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In the textbook that I'm using, it explains that bacteria does not digest its own chromosomal DNA because the sites that would be cut by its own endonuclease are methylated. Is there a similar mechanism involved for protecting its own plasmids? If so, how are plasmids cut while serving as vectors?
Laboratory strains used for the purposes of cloning have been genetically engineered to address this issue, typically by deleting genes of the various restriction-modification systems. There are four broad classes of restriction modification systems, which I will discuss individually. Unless individually referenced, most information below is based on the following supplier technical notes:
Promega: What are the effects of the bacterial DNA restriction-modification systems on cloning?
New England Biolabs: Restriction of Foreign DNA by E. coli K-12
Type I
In Escherichia coli, this system consists of the hsdR, hsdM and hsdS genes and is capable of both restricting (digesting) unmethylated DNA and methylating hemimethylated DNA. Knockout of hsdR and/or hsdM prevents restriction and methylation, respectively. This allows engineering of strains for specific purposes. For example, a common E. coli strain used for cloning, DH5α, has the genotype hsdR17 (rK-, mK+) which abrogates restriction activity but maintains methylation activity. This means that exogenous DNA will not be restricted, but will be methylated and therefore able to be subsequently transformed to r+ strains without worry of degradation, if the need may arise.
Type II
This is the class of restriction enzyme commonly used in the lab to cut DNA because they generally recognize short, palindromic sequences and cut within or near the recognition site. The E. coli background strains K and B, from which most lab strains are derived, do not contain Type II enzymes. The Type II enzyme EcoRI, for example, was isolated from the E. coli RY13 strain (Yoshimori, 1971; PhD thesis).
Type III
Similarly to Type II, the K and B strains do not possess this restriction-modification system, which was first characterized in E. coli 15T-.
Type IV
This system differs from the others in that it cleaves methylated and hemimethylated DNA, though it does not recognize methylation by Type I or Type II modification systems, nor Dam or Dcm methylation. In E. coli, this system consists of the mcrA, mcrBC and mrr genes and is most problematic when attempting transformation of DNA from organisms with ubiquitous cytosine or adenine methylation (eg plants and mammals). For example, the lab strain T7 Express, which is derived from the common strain BL21, has the genotype Δ(mcrC-mrr)114::IS10 which deletes mcrBC, hsdRMS and mrr.
Note on Dam and Dcm Methylases
These bacterial enzymes methylate adenine and cytosine residues, respectively, but are not directly involved in the restriction-modification systems discussed above. However, some Type II enzymes used for cloning are sensitive to this methylation and so lab strains are available with these enzymes deleted.
Restriction Endonucleases
With > 45 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.
NEB scientists continue to improve its portfolio of restriction enzymes, as well as explore their utility in new technologies. As a result, NEB scientists continue to publish scientific papers and to be awarded grants in this area. With the industry&rsquos largest research and development group dedicated to restriction enzymes, we are proud to have been there first: the first to commercialize a recombinant enzyme, the first to introduce a nicking enzyme. In addition, NEB has an ongoing history of innovation by engineering restriction enzymes with altered specificities and improved performance. Through continued research in these areas, we are committed to driving the innovations that allow us to offer maximum convenience and performance.
NEB feels that moving away from animal-containing products is a step in the right direction. We are excited to announce that we are in the process of switching all reaction buffers to be BSA-free! Learn more at www.neb.com/BSA-free.
For details on NEB&rsquos quality controls for restriction endonucleases, visit our Restriction Enzyme Quality page.
All of NEB's Restriction enzymes have transitioned to a new buffer system. Visit NEBCutSmart.com for further details.
Convenience
- >215 restriction enzymes are 100% active in a single buffer &ndash rCutSmart&trade Buffer.
- >195 restriction enzymes are Time-Saver qualified, meaning you can digest DNA in 5-15 minutes, or digest DNA safely overnight.
- Choose from >285 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 rCutSmart Buffer. A vial of 6X Purple Loading Dye is included with most restriction enzymes.
- 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 enzyme by name, sequence, overhang or type.
Restriction Enzyme Troubleshooting Guide
The following guide can be used for troubleshooting restriction enzyme digestions. You may also be interested in reviewing additional tips for optimizing digestion reactions.
We are excited to announce that we are in the process of switching all reaction buffers to be BSA-free. Beginning April 2021, NEB will be switching our current BSA-containing reaction buffers (NEBuffer&trade 1.1, 2.1, 3.1 and CutSmart ® Buffer) to Recombinant Albumin (rAlbumin)-containing buffers (NEBuffer r1.1, r2.1, r3.1 and rCutSmart&trade Buffer). We anticipate that this switch may take as long as 6 months to complete. We feel that moving away from animal-containing products is a step in the right direction and are able to offer this enhancement at the same price. Find more details at www.neb.com/BSA-free.
