyme cuts at a site about 1000 bp away from the recognition site.
The recognition site is composed of two specific portions: one with 3 to 4 nucleotides and other with 4 to 5 nucleotides which is separated by 6 to 8 nucleotides which is non specific and this site is asymmetrical.
It posses both restriction digestion and modification methylase activities.
Their cofactor includes adenosine triphosphate (ATP), S-Adenosyl methionine (AdoMet) and magnesium (Mg2+).
Type I enzyme involves three subunits: HsdR for restriction digestion,
HsdM for adding methyl groups (methyltransferase activity) and
HsdS for recognition of DNA binding site (specificity).
Some of the examples of Type I enzymes are EcoB and EcoK.
Type 2 restriction enzyme :
the common Type II endonucleases
are homodimers (most between 25 and 35 kDa for
the monomeric subunit), require only Mg2-, and
cleave within palindromes, partial palindromes, or interrupted palindromes. Despite dissimilar primary sequence,
Type II enzyme cleave within a short specific distance of about 4 to 8 nucleotides in length and the sites are usually undivided.
They cleave the phosphodiester bond of DNA either as a blunt end or sticky end.
They do not use ATP or AdoMet but require Mg2+ as a cofactor.
Type II enzyme is a homodimeric enzyme and are most commonly used enzymes.
After cutting two types of ands are formed by the restriction enzymes.
1 . Sticky or overhang or cohesive ends
3' over hang
5' overhang
2 . Blunt ends
There are seven subtypes of type II enzyme with different characteristics:
Type IIS (Eg: Fok I)
Type IIE (Eg: Nae I)
Type IIF (Eg: NgoM IV)
Type IIT (Eg: Bpu10 I)
Type IIG (Eg: Eco 571)
Type IIB (Eg: Bcg I)
Type IIM (Eg: DPNI-RO)
Type IIS
Cleavage at a defined distance from the recognition site
Asymmetric recognition site
Fok I
Type IIE
Interact with two recognition sites
One serves as the allosteric effector and the other site is for cleavage
Nae I
Type IIF
Interact with two recognition sites
Both are required for cleavage
Homotetrameric enzyme
NgoM IV
Type IIT
Contain different subunits with restriction and modification activities
Bpu10 I
Type IIG
Comprised of a single polypeptide chain with restriction and modification activity
Eco571
Type IIB
Cleave both sides of the recognition site
Bcg I
Type IIM
Recognize methylated sites DPNI-RO
Star activity of an enzyme :
Under extreme conditions e.g. elevated pH or low ionic strength, restriction endonucleases are capable of cleaving sequences which are similar but not identical to their defined recognition sequence.This altered specificity is known as star activity.
To avoid star activity one can have to maintain optimum environment of reaction
Type 3 restriction enzyme :
Type III enzyme recognizes two separate non-palindromic sequences and cleaves DNA at the site of 25 to 27 bp from the recognition site.
Type II enzymes are composed of two subunits: Res and Mod.
The Res subunit is required for restriction digestion and the Mod subunit is for the recognition of DNA sequence and methyltransferase modification. Therefore, Type III enzymes are multifunctional and hetero-oligomeric.
Type III enzyme requires ATP and Mg2+ cofactor for their role in DNA methylation and restriction digestion.
Type III enzyme methylate only one strand of DNA at the N-6 position of adenosine which is enough to protect it against restriction digestion.
Some of the examples of Type III enzymes are EcoP I and Hinf III.
Type 4 restriction enzymes:
Type IV enzyme cleaves near or close to the recognition sequence.
It targets methylated DNA, hydromethylated DNA and glucosyl hydromethylated DNA which are all modified types of DNA.
Some of the examples of Type IV enzymes in the systems of E. coli are McrBC and Mrr.
There are also other type of restriction enzyme that are artificial restriction enzyme.
Artificial restriction enzymes :
In addition to the many natural restriction enzymes isolated from bacteria and archaea, it is now possible to synthesize artificial restriction enzymes that cut DNA at any desired sequence. Examples:
zinc-finger nucleases
TALENs
CRISPR RNA molecules
APPLICATIONS
1. Restriction fragment length polymorphism (RFLP)
2. Restriction mapping
3. Molecular cloning
4. Restriction enzyme mediated integration
5. DNA or gene sequencing
6. Pulse field gel electrophoresis etc.,
---Endonucleases are proteinitious enzymes
---enzymes are worked as catalysts. We all know about catalysts that they enhance the rate of reaction and reduce time taken for a successive reaction
--- for example all the living organisms are blessed with a life. Right?
