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8.5: Restriction Enzymes - Biology

8.5: Restriction Enzymes - Biology


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DNA can be cut by restriction endonucleases (RE). Endonucleases are enzymes that can hydrolyze the nucleic acid polymer by breaking the phosphodiester bond between the phosphate and the pentose on the nucleic acid backbone. This is a very strong covalent bond while the weaker hydrogen bonds maintain their interactions and double strandedness.

As the name implies, restriction endonucleases (or restriction enzymes) are “restricted” in their ability to cut or digest DNA. The restriction that is useful to biologists is usually palindromic DNA sequences. Palindromic sequences are the same sequence forwards and backwards. Some examples of palindromes: RACE CAR, CIVIC, A MAN A PLAN A CANAL PANAMA. With respect to DNA, there are 2 strands that run antiparallel to each other. Therefore, the reverse complement of one strand is identical to the other. Molecular biologists also tend to use these special molecular scissors that recognize palindromes of 6 or 8. By using 6-cutters or 8-cutters, the sequences occur throughout large stretches rarely, but often enough to be of utility.

Restriction enzymes hydrolyze covalent phosphodiester bonds of the DNA to leave either “sticky/cohesive” ends or “blunt” ends. This distinction in cutting is important because an EcoRI sticky end can be used to match up a piece of DNA cut with the same enzyme in order to glue or ligate them back together. While endonucleases cut DNA, ligases join them back together. DNA digested with EcoRI can be ligated back together with another piece of DNA digested with EcoRI, but not to a piece digested with SmaI. Another blunt cutter is EcoRV with a recognition sequence of GAT | ATC.

EcoRI generates sticky of cohesive ends SmaI generates blunt ends

Restriction Digestions


Restriction Enzymes

Restriction enzymes, restriction endonucleases, or molecular scissors are bacteria-produced enzymes that can slice between two DNA strands at areas called recognition sites. Restriction enzymes were first discovered during Enterobacteria coli research. Type II restriction enzymes (REs) are of particular importance in the fields of molecular cloning, gene sequencing, and DNA mapping as this group can cut DNA very close to specific recognition sites and does not require energy in the form of ATP.


Pacific Northwest National Laboratory, Richland, Washington

A. Massey & Associates, Chapel Hill, North Carolina

Pacific Northwest National Laboratory, Richland, Washington

A. Massey & Associates, Chapel Hill, North Carolina

Summary

Students are introduced to restriction enzymes and simulate the activity of restriction enzymes with scissors. They are also introduced to restriction maps and asked to make simple predictions based on a map. Restriction enzymes were originally discovered through their ability to break down foreign DNA. Restriction enzymes can distinguish between the DNA normally present in the cell and foreign DNA, such as infecting bacteriophage DNA. They defend the cell from invasion by cutting foreign DNA into pieces and thereby rendering it nonfunctional. Restriction enzymes appear to be made exclusively by prokaryotes. The action of restriction enzymes is introduced and modeled in the activity DNA Scissors. The idea of rejoining restriction fragments and the need for complementarity in the single-stranded “tails” are introduced in the activity DNA Scissors. Restriction enzymes and DNA ligase play starring roles in DNA cloning. The discovery of restriction enzymes gave scientists a way to cut DNA into defined pieces. Every time a given piece of DNA was cut with a given enzyme, the same fragments were produced. These defined pieces could be put back together in new ways. A new phrase was coined to describe a DNA molecule that had been assembled from different starting molecules: recombinant DNA. After restriction digestion, the fragments of DNA are often separated by gel electrophoresis. The chapter also presents answers to exercise questions.


Relative activity of restriction enzymes in universal and basal buffers

Our restriction enzymes are supplied with an optimal universal buffer (one of five universal buffers indicated in blue in the table below). The relative activity in each of the other universal buffers is normalized to the optimal buffer, where the activity of each enzyme in the optimal buffer is expressed as 100%. Values in ( ) indicate buffers that are likely to be affected by star activity. In order to avoid these effects, use of buffers highlighted in blue or pink is recommended.