During this transition period, you may receive product with BSA or rAlbumin-containing buffers. NEB has rigorously tested both and has not seen any difference in enzyme performance when using either buffer. Either buffer can be used with your enzyme. All website content will be switched in April to reflect the changes, although you may not receive the new buffer with your product immediately.
Videos
Restriction Enzyme Digestion Problem: DNA Smear on Agarose Gel
Learn more about what causes this common problem, and how NEB's enzymes are QC'd to avoid DNA smearing.
Why is My Restriction Enzyme Not Cutting DNA?
Not getting the cleavage you expected? Let an NEB scientist help you troubleshoot your reaction.
Restriction Enzyme Digest Problem: Too Many DNA Bands
Are you finding unexpected bands in your digestion reaction? Here are some tips to help you determine the cause.
Restriction Enzyme Digest Protocol: Cutting Close to DNA End
When cutting close to the end of a DNA molecule, make sure you know how many bases to add to the ends of your PCR primers.
TIME-SAVER &trade Protocol for Restriction Enzyme Digests
Need a protocol to digest quickly and completely? Try this protocol for Time-Saver&trade qualified enzymes from NEB.
Reduce Star Activity with High-Fidelity Restriction Enzymes
NEB has engineered HF® enzymes to eliminate star activity. Learn how, and what this means for your digests.
Standard Protocol for Restriction Enzyme Digests
Let one of NEB's restriction enzyme experts help you improve your technique and avoid common mistakes in digest setup.
- Check the methylation sensitivity of the enzyme(s) to determine if the enzyme is blocked by methylation of the recognition sequence
- Use the recommended buffer supplied with the restriction enzyme
- Clean up the DNA to remove any contaminants that may inhibit the enzyme
- When digesting a PCR fragment, make sure to have at least 6 nucleotides between the recognition site and the end of the DNA molecule
- Lower the number of units
- Add SDS (0.1&ndash0.5%) to the loading buffer to dissociate the enzyme from the DNA or use Gel Loading Dye, Purple (6X) (NEB #B7024)
- Use fresh, clean running buffer
- Use a fresh agarose gel
- Clean up the DNA (NEB #T1030)
- Check the methylation sensitivity of the enzyme(s) to determine if the enzyme is blocked by methylation of the recognition sequence
- DNA isolated from a bacterial source may be blocked by Dam and Dcm methylation
- If the enzyme is inhibited by Dam or Dcm methylation, grow the plasmid in a dam-/dcm- strain (NEB #C2925)
- DNA isolated from eukaryotic source may be blocked by CpG methylation
- Enzymes that have low activity in salt-containing buffers (NEBuffer r3.1) may be salt sensitive, so clean up the DNA (NEB #T1030) prior to digestion
- DNA purification procedures that use spin columns can result in high salt levels, which inhibit enzyme activity. 1 To prevent this, DNA solution should be no more than 25% of total reaction volume.
- Clean up the PCR fragment prior to restriction digest (NEB #T1030)
- Use the recommended buffer supplied with the restriction enzyme
- Use at least 3&ndash5 units of enzyme per &mug of DNA
- Increase the incubation time
- Some enzymes have a lower activity on supercolied DNA. Increase the number of enzyme units in the reaction.
- Some enzymes can exhibit slower cleavage towards specific sites. Increase the incubation time, 1&ndash2 hours is typically sufficient.
- Some enzymes require the presence of two recognition sites to cut efficiently
- Assay substrate DNA in the presence of a control DNA. Control DNA will not cleave if there is an inhibitor present. Mini prep DNA is particularly susceptible to contaminants.
- Clean DNA with a spin column (NEB #T1030) or increase volume to dilute contaminant
- Lower the number of units in the reaction
- Add SDS (0.1&ndash0.5%) to the loading buffer to dissociate the enzyme from the substrate
- Use the recommended buffer supplied with the restriction enzyme
- Decrease the number of enzyme units in the reaction
- Make sure the amount of enzyme added does not exceed 10% of the total reaction volume. This ensures that the total glycerol concentration does not exceed 5% v/v
- Decrease the incubation time. Using the minimum reaction time required for complete digestion will help prevent star activity.
- Try using a High-Fidelity (HF) restriction enzyme. HF enzymes have been engineered for reduced star activity.