But most us ( humans) lead life in routine manner just like a normal chemical reaction.
--- when we add some goals, aims, aspirations to our life, just like a catalyst to reaction this will enhance the efficiency of our life.
Enzymes with somewhat similar activity of restriction endonucleases
Enzymes those perform degradation of DNA similar to endonucleases are as follows
1. S1 nucleases
2 . Exonucleases
S1 nucleases act upon only single standard genetic materials without out any recognition and accuracy.
Exonucleases also removes nucleotides from the single standard DNA. Exonuclease is a co enzymes of DNA POLYMERASE 1. which removes only 1 to few nucleotides, starts its activity either from 5' or from 3' end, not from both ends.
RestrictionEndonucleases
History
---once upon a time, before the discovery of R.Eases a technique named SONICATION is used to agitate the DNA.
Restriction enzymes, a Revolutionary discovery in molecular biology.
Why they are called so??
A name RESTRICTION is given because of their ability of restricting (host restriction and modification phenomenon)
restriction enzymes are found in bacterium. This enzymes acts as defense system, just like our immune system
--- we are infected by bacteria where as bacteria are infected by bacteriophage.
( virus ) when a bacteriophage invade on bacteria and injects its genetic material into the bacterium these restrication enzymes acts as defensive barriers. R.E cuts the viral dna and meanwhile protects own genetic material by methylating ( adding methyle group) specific or recognition sequence.
This idea of restriction enzymes started as a hypothesis by Werner Arber who noticed that certain bacterial strains fought off bacteriophage infection by chopping off its DNA. But bacterial DNA remains unchanged why?
Arber and meselson hypothesized that bacterial cells produce two types of enzymes: one called a “restriction” enzyme that can identify and cut foreign DNA, and a “modification” enzyme that recognizes the host DNA and protects it from cleavage. This hypothesis was proven in an experiment in which two enzymes were isolated from E. coli. The modification enzyme, methylase, protected DNA of the bacterium, while the restriction enzyme chopped off phage (non-methylated) DNA.
Chronological order of discovery:
1969 Werner Arber and Stewart Linn discovered that most
bacteria contained a class of enzymes which modify the bases in
DNA termed methylases.
The phenomena of restriction and modification were illustrated by experimenting with bacteriophage λ on two E. coli host strains. C and K
In 1970, Hamilton O. Smith, Thomas Kelly and Kent Wilcox isolated
and characterized the first type II restriction enzyme, HindII, from
the bacterium Haemophilus influenzae.
In 1972 Daniel Nathans and Kathleen Danna showed that cleavage of SV40
DNA by restriction enzymes yielded specific fragments which can
be separated using polyacrylamide gel electrophoresis, thus
showing that restriction enzymes cut at specific sites and can be
used for mapping of the DNA.
Arber discovered restriction enzymes.
Smith verified Arber's hypothesis.
Nathans pioneered the applications of restriction enzymes
1n 1978 three of these collectively awarded with noble prize.
Although primarily found in bacterial genomes
and plasmids, restriction endonucleases also exist
in archaea, viruses, and eukaryotes.
As manimum as 7 and maximum of 14 genes are found in bacteria, but some of them are inactive.
Including all types, there are 3500 restric-
tion enzymes found in 10000 speices that recognize 259 different DNA
sequences are now known.
Data of enzymes discovered are updated every month in follow website
www.neb.com
Host restriction modification phenomenon:
Restriction endonucleases were originally named for their ability to restrict the growth of phage in a host bacterial cell by cleavage of the invading DNA. The endonucleases recognize specific sites for action called Restriction sites.
In this manner, they may be acting as bacterial protection systems. The DNA of the host is protected from restriction by the activity of a methylase(s),which recognizes the same sequence as the restriction enzyme and methylates a specific nucleotide (4-methylcytosine, 5-methylcytosine, 5-hydroxymethylcytosine, or 6-methyladenine) on each strandwithin this sequence. Once methylated, the host DNA is no longer a substrate for the endonuclease.
Because both strands of the host DNA are methylated and even hemi-methylated DNA is protected,
freshly replicated host DNA is not digested by the endonucleases.