A few specific enzymes (AccIII, BalI, BcnI, BglI, Bpu1102I, Cfr10I, Eco52I, NruI, PshBI, SnaBI, SspI, TaqI, and VpaK11BI), are each supplied with a basal buffer specialized for the particular enzyme. The compositions of these basal buffers vary depending on the enzyme.

Restriction enzyme Relative activities (%)
LMHKT + BSABasal***
AatII <20 <20 <20 <20 100 120
AccI 20 100 <20 (<20) 160 80
AccII (260) 100 <20 20 200 160
AccIII (<20) (<20) 20 (80) (<20) 100
AfaI 60 60 40 60 100 100
AflII 20 80* <20 <20 140 120
AluI 100 100 <20 40 200 120
Aor13HI <20 20 <20 80 * 80 100
Aor51HI 80 100 <20 20 120 120
ApaI 100 <20 <20 <20 <20 120
ApaLI 100 20 <20 <20 120 120
AvaI (<20) 100 20 40 100 120
AvaII 80 100 <20 20 100 100
BalI 20 20 <20 <20 40 100
BamHI (<20) <20 40 100 (<20) 80
BanII (120) (120) 100 80 (100) 100
BcnI <20 20 40 60 60 100
BglI <20 <20 20 40 <20 100
BglII <20 20 100 (100) (60) 100
BlnI <20 20 40 100 20 120
BmeT110I <20 <20 20 100 <20 140
BmgT120I <20 <20 100 40 <20 240
Bpu1102I <20 <20 <20 40 60 100
BspT104I 100 60 <20 <20 100 120
BspT107I <20 20 80 100 20 100
Bsp1286I 100 20 <20 <20 60 100
Bsp1407I 20 60 20 20 100 100
BssHII 100 100 60 20 140 100
BstPI (<20) (60) 100 (100) (100) 100
BstXI <20 40 100 <20 <20 120
Bst1107I (<20) 60 100 100 40 100
Cfr10I (<20) (<20) (<20) 40 (20) 100
ClaI 40 100 120 100 60 100
CpoI <20 <20 80 100 <20 100
DraI 100 100 60 100 80 80
EaeI 60 100 <20 <20 120 160
EcoO65I (20) (60) 60 * 40 40 100
EcoO109I 100 60 <20 <20 100 160
EcoRI (20) (100) 100 (120) (80) 120
EcoRV (<20) (40) 100 (120) (40) 100
EcoT14I (<20) (40) 100 120 (60) 100
EcoT22I <20 20 100 (140) (20) 120
Eco52I <20 <20 <20 <20 <20 100
Eco81I <20 100 <20 <20 100 160
FbaI (<20) (<20) (80) 100 (20) 100
FokI (20) (60) <20 <20 (200) 100
HaeII 80 100 <20 80 140 100
HaeIII 60 100 100 60 100 100
HapII 100 60 <20 <20 100 80
HhaI 80 100 100 120 120 100
HincII 20 100 20 40 100 80
HindIII (60) 100 <20 200 (100) 80
HinfI 80 100 100 160 60 100
Hin1I 40 80* <20 20 60 160
HpaI <20 (40) 20 100 (80) 100
KpnI 100 60 <20 <20 (100) 80
MboI 20 40 60 100 40 100
MboII 100 60 <20 <20 60 100
MflI 100 80 <20 <20 80 100
MluI 60 60 100 (100) 60 100
MspI 80 80 <20 100 100 80
MunI (200) 100* <20 <20 160 100
MvaI (<20) (40) 80 100 (20) 120
NaeI 100 <20 <20 <20 100 120
NcoI (40) (60) 20 60* (60) 160
NdeI <20 40 100 100 80 100
NheI (120) 100 <20 <20 (160) 100
NotI (<20) (<20) 20** <20 (<20) 100
NruI 0 <20 20 20 <20 100
NsbI 40 20 <20 60 100 100
PmaCI 100 80 <20 <20 100 100
PshAI 20 40 <20 100 60 160
PshBI (20) (40) 20 40 40 100
Psp1406I 20 60 <20 <20 100 100
PstI (<20) (60) 100 80 (20) 80
PvuI (<20) (20) (40) 80* (40) 120
PvuII (80) 100 40 <20 (40) 100
SacI 100 60 <20 <20 80 80
SacII 40 20 <20 <20 100 40
SalI <20 <20 100 (20) <20 120
Sau3AI (60) 80 100 <20 (80) 100
ScaI (<20) (<20) 100 (60) (<20) 100
SfiI (40) 100 <20 <20 100 100
SmaI <20 <20 <20 <20 100 100
SmiI <10 <20 100 40 <10 100
SnaBI (20) (40) <20 <20 (40) 100
SpeI (80) 100 80 100 (80) 100
SphI (20) (40) 100 120 (20) 100
Sse8387I (120) 60* <20 <20 (60) 100
SspI (<20) (60) 40 (100) (80) 100
StuI 60 100 60 80 140 100
TaqI 40 80 60 60 80 100
Tth111I (20) 80 40 100 (80) 120
Van91I <20 (20) 60 100 (60) 100
VpaK11BI <20 <20 60 (40) <20 100
XbaI <20 80* 20 <20 120 120
XhoI <20 60 100 160 60 100
XspI <20 60 <20 100 160 100