- Enzymes that have low activity in salt-containing buffers (e.g., NEBuffer r3.1) may be salt sensitive. Make sure to clean up the DNA (NEB #T1030) prior to digestion.
- DNA purification procedures that use spin columns can result in high salt levels, which inhibit enzyme activity. 1 To prevent this, DNA solution should be no more than 25% of total reaction volume.
- Clean-up the PCR fragment prior to restriction digest (NEB #T1030)
- Use the recommended buffer supplied with the restriction enzyme
- Use at least 5&ndash10 units of enzyme per &mug of DNA
- Digest the DNA for 1&ndash2 hours
1 Monarch Kits (NEB #T1010, NEB #T1020, and NEB #T1030) use columns that have been designed to minimize salt carry over into the eluted DNA, so using them can minimize this issue.
How can I tell if my enzyme will cut?
Whether performing a digest for cloning purposes or for diagnostics, we suggest double checking to make sure your results will not be affected by methylation. Conveniently, the majority of restriction enzymes commonly used in the lab do not have recognition sites that could overlap with a methylation site. The quick-reference table below lists 10 common enzymes that may be affected by methylation. This table is by no means exhaustive, so you may want to consult the REBASE database for more detailed information.
Enzyme | Dam methylation | Dcm methylation | EcoKI methylation |
ApaI | Not affected | Blocked by overlapping methylation | Not affected |
BsaI | Not affected | Blocked by overlapping methylation | Not affected |
ClaI | Blocked by overlapping methylation | Not affected | Not affected |
DraI | Not affected | Not affected | Blocked by overlapping methylation |
HpaI | Not affected | Not affected | Blocked by overlapping methylation |
MboI | Blocked by overlapping methylation | Not affected | Not affected |
MscI | Not affected | Blocked by overlapping methylation | Not affected |
PmeI | Not affected | Not affected | Blocked by overlapping methylation |
XbaI | Blocked by overlapping methylation | Not affected | Not affected |
DpnI | Requires methylation for activity | Not affected | Not affected |
If your enzyme is affected by methylation, knowing the sequence of the DNA surrounding your restriction site(s) can help you quickly predict which (if any) of those sites will be methylated. Using the same example plasmid as above (which has the XbaI site blocked by methylation), the figure below illustrates how sequence visualization software (we used Snapgene here) can help you assess whether your restriction site will be blocked by methylation and how that may change your expected digest results. If Dam methylation is not taken into account, the digest pattern depicted in Lane 1 would be expected for a XbaI/NotI digest. The program, however, allows you to choose whether to look for Dam, Dcm, and/or EcoKI methylation sites and warns you when a chosen enzyme site is blocked. Further, the program takes this into account when simulating a digest as depicted in Lane 2.
Restriction enzymes are powerful tools of molecular genetics used to:
&bull Map DNA molecules
&bull Analyze population polymorphisms
&bull Rearrange DNA molecules
&bull Prepare molecular probes
&bull Create mutants
Temperature: Most digestions are carried out at 37°C. However, there are a few exceptions e.g., digestion with Sma I is carried out at lower temperatures (
25°C), while with Taq I at higher temperature i.e., 65°C.
Buffer Systems: Tris-HCl is the most commonly used buffering agent in incubation mixtures, which is temperature dependent. Most restriction enzymes are active in the pH range 7.0-8.0.
Ionic Conditions: Mg2+ is an absolute requirement for all restriction endonucleases, but the requirement of other ions (Na+/K+) varies with different enzymes.
Methylation of DNA: Methylation of specific adenine or cytidine residues within the recognition sequence of the restriction enzyme affects the digestion of DNA.
Restriction Enzyme Digestion
Read about Type II restriction enzymes and the distinguishing properties of the four principle subtypes.
High throughput sequencing methods have revolutionized genomic analysis by producing millions of sequence reads from an organism’s DNA at an ever decreasing cost.
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Usage Guidelines
This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).
While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.
For more information about commercial rights, please contact NEB's Global Business Development team at [email protected].
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
Videos
What is a Type II Restriction Enzyme?
Type II restriction enzymes are most commonly used for molecular biology applications, as they recognize stereotypical sequences and produce a predictable cleavage pattern. Learn more about how Type II REs work.
What is a Type I Restriction Enzyme?
Type I restriction enzymes are a group of endonucleases that recognize a bipartite sequence, but do not produce a predictable cleavage pattern. Learn more about how Type I REs work.
What is a Type III Restriction Enzyme?
Type III restriction enzymes are a group of endonucleases that recognize a non-pallindromic sequence, comprising two inversely oriented sites. Learn more about these poorly understood enzymes.