Digested protected
CH3
|
5' G| A A T T C 3' 5' G A A T T C 3'
|________
3' C T T A A |G 5' 3' C T T A A G 5'
|
CH3
Staggered cut methylated
EcoR1 modification
As so often in biology exceptions exists
Here in some cases instead of methyl group DNA is modified by inserting sulfur.
Recently discovered dnd system illustrate about the insertion of sulfur into the PHOSPHORUS backbone forms a PHOSPHOROTHIOATE group one of the non- bridging oxygen bonds in the phosphate backbone can be replaced by sulfur.
This type of modification are carried out by five types dnd's I.e., dnd A B C D E
or 4 ssp enzymes I.e., ssp A B C D
L- cysteine desulfarse provides sulfur atoms to dnd A, which is needed for modification.
dnd's have operons and other activities
This is a very vast Topic (dnd and ssp ) . if you want more data you may work on it by books
Nomenclature:
Restriction enzymes are named according to the proposal of Smith and Nathans. Enzymes are named with three letters in italics are derived from the
first letter of the genus and the first two letters of the microbial species from which the enzyme was
derived. An additional letter without italics may be used to designate a particular strain. This is followed by a roman numeral to signify the first, sec-
ond, and so on, enzyme discovered from the organism.
Enzyme Source Organism Recognition Sequence
HpaII Haemophilus parainfluenzae C/CGG GGC/C
MboI Moraxella bovis /GATC CTAG/
NdeII Neisseria denitrificans /GATC CTAG/
EcoRI Escherichia coli RY13 G/AATTC CTTAA/G
EcoRII Escherichia coli RY13 /CC(A or T)GGGG(T or A)CC/
EcoRV Escherichia coli J62/pGL74 GAT/ATC CTA/TAG
BamHI Bacillus amyloliquefaciens G/GATCC CCTAG/G
SauI Staphylococcus aureus CC/TNAGG GGANT/CC
BglI Bacillus globigii GCCNNNN/NGGC
CGGN/NNNNCCG
NotI Nocardia otitidis-caviarum GC/GGCCGC CGCCGG/CG
DraII Deinococcus radiophilus RG/GNCCY YCCNG/GR
/ = position where enzyme cuts; N = any base, R = any purine, Y = any pyrimidine
Isoschizomers
Different types of restriction enzymes isolated from different bacteria which
have same recognition sequence.
Ex. SphI (CGTAC/G) and BbuI (CGTAC/G) are isoschizomers of each
other
Neoschizomers
Different R. enzymes having same recognition sequence but cutting site (base) is not same.
Working of restriction enzymes in simply in 3 words
Finding కనిపెట్టుట recognition
Covering కాపాడుట methylation
Cutting కత్తిరించుట digestion
Strategies followed by restriction enzymes for recognition:
Cutting of DNA by Restriction Enzymes:
It might seem logical for the DNA to be cut at the recognition site where the restriction enzyme binds. This is often true, but not always. There are two major types of restriction enzyme that differ in where they cut the DNA, relative to the recognitionsite. How restriction enzymes find their binding sites in vast lengths of DNA is considered
When the restriction enzymes diffused to the target DNA, check the site of contact for a match to their recognition sequence, and released the DNA if it did not match. This procedure would take so long that invading viral DNA would be expressed and take over the cell before the restriction enzyme destroyed it.
Two major strategies have been proposed for proteins that must locate target sequences within long DNA molecules in a reasonable time. This applies not only to restriction and modification enzymes but to regulatory proteins that must locate promoters or other specific sites on DNA.
One way is for the protein to slide along the DNA strand without letting go.
The protein monitors the sequence rapidly, but without extreme accuracy, as it moves along the DNA strand. If a possible match is found, the enzyme stops and tests if the match is accurate. The alternative is hopping from one site on the DNA sequence to another that is usually reasonably close but may sometimes be far away . The enzyme does not usually lose contact with the DNA but relies on DNA looping to bring the sites together . After hopping, the protein will prescan the DNA by local sliding for a while before hopping again.
● ---- protein or enzyme reading DNA by sliding or by hopping
Classification of restriction enzymes:
Type 1
Type 2 (orthodox)
Type 3
Type 4
Type 1 enzyme :
It is the first identified enzyme in two strains of E. coli (K-12 and B).