Blue: buffer supplied with the restriction enzyme
Pink: alternative buffer recommended for use

*+0.01% BSA &rarr 100% AflII, EcoO65I, FokI, Hin1I, MunI, NcoI, PvuI, SplI, Sse8387I, XbaI
**+0.01% BSA + 01% Triton X-100 &rarr 100% NotI
*** The compositions of the basal buffers are enzyme-specific.


Restriction Endonucleases

With over 40 years of offering restriction enzymes to the research community, NEB has earned the reputation of being a leader in enzyme technologies. Working continuously to be worthy of that distinction, NEB strives to develop enzymes of the highest purity and unparalleled performance.

All of NEB's Restriction enzymes have transitioned to a new buffer system. Visit NEBCutSmart.com for further details.

Convenience

  • A vial of 6X Purple Load Dye is included with most restriction enzymes.
  • Over 210 restriction enzymes are 100% active in a single buffer &ndash CutSmart&trade Buffer.
  • >190 restriction enzymes are Time-Saver qualified, meaning you can digest DNA in 5-15 minutes, or digest DNA safely overnight.
  • Choose from >280 restriction enzymes, the largest selection commercially available.

Performance

  • Choose a High-Fidelity (HF®) restriction enzyme, which has been engineered for reduced star activity, rapid digestion (5-15 minutes) and 100% activity in CutSmart Buffer. A vial of 6X Purple Load Dye is included with every HF restriction enzyme.
  • All of our restriction enzymes undergo stringent quality control testing, ensuring the highest levels of purity and lot-to-lot consistency.

Use Enzyme Finder to select restriction enzymes by name, sequence, overhang or type.


8.5: Restriction Enzymes - Biology

Examples of class II restriction enzymes

from
Haemophilus aegytius

from
Haemophilus influenzae Rd

Restriction enzymes are obtained from many prokaryotes and about 1500 enzymes with known sequence recognition sites have been isolated. Naming these endonucleases follows a system proposed by Nathans and Smith. Each name contains at least one capital letter and two small letters followed by a Roman numeral. The letters are initials of the genus and species of origin and the number represents the number of enzymes discovered in the organism. (Historically the numeral identified the protein peak in which the enzyme eluted during chromatography.) Additional information may be added as a letter. For Eco RI, the R indicates the particular strain of E. coli.

Restriction enzymes from different organisms may recognize the same DNA sequence. If the enzymes recognize the same site and cleave at the same position, they are labeled isoschizomers. Ones that recognize the same site but cleave in different positions are heteroschizomers or neoschizomers.

Isoschizomer example

from
Streptomyces phaeochromogenes

from
Bacillus sp. Bu 17091

Heteroisoschizomer or neoschizomer example

from
Xanthomonas malvacea rum

Iso- or heteroisoschizomers add flexibility to experimental design. Cost, methylation sensitivities, and types of "ends" are considerations as well as buffer conditions for optimal activity.