Cloning With Restriction Enzymes
Restriction enzymes are an integral part of the cloning workflow, for generating compatible ends on fragments and vectors. This animation discusses three guidelines for determining which restriction enzymes to use in your cloning experiment.
Standard Protocol for Restriction Enzyme Digests
Let one of NEB's restriction enzyme experts help you improve your technique and avoid common mistakes in digest setup.
Why is My Restriction Enzyme Not Cutting DNA?
Not getting the cleavage you expected? Let an NEB scientist help you troubleshoot your reaction.
Restriction Enzyme Digest Problem: Too Many DNA Bands
Are you finding unexpected bands in your digestion reaction? Here are some tips to help you determine the cause.
What is Restriction Enzyme Star Activity?
Learn what Star Activity is, why it is detrimental to accurate restriction enzyme digestion, and how NEB's HF enzymes are engineered to avoid it.
Reduce Star Activity with High-Fidelity Restriction Enzymes
NEB has engineered HF® enzymes to eliminate star activity. Learn how, and what this means for your digests.
NEB ® Restriction Enzyme Double Digest Protocol
Double digestions can save you time, and this video can offer tips for how to achieve the best results, no matter which of NEB's restriction enzymes you're using.
Restriction Enzyme Digest Protocol: Cutting Close to DNA End
When cutting close to the end of a DNA molecule, make sure you know how many bases to add to the ends of your PCR primers.
Restriction Enzyme Digestion Problem: DNA Smear on Agarose Gel
Learn more about what causes this common problem, and how NEB's enzymes are QC'd to avoid DNA smearing.
Restriction Enzymes in Isothermal Amplification
Isothermal amplification generates many copies of a target sequence in a short period of time, at a constant temperature. Learn more about isothermal amplification.
Restriction Enzymes in Optical Mapping
Optical mapping is a method that allows production of restriction maps of whole chromosomes or genomes. Learn more about optical mapping.
Restriction Endonucleases: Molecular Cloning and Beyond
The sequence-specific DNA cleavage activity of restriction endonucleases (REases), combined with other enzymatic activities that amplify and ligate nucleic acids, have enabled modern molecular biology. After more than half a century of research and development, the applications of REases have evolved from the cloning of exogenous DNA and genome mapping to more sophisticated applications, such as the identification and mapping of epigenetic modifications and the high-throughput assembly of combinatorial libraries. Furthermore, the discovery and engineering of nicking endonucleases (NEases) has opened the door to techniques such as isothermal amplification of DNA among others. In this review, we will examine the major breakthroughs of REase research, applications of REases and NEases in various areas of biological research and novel technologies for assembling large DNA molecules.
Siu-Hong Chan, Ph.D., New England Biolabs, Inc.
INTRODUCTION
In the 1950s, a phenomenon known as &ldquohost controlled/induced variation of bacterial viruses&rdquo was reported, in which bacteriophages isolated from one E. coli strain showed a decrease in their ability to reproduce in a different strain, but regained the ability in subsequent infection cycles (1,2). In 1965, Werner Arber&rsquos seminal paper established the theoretical framework of the restriction-modification system, functioning as bacterial defense against invading bacteriophage (3). The first REases discovered recognized specific DNA sequences, but cut at variable distances away from their recognition sequence (Type I) and, thus were of little use in DNA manipulation. Soon after, the discovery and purification of REases that recognized and cut at specific sites (Type II REases) allowed scientists to perform precise manipulations of DNA in vitro, such as the cloning of exogenous genes and creation of efficient cloning vectors. Now, more than 4,000 REases are known, recognizing more than 300 distinct sequences (for a full list, visit REBASE® at rebase.neb.com). With the advent of the Polymerase Chain Reaction (PCR), RT-PCR, and PCR-based mutagenesis methodologies, the traditional cloning workflow transformed biological research in the decades that followed.
DEVELOPMENT OF RESTRICTION ENZYMES AND GENE EDITING TECHNIQUES
ENGINEERING OF RESTRICTION ENZYMES
Traditionally, REases were purified from the native organism. The development of gene cloning vectors and selection methodologies enabled the cloning of REases. Cloning not only allowed the production of large quantities of highly purified enzymes, but also made the engineering of REases possible. Currently, > 250 of the restriction enzymes supplied by New England Biolabs (NEB) are recombinant proteins.