Type I enzyme cuts at a site about 1000 bp away from the recognition site.
The recognition site is composed of two specific portions: one with 3 to 4 nucleotides and other with 4 to 5 nucleotides which is separated by 6 to 8 nucleotides which is non specific and this site is asymmetrical.
It posses both restriction digestion and modification methylase activities.
Their cofactor includes adenosine triphosphate (ATP), S-Adenosyl methionine (AdoMet) and magnesium (Mg2+).
Type I enzyme involves three subunits: HsdR for restriction digestion,
HsdM for adding methyl groups (methyltransferase activity) and
HsdS for recognition of DNA binding site (specificity).
Some of the examples of Type I enzymes are EcoB and EcoK.
Type 2 restriction enzyme :
the common Type II endonucleases
are homodimers (most between 25 and 35 kDa for
the monomeric subunit), require only Mg2-, and
cleave within palindromes, partial palindromes, or interrupted palindromes. Despite dissimilar primary sequence,
Type II enzyme cleave within a short specific distance of about 4 to 8 nucleotides in length and the sites are usually undivided.
They cleave the phosphodiester bond of DNA either as a blunt end or sticky end.
They do not use ATP or AdoMet but require Mg2+ as a cofactor.
Type II enzyme is a homodimeric enzyme and are most commonly used enzymes.
After cutting two types of ands are formed by the restriction enzymes.
1 . Sticky or overhang or cohesive ends
3' over hang
5' overhang
2 . Blunt ends
There are seven subtypes of type II enzyme with different characteristics:
Type IIS (Eg: Fok I)
Type IIE (Eg: Nae I)
Type IIF (Eg: NgoM IV)
Type IIT (Eg: Bpu10 I)
Type IIG (Eg: Eco 571)
Type IIB (Eg: Bcg I)
Type IIM (Eg: DPNI-RO)
Type IIS
Cleavage at a defined distance from the recognition site
Asymmetric recognition site
Fok I
Type IIE
Interact with two recognition sites
One serves as the allosteric effector and the other site is for cleavage
Nae I
Type IIF
Interact with two recognition sites
Both are required for cleavage
Homotetrameric enzyme
NgoM IV
Type IIT
Contain different subunits with restriction and modification activities
Bpu10 I
Type IIG
Comprised of a single polypeptide chain with restriction and modification activity
Eco571
Type IIB
Cleave both sides of the recognition site
Bcg I
Type IIM
Recognize methylated sites DPNI-RO
Star activity of an enzyme :
Under extreme conditions e.g. elevated pH or low ionic strength, restriction endonucleases are capable of cleaving sequences which are similar but not identical to their defined recognition sequence.This altered specificity is known as star activity.
To avoid star activity one can have to maintain optimum environment of reaction
Type 3 restriction enzyme :
Type III enzyme recognizes two separate non-palindromic sequences and cleaves DNA at the site of 25 to 27 bp from the recognition site.
Type II enzymes are composed of two subunits: Res and Mod.
The Res subunit is required for restriction digestion and the Mod subunit is for the recognition of DNA sequence and methyltransferase modification. Therefore, Type III enzymes are multifunctional and hetero-oligomeric.
Type III enzyme requires ATP and Mg2+ cofactor for their role in DNA methylation and restriction digestion.
Type III enzyme methylate only one strand of DNA at the N-6 position of adenosine which is enough to protect it against restriction digestion.
Some of the examples of Type III enzymes are EcoP I and Hinf III.
Type 4 restriction enzymes:
Type IV enzyme cleaves near or close to the recognition sequence.
It targets methylated DNA, hydromethylated DNA and glucosyl hydromethylated DNA which are all modified types of DNA.
Some of the examples of Type IV enzymes in the systems of E. coli are McrBC and Mrr.
There are also other type of restriction enzyme that are artificial restriction enzyme.
Artificial restriction enzymes :
In addition to the many natural restriction enzymes isolated from bacteria and archaea, it is now possible to synthesize artificial restriction enzymes that cut DNA at any desired sequence. Examples:
zinc-finger nucleases
TALENs
CRISPR RNA molecules
APPLICATIONS
1. Restriction fragment length polymorphism (RFLP)
2. Restriction mapping
3. Molecular cloning
4. Restriction enzyme mediated integration
5. DNA or gene sequencing
6. Pulse field gel electrophoresis etc.,