A few buffer conditions suit nearly all the restriction enzymes but no single buffer allows activity of every enzyme. Suppliers of enzymes always provide a reaction buffer (10x concentrate) that is optimum for the enzyme. Components of the 1x buffer usually are 10-100 mM Tris at pH 7.3 to 8.5, various levels of salts like KCl and NaCl (10 to 150 mM), 10 mM Mg(2+), 2 mM beta-mercaptoethanol. Sometimes 0.01% Triton- X100 (a detergent) and bovine serum albumin are included as a stabilizers. (Alternatively, swine skin gelatin can be used and offers the advantages that it is stable to autoclaving and costs about 1/15 as much as BSA.)

Since restriction enzymes can require different buffer conditions, some strategy must be used to do double digests. The preferred method is to simultaneously digest with both enzymes in a compatible buffer. This method can be used even if one enzyme is not fully active (e.g., 75% active). More of one enzyme can be added (e.g., 1 U of enzyme A + 1.33 U enzyme B) for equal cutting efficiency. There are limits to the excess enzyme due to increased glycerol in the reaction that can reduce specificity of some enzymes.

An alternative method is to digest with the "low salt" enzyme then add more buffer and the "high salt" enzyme to complete the digest. This obviously doubles the time required for digestion. In extreme cases the DNA can be precipitated after one digest and dissolved in the second digest buffer. Digests are carried out at 37 degrees C unless otherwise noted for the enzyme.

Non-specific or relaxed specificity cleavage or "star" activity can occur if non-optimal conditions are used. Conditions that encourage star activity include:


Restriction enzyme

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Restriction enzyme, also called restriction endonuclease, a protein produced by bacteria that cleaves DNA at specific sites along the molecule. In the bacterial cell, restriction enzymes cleave foreign DNA, thus eliminating infecting organisms. Restriction enzymes can be isolated from bacterial cells and used in the laboratory to manipulate fragments of DNA, such as those that contain genes for this reason they are indispensible tools of recombinant DNA technology (genetic engineering).

A bacterium uses a restriction enzyme to defend against bacterial viruses called bacteriophages, or phages. When a phage infects a bacterium, it inserts its DNA into the bacterial cell so that it might be replicated. The restriction enzyme prevents replication of the phage DNA by cutting it into many pieces. Restriction enzymes were named for their ability to restrict, or limit, the number of strains of bacteriophage that can infect a bacterium.

Each restriction enzyme recognizes a short, specific sequence of nucleotide bases (the four basic chemical subunits of the linear double-stranded DNA molecule—adenine, cytosine, thymine, and guanine). These regions are called recognition sequences, or recognition sites, and are randomly distributed throughout the DNA. Different bacterial species make restriction enzymes that recognize different nucleotide sequences.

When a restriction endonuclease recognizes a sequence, it snips through the DNA molecule by catalyzing the hydrolysis (splitting of a chemical bond by addition of a water molecule) of the bond between adjacent nucleotides. Bacteria prevent their own DNA from being degraded in this manner by disguising their recognition sequences. Enzymes called methylases add methyl groups (—CH3) to adenine or cytosine bases within the recognition sequence, which is thus modified and protected from the endonuclease. The restriction enzyme and its corresponding methylase constitute the restriction-modification system of a bacterial species.

Traditionally, four types of restriction enzymes are recognized, designated I, II, III, and IV, which differ primarily in structure, cleavage site, specificity, and cofactors. Types I and III enzymes are similar in that both restriction and methylase activities are carried out by one large enzyme complex, in contrast to the type II system, in which the restriction enzyme is independent of its methylase. Type II restriction enzymes also differ from types I and III in that they cleave DNA at specific sites within the recognition site the others cleave DNA randomly, sometimes hundreds of bases from the recognition sequence. Several thousand type II restriction enzymes have been identified from a variety of bacterial species. These enzymes recognize a few hundred distinct sequences, generally four to eight bases in length. Type IV restriction enzymes cleave only methylated DNA and show weak sequence specificity.