Engineering Improved Performance
Cleavage activity at non-cognate sites (i.e., star activity) had been observed and well-documented for some REases. Of those, some exhibit star activity under sub-optimal reaction conditions, while others have a very narrow range of enzyme units that completely digest a given amount of substrate without exhibiting star activity (4). Through intensive research, scientists at NEB began engineering restriction enzymes that exhibit minimal, if any, star activity with extended reaction times and at high enzyme concentrations. This research enabled the introduction of High Fidelity (HF&trade) REases that have improved performance under a wider range of reaction conditions (for more information, visit www.neb.com/HF).
Engineering New Sequence Specificities
Attempts to alter the sequence specificities of Type IIP REases have been largely unsuccessful, presumably because the sequence specificity determinant is structurally integrated with the active sites of Type IIP REases. MmeI, a Type IIG REase with both methyltransferase (MTase) and REase activities in the same polypeptide, recognizes the target sequence TCCRAC using the target recognition domain (TRD) within its MTase component. This represented an excellent opportunity to engineer altered sequence specificity into the REase. As an added advantage, the sharing of the TRD between the REase and MTase activities resulted in an equivalent change in MTase activity for any change in target sequence cleavage specificity, protecting the new target site from cleavage in recombinant host cells. Through bioinformatics analysis of homologous protein sequences, scientists at NEB identified the amino acid residues that recognized specific bases within the target sequences and created MmeI mutants with altered sequence specificities (5). Rational design of MmeI mutants and homologs unlocked the potential for the creation of REases with hundreds of new sequence specificities.
Figure 1. Nicking Enzyme Engineering
Type IIS REases, such as FokI (light and dark brown) and BstNBI (isoschizomer of BspD6I, light and dark purple), and homing endonuclease I-AniI (cyan), have been engineered to posses nicking enzyme activities.
Engineering Nicking Endonucleases
Basic research involving REases led to surprising findings about the seemingly straightforward mechanism of cleavage. Prototypical Type IIP REases normally act as homodimers, with each of the monomers nicking half of the palindromic site. Type IIS REases, on the other hand, exhibit a broad range of double-stranded cleavage mechanisms, namely heterodimerization, as by BtsI and BbvCI, and sequential cleavage of the dsDNA as monomer, as by FokI. These properties have been exploited to create strand-specific nicking enzymes (NEases) (for more information about nicking enzymes, see review in (6)).
APPLICATIONS UTILIZING RESTRICTION ENZYMES
Traditional Cloning
In combination with DNA ligases, REases facilitated a robust &ldquocut and paste&rdquo workflow where a defined DNA fragment could be moved from one organism to another (Fig. 2). Using this methodology, Stanley Cohen and his colleagues incorporated exogenous DNA into natural plasmids to create the vehicle for cloning-plasmid vectors that self-propagate in E. coli (7). These became the backbone of many present-day vectors, and enabled the cloning of DNA for the study and production of recombinant proteins. Restriction enzymes are also useful as post-cloning confirmatory tools, to ensure that insertions have taken place correctly. The traditional cloning workflow, along with DNA amplification technologies, such as PCR and RT-PCR, has become a mainstream application for REases and facilitated the study of many molecular mechanisms.
Figure 2. Traditional Cloning Workflow
Using PCR, restriction sites are added to both ends of a dsDNA, which is then digested by the corresponding REases. The cleaved DNA can then be ligated to a plasmid vector cleaved by the same or compatible REases with T4 DNA ligase. DNA fragments can also be moved from one vector into another by digesting with REases and ligating to compatible ends of the target vector.
DNA Mapping
Armed with only a handful of REases in the early 1970s, Daniel Nathans mapped the functional units of SV40 DNA (8), and commenced the era of &ldquorestriction mapping&rdquo and comparison of complex genomes. It has since evolved into sophisticated methodologies that allow the detection of single nucleotide polymorphisms (SNP) and insertions/deletions (Indels) (9), driving applications that include identifying genetic disorder loci, assessing the genetic diversity of populations and parental testing.
Understanding Epigenetic Modifications
REases&rsquo sensitivity to the methylation status of target bases has been exploited to map modified bases within genomes. Restriction Landmark Genome Scanning (RLGS) is a 2-dimensional gel electrophoresis-based mapping technique that employs NotI (GC^GGCCGC), AscI (GG^CGCGCC), EagI (C^GGCCG) or BssHII (G^CGCGC) to interrogate changes in the methylation patterns of the genome during the development of normal and cancer cells. Methylation-Sensitive Amplification Polymorphism (MSAP) takes advantage of the differential sensitivity of MspI and HpaII toward the methylation status of the second C of quadruplet CCGG to identify 5-methylcytosine (5-mC) or 5-hydroxymethylcytosine (5-hmC) (10,11). Scientists at NEB further exploited the property of MspI and HpaII on 5-glucosyl hydroxymethylcytosine (5-ghmC) in the EpiMark® 5-hmC and 5-mC Analysis Kit (NEB #E3317S)(12), which differentiates 5-hmC from 5-mC for more refined epigenetic marker identification and quantitation (for more information, visit EpiMark.com). Additionally, the recently discovered REases that recognize and cleave DNA at 5-mC and 5-hmC sites (e.g., MspJI, FspEI and LpnPI), as well as those that preferentially cleave 5-hmC or 5-ghmC over 5-mC or C (e.g., PvuRts1I, AbaSI) (13), are potential tools for high-throughput mapping of the cytosine-based epigenetic markers in cytosine-methylated genomes (14,15).