Restriction enzymes were discovered and characterized in the late 1960s and early 1970s by molecular biologists Werner Arber, Hamilton O. Smith, and Daniel Nathans. The ability of the enzymes to cut DNA at precise locations enabled researchers to isolate gene-containing fragments and recombine them with other molecules of DNA—i.e., to clone genes. The names of restriction enzymes are derived from the genus, species, and strain designations of the bacteria that produce them for example, the enzyme EcoRI is produced by Escherichia coli strain RY13. It is thought that restriction enzymes originated from a common ancestral protein and evolved to recognize specific sequences through processes such as genetic recombination and gene amplification.

The Editors of Encyclopaedia Britannica This article was most recently revised and updated by Kara Rogers, Senior Editor.


Restriction Enzymes: Types & Examples

One of the most important steps in molecular biology, especially molecular genetics and analysis, is the isolation of DNA from the human genome and make many copies of it. Now, these copies can be utilized for further analysis of whatsoever type.

A key event in the development of molecular genetics methodology has been the discovery of Restriction Enzymes, also known as Restriction Endonucleases.

Introduction

A restriction enzyme is a kind of nuclease enzyme which is capable of cleaving double-stranded DNA. The enzymes may cleave DNA at random or specific sequences which are referred to as restriction sites. The recognition sites are palindromic in origin, that is, they are the sequences which are read the same forward and backward.

These restriction enzymes are produced naturally by bacteria. The bacterial species use it as a form of defense mechanism against viruses. However, in bacteria, restriction enzymes are present as a part of a combined system called the restriction modification system. The bacterial species modify their own DNA with the help of enzymes which methylate it. This particular process of methylation of bacterial DNA protects it from cleavage from its own restriction endonucleases.

Types

There are two different kinds of restriction enzymes:

  1. Exonucleases: restriction exonucleases are primarily responsible for hydrolysis of the terminal nucleotides from the end of DNA or RNA molecule either from 5’ to 3’ direction or 3’ to 5’ direction for example- exonuclease I, exonuclease II, etc.
  2. Endonuclease: restriction endonucleases recognize particular base sequences (restriction sites) within DNA or RNA molecule and catalyze the cleavage of internal phosphodiester bond for exEcoRI, Hind III, BamHI, etc.

History

The first restriction enzyme to be discovered was Hind II in the year 1970. In 1978, Daniel Nathans, Werner Arber, and Hamilton O. Smith were awarded the Nobel Prize for Physiology or Medicine.

Restriction Enzyme Nomenclature

The very name of the restriction enzymes consists of three parts:

  1. An abbreviation of the genus and the species of the organism to 3 letters, for example- Eco for Escherichia coli identified by the first letter, E, of the genus and the first two letters, co, of the species.
  2. It is followed by a letter, number or combination of both of them to signify the strain of the species.
  3. A Roman numeral to indicate the order in which the different restriction-modification systems were found in the same organism or strain per se.

Classification of Restriction Endonucleases

Based on the types of sequences identified, the nature of cuts made in the DNA, and the enzyme structure, there are three classes:

  1. Type I restriction enzymes,
  2. Type II restriction enzymes, and
  3. Type III restriction enzymes.

A. Type I Restriction Enzymes

  • Type I restriction enzymes possess both restriction and modification activities. In this case, the restriction will depend upon the methylation status of the target DNA sequence.
  • Cleavage takes place nearly 1000 base pairs away from the restriction site.
  • The structure of the recognition site is asymmetrical. It is composed of 2 parts. One part of the recognition site is composed of 3-4 nucleotides while the other one contains 4-5 nucleotides. The two parts are separated by a non-specific spacer of about 6-8 nucleotides.
  • For their function, the type I restriction enzymes require S- adenosylmethionine (SAM), ATP, and Mg 2+
  • They are composed of 3 subunits, a specificity subunit which determines the recognition site, a restriction subunit, and a modification subunit.

B. Type II Restriction Enzymes

  • Two separate enzymes mediate restriction and modification. Henceforth, DNA can be cleaved in the absence of modifying enzymes. Although the target sequence identified by the two enzymes is the same, they can be separately purified from each other.
  • The nucleotides are cleaved at the restriction site only. The recognition sequence is rotationally symmetrical, called palindromic sequence. The specific palindromic site can either be continuous (e.g., KpnI identifies the sequence 5´-GGTACC-3´) or non-continuous (e.g., BstEII recognizes the sequence 5´-GGTNACC-3´, where N can be any nucleotide)
  • These require Mg 2+ as a cofactor but not ATP.
  • They are required in genetic mapping and reconstruction of the DNA in vitro only because they identify particular sites and cleave at those sites only.