In vitro DNA Assembly Technologies
Synthetic biology is a rapidly growing field, in which defined components are used to create biological systems for the study of biological processes and the creation of useful biological devices (16). Novel technologies such as BioBrick&trade originally emerged to facilitate the building of such biological systems. Recently, more robust approaches, such as Golden Gate Assembly and Gibson Assembly&trade, have been widely adopted by the synthetic biology community. Both approaches allow for the parallel and seamless assembly of multiple DNA fragments without resorting to non-standard bases.
BioBrick: The BioBricks community sought to create thousands of &ldquostandardized parts&rdquo of DNAs for rapid gene assembly. With the annual International Genetically Engineered Machines (iGEM) competition (igem.org), the BioBricks community grew and elicited broad interest from many university students in synthetic biology. Based on traditional REase-ligation methodology, BioBrick and its derivative methodologies (BioBrick Assembly Kit, NEB #E0546, and its derivative, BglBricks (17)) are easy to use, but they introduce scar sequences at the junctions. They also require multiple cloning cycles to create a working biological system.
Golden Gate Assembly: Golden Gate Assembly and its derivative methods (19,20) exploit the ability of Type IIS REases to cleave DNA outside of the recognition sequence. The inserts and cloning vectors are designed to place the Type IIS recognition site distal to the cleavage site, such that the Type IIS REase can remove the recognition sequence from the assembly (Fig. 3). The advantages of such an arrangement are three-fold: 1. the overhang sequence created is not dictated by the REase, and therefore no scar sequence is introduced 2. the fragment-specific sequence of the overhangs allows orderly assembly of multiple fragments simultaneously and 3. the restriction site is eliminated from the ligated product, so digestion and ligation can be carried out simultaneously. The net result is the ordered and seamless assembly of DNA fragments in one reaction. The accuracy of the assembly is dependent on the length of the overhang sequences. Therefore, Type IIS REases that create 4-base overhangs (such as BsaI/BsaI-HF, BbsI, BsmBI and Esp3I) are preferred. The downside of these Type IIS REase-based methods is that the small number of overhanging bases can lead to the mis-ligation of fragments with similar overhang sequences (21). It is also necessary to verify that the Type IIS REase sites used are not present in the fragments for the assembly of the expected product. Nonetheless, Golden Gate Assembly is a robust technology that generates multiple site-directed mutations (22) and assembles multiple DNA fragments (23,24). As open source methods and reagents have become increasingly available (see www.addgene.org), Golden Gate Assembly has been widely used in the construction of custom-specific TALENs for in vivo gene editing (25), among other applications.
Figure 3. Golden Gate Assembly Workflow
In its simplest form, Golden Gate Assembly requires a BsaI recognition site (GGTCTC) added to both ends of a dsDNA fragment distal to the cleavage site, such that the BsaI site is eliminated by digestion with BsaI or BsaI-HF (GGTCTC 1/5). Upon cleavage, the overhanging sequences of the adjoining fragments anneal to each other. DNA ligase then seals the nicks to create a new covalently linked DNA molecule. Multiple pieces of DNA can be cleaved and ligated simultaneously. Gibson Assembly: Daniel G. Gibson, of the J. Craig Venter Institute, described a robust exonuclease-based method to assembly DNA seamlessly and in the correct order. The reaction is carried out under isothermal conditions using three enzymatic activities: a 5&rsquo exonuclease generates long overhangs, a polymerase fills in the gaps of the annealed ss regions, and a DNA ligase seals the nicks of the annealed and filled-in gaps (26) (Fig. 4). Applying this methodology, the 16.3 kb mouse mitochondrial genome was assembled from 600 overlapping 60-mers (26). In combination with in vivo assembly in yeast, Gibson Assembly was used to synthesize the 1.1 Mbp Mycoplasma mycoides genome. The synthesized genome was transplanted to a M. capricolum recipient cell creating new self-replicating M. mycoides cells (27).