How they work:

  • The type II restriction enzymes first establish non-specific contact with DNA and bind to them in the form of dimmers.
  • The target sequence is then detected by a combination of two processes. Either the enzyme diffuses linearly/ slides along the DNA sequence over short distances or hops/ jumps over long distances.
  • Once the target sequence is located, various conformational changes occur in the enzyme as well as the DNA. These conformational changes, in turn, activate catalytic center.
  • The phosphodiester bond is hydrolyzed, and the product is released.

Structures of free, nonspecific, and specific DNA-bound forms of BamHI

C. Type III Restriction Enzyme

  • The type III enzymes recognize and methylate the same DNA sequence. However, they cleave nearly 24-26 base pairs away.
  • They are composed of two different subunits. The recognition and modification of DNA are carried out by the first subunit- ‘M’ and the nuclease activity is rendered by the other subunit ‘R’.
  • DNA cleavage is aided by ATP as well as Mg 2+ whereas SAM is responsible for stimulating cleavage.
  • Only one of the DNA strand is cleaved. However, to break the double-stranded DNA, two recognition sites in opposite directions are required.

Star Activity

Some restriction enzymes are capable of cleaving recognition sites which are similar to but not identical to the defined recognition sequence under non-standard reaction conditions (low ionic strength, high pH).

Isoschizomers, Neoschizomers, and Isocaudomers

  • Isoschizomers are the restriction enzymes which recognize and cleave at the same recognition site. For example, SphI (CGTAC/G) and BbuI (CGTAC/G) are isoschizomers of each other.
  • Neoschizomers are the restriction enzymes which recognize the same site and have a different cleavage pattern. For example, SmaI (GGG/CCC) and XmaI (G/GGCCC) are neoschizomers of each other.
  • Isocaudomers are the restriction enzymes which recognize slightly different sequences but produce the same ends. For example, both Sau3a and BamHI render a 5’-GATC-3’ sticky end although both have different recognition sequences.

Cleavage Patterns

Cleavage patterns of HindIII, SmaI, EcoRI, and BamHI are described as below. Most of the enzymes recognize sequences which are 4 to 6 base pairs long. However, they can also be up to 8 base pairs in length.

The process of cleavage of DNA by the restriction enzyme culminates with the formation of either sticky ends or blunt ends.

The blunt-ended fragments can be joined with the DNA fragment only with the aid of linkers and adapters.


Pacific Northwest National Laboratory, Richland, Washington

A. Massey & Associates, Chapel Hill, North Carolina

Pacific Northwest National Laboratory, Richland, Washington

A. Massey & Associates, Chapel Hill, North Carolina

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Summary

Students are introduced to restriction enzymes and simulate the activity of restriction enzymes with scissors. They are also introduced to restriction maps and asked to make simple predictions based on a map. Restriction enzymes were originally discovered through their ability to break down foreign DNA. Restriction enzymes can distinguish between the DNA normally present in the cell and foreign DNA, such as infecting bacteriophage DNA. They defend the cell from invasion by cutting foreign DNA into pieces and thereby rendering it nonfunctional. Restriction enzymes appear to be made exclusively by prokaryotes. The action of restriction enzymes is introduced and modeled in the activity DNA Scissors. The idea of rejoining restriction fragments and the need for complementarity in the single-stranded “tails” are introduced in the activity DNA Scissors. Restriction enzymes and DNA ligase play starring roles in DNA cloning. The discovery of restriction enzymes gave scientists a way to cut DNA into defined pieces. Every time a given piece of DNA was cut with a given enzyme, the same fragments were produced. These defined pieces could be put back together in new ways. A new phrase was coined to describe a DNA molecule that had been assembled from different starting molecules: recombinant DNA. After restriction digestion, the fragments of DNA are often separated by gel electrophoresis. The chapter also presents answers to exercise questions.