Gibson Assembly can also be used for cloning the assembly of a DNA insert with a restriction-digested vector, followed by transformation, can be completed in a little less than two hours with the Gibson Assembly Cloning Kit (NEB #E5510S, for more information, visit NEBGibson.com). Other applications of Gibson Assembly include the introduction of multiple mutations, assembly of plasmid vectors from chemically synthesized oligonucleotides, and creating combinatorial libraries of genes and pathways.
Figure 4. Gibson Assembly Workflow
Gibson Assembly employs three enzymatic activities in a single-tube reaction: 5´ exonuclease, the 3´ extension activity of a DNA polymerase and DNA ligase activity. The 5´ exonuclease activity chews back the 5´ end sequences and exposes the complementary sequence for annealing. The polymerase activity then fills in the gaps on the annealed regions. A DNA ligase then seals the nick and covalently links the DNA fragments together. The overlapping sequence of adjoining fragments is much longer than those used in Golden Gate Assembly, and therefore results in a higher percentage of correct assemblies. The NEB Gibson Assembly Master Mix (NEB #E2611) and Gibson Assembly Cloning Kit (NEB #E5510S) enable rapid assembly at 50˚C.
Construction of DNA Libraries
SAGE (Serial Analysis of Gene Expression) has allowed the identification and quantification of a large number of mRNA transcripts. It has been widely used in cancer research to identify mutations and study gene expression. REases are key to the SAGE workflow. NlaIII is instrumental as an anchoring enzyme, because of its unique property of recognizing a 4-bp sequence CATG and creating a 4 nucleotide overhang of the same sequence. The use of Type IIS enzymes as tagging enzymes that cleave further and further away from the recognition sequence allows for the higher information content of SAGE analyses (e.g., FokI and BsmFI in SAGE (28), MmeI in LongSAGE (29) and EcoP15I in SuperSAGE (30) and DeepSAGE (31)).
Chromosome conformation capture (3C) and derivative methods allow the mapping of the spatial organizations of genomes in unprecedentedly high resolution and throughput (32). REases plays an indispensible role in creating the compatible ends of the DNA cross-linked to its interacting proteins, such that spatially associated sequences can be ligated and, hence, identified through high-throughput sequencing.
Although REases do not allow for the random fragmentation of DNA that most next-generation DNA sequencing technologies require, they are being used in novel target enrichment methodologies (hairpin adaptor ligation (33) and HaloPlex&trade enrichment (Agilent)). The long-reach REase, AcuI, and USER&trade Enzyme are also used to insert tags into sample DNA, which is then amplified by rolling circle amplification (RCA) to form long, single-stranded DNA &ldquonanoballs&rdquo that serve as template in the high density, chip-based sequencing-by-ligation methodology, developed by Complete Genomics (34). ApeKI was also used to generate the DNA library for a genotyping-by-sequencing technology for the study of sequence diversity of maize (35).
Creation of Nicks in DNA
Before NEases were available, non-hydrolyzable phosphorothioate groups were incorporated into a specific strand of the target DNA such that REases can introduce sequence- and strand-specific nicks into the DNA for applications such as strand displacement amplification (SDA), where a strand-displacing DNA polymerase (e.g., Bst 2.0 DNA Polymerase, NEB #M0537) extends from the newly created 3&rsquo-hydroxyl end, and essentially replicates the complementary sequence (36). Because the nicking site is regenerated, repeated nicking-extension cycles result in amplification of specific single-stranded segments of the sample DNA without the need for thermocycling. NEases greatly streamline the workflow of such applications and open the door to applications that cannot be achieved by REases. Nicking enzyme-based isothermal DNA amplification technologies, such as RCA, NESA, EXPAR and related amplification schemes, have been shown to be capable of detecting very low levels of DNA (37,38). Nicking-based DNA amplification had also been incorporated into molecular beacon technologies to amplify signal (39). The implementation of these sample and/or signal amplification schemes can lead to simple, but sensitive and specific, methods for the detection of target DNA molecules in the field (NEAR, EnviroLogix&trade). By ligating adaptors containing nicking sites to the ends of blunt-ended DNA, the simultaneous actions of the NEase(s) and strand-displacing DNA polymerase can quickly amplify a specific fragment of dsDNA (40). Amplification by nicking-extension cycling is amenable to multiplexing and can potentially achieve a higher fidelity than PCR. The combined activity of NEases and Bst DNA polymerase have also been used to introduce site-specific fluorescent labels into long/chromosomal DNA in vitro for visualization (nanocoding) (41). Innovative applications of nicking enzymes include the generation of reporter plasmids with modified bases or structures (42) and the creation of a DNA motor that transports a DNA cargo without added energy (43). A review of NEases and their applications has been published elsewhere (6).
In vivo Gene Editing
The ability to &ldquocut and paste&rdquo DNA using REases in vitro has naturally led to the quest for performing the art in vivo to correct mutations that cause genetic diseases. Direct use of REases and homing endonucleases in Restriction Enzyme Mediated Integration (REMI) facilitated the generation of transgenic embryos of higher organisms (44,45). There is, however, no control over the integration site. The concept of editing genes through site-specific cleavage has been realized using Zinc Finger Nucleases (ZFNs) and Transcription Activator-like Effector Nucleases (TALENs), due to their ability to create customizable double stranded breaks in complex genomes. With the great success of gene editing in model organisms and livestock (46-50), the therapeutic potential of these gene editing reagents is being put to the first test in the Phase I/II clinical trials of a regime that uses a ZFN to improve CD4+ T-cell counts by knocking out the expression of the CCR5 gene in autologous T-cells from HIV patients (ClinicalTrials.gov indentifier NCT00842634) (51). Recent research on CRISPR, the adaptive defense system of bacteria and archaea, has shown the potential of the Cas9-crRNA complex as programmable RNA-guided DNA endonucleases and strand-specific nicking endonucleases for in vivo gene editing (52,53).
MOVING FORWARD
Restriction enzymes have been one of the major forces that enabled the cloning of genes and transformed molecular biology. Novel technologies, such as Golden Gate Assembly and Gibson Assembly, continue to emerge and expand our ability to create new DNA molecules. The potential to generate new recognition specificity in the MmeI family REases, the engineering of more NEases and the discovery of ever more modification-specific REases continues to create new tools for DNA manipulation and epigenome analysis. Innovative applications of these enzymes will take REases&rsquo role beyond molecular cloning by continuing to accelerate the development of biotechnology and presenting us with new opportunities and challenges.
How do restriction endonucleases cut DNA?
Read rest of the answer. Accordingly, where do restriction endonucleases cut DNA?
Restriction Enzyme Types Generally, Type I enzymes cut DNA at locations distant to the recognition sequence Type II cut DNA within or close to the recognition sequence Type III cut DNA near recognition sequences and Type IV cleave methylated DNA.
what is DNA restriction? DNA restriction enzymes break DNA strands at specific sites based on the nucleic acid sequence. Thus, digestion with a given restriction enzyme or combination of restriction enzymes will produce fragments of different lengths that are directly related to the DNA sequence.
Similarly, it is asked, why do restriction enzymes not cut their own DNA?
Bacteria have restriction enzymes, also called restriction endonucleases, which cut double stranded DNA at specific points into fragments. Interestingly, restriction enzymes don't cleave their own DNA. Bacteria prevent their own DNA from chop down by restriction enzyme through methylation of the restriction sites.
Which bonds do restriction enzymes cut?
Restriction enzymes cut DNA bonds between 3&prime OH of one nucleotide and 5&prime phosphate of the next one at the specific restriction site. Adding methyl groups to certain bases at the recognition sites on the bacterial DNA blocks the restriction enzyme to bind and protects the bacterial DNA from being cut by themselves.
Traditional Cloning
Traditional Cloning usually refers to the use of restriction endonucleases to generate DNA fragments with specific complementary end sequences that can be joined together with a DNA ligase, prior to transformation. This typically involves preparing both a DNA fragment to be cloned (insert) and a self-replicating DNA plasmid (vector) by cutting with two unique restriction enzymes that flank the DNA sequence and are present at the preferred site of insertion of the vector, often called the multiple cloning site (MCS). By using two different REs, two non-compatible ends are generated, thus forcing the insert to be cloned directionally, and lowering the transformation background of re-ligated vector alone. Directional cloning is often useful to maintain an open reading frame or another positional requirement with cis-acting regulatory elements. Non-directional cloning can also be performed with compatible ends generated by a single restriction enzyme in this case, the clones will need to be screened to determine that the gene orientation is correct. Typically, the vector needs to be dephosphorylated to prevent self-ligation, which directly competes with the insert and lowers the efficiency of the cloning reaction.
In the early years of cloning, genomic DNA was often cloned into plasmid vectors using DNA adapters to add the required restriction sites to a gene of interest, prior to ligation. Additionally, genes, or other DNA elements, were swapped between vectors using compatible ends contained by both vectors