8.5: Restriction Enzymes - Biology

Article Summary:

What are Restriction Enzymes?

Restriction enzymes or to use their correct name, restriction endonucleases, are a type of enzyme which have the ability to "cut" molecules of DNA. They are often referred to as "genetic scissors".

The restriction enzyme recognises a unique sequence of nucleotides in the DNA strand, which is usually between four to six base-pairs in length. The complimentary DNA strand has the same sequence but in the reverse direction, thus ensuring both strands of DNA are cut at the same location.

Where are Restriction Enzymes found?

Restriction enzymes are found in many different strains of bacteria and their biological purpose is to participate and assist actively in cell defence. These enzymes prevent and "restrict" (hence their name) any foreign, i.e. viral DNA that may enter the cell, by destroying it. The host cell has an inbuilt restriction-modification system that methylates its own DNA at sites specific for its respective restriction enzymes, thereby protecting it from self-cleavage.

How are Restriction Enzymes used in Biotechnology?

In relation to biotechnology, restriction enzymes are used to cut DNA into smaller strands in order to study differences and similarities in fragment length amongst individuals such as in Restriction Fragment Length Polymorphism (RFLP) or in gene cloning. Such techniques have been used to determine that individuals or groups of individuals have distinctive differences in gene sequences and restriction cleavage patterns in specific areas of the genome. The basis for DNA fingerprinting is taken from this knowledge. Both RFLP and gene cloning processes are dependent on the use of agarose gel electrophoresis to allow complete and precise separation of the DNA fragments.

What types of Restriction Enzymes are there?

There are three different types of restriction enzymes simply named Type I, Type II and Type III.

Type I restriction enzymes cut DNA at random locations as remote as 1000 or more base-pairs from the recognition site.

Type III restriction enzymes cut at approximately 25 base-pairs from the recognition site.

Both Type I and Type III restriction enzymes require energy in the form of Adenosine Triphosphate (ATP) and may exist as larger enzymes consisting of multiple subunits.

However, Type II enzymes, such as those predominantly used in biotechnology, cut DNA within the recognised sequence without the need for ATP, and are smaller and less complex than Types I and III. Type II restriction enzymes are given specific names according to which bacterial species they are isolated from. By way of example, the Type II restriction enzyme isolated from E. Coli is named EcoR1.

Type II restriction enzymes are able to create two different types of cut, which is dependent on whether they cut both strands at the centre of the recognition sequence, or each strand closer to one end of the recognition sequence. The former cut will generate "blunt ends" with no nucleotide overhangs, while the latter cut generates "sticky" or "cohesive" ends, due to the fact that each resulting fragment of DNA has an overhang that compliments the other fragments. Both types are widely used in biotechnology, particularly in the fields of molecular genetics and protein engineering processes.

Biotechnology exploits the ability of restriction enzymes to reliably and precisely cleave DNA at specific sequences, which has led to the widespread use of these genetic tools in many molecular genetics techniques. Restriction enzymes can be used to map DNA fragments or the entire genome, thus determining the specific order of the restriction enzyme sites in the genome. Restriction enzymes are also frequently used to verify the identity of a specific DNA fragment, based on the known restriction enzyme sites sequence that it contains.

An extremely important use of restriction enzymes has been in the generation of recombinant DNA molecules. These are DNA molecules which consist of consist of genes or DNA fragments from two different organisms. Typically, a small circular DNA molecule known as a plasmid, obtained from bacteria, is joined to another piece of DNA from another gene of interest.

Type II restriction enzymes are used at several points during this process. They are used to digest the DNA from the experimental organism, in order to prepare the DNA for cloning. Thereafter, a bacterial plasmid or bacterial virus is cleaved with an enzyme that yields compatible ends. These compatible ends could be blunt with no overhang, or have complementary overhanging sequences. DNA from the gene of interest is combined with DNA from the plasmid or virus, and both types of DNA are joined with an enzyme called DNA ligase.

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Watch the video: Restriction Endonuclease: Types. Mechanism. Nomenclature (October 2022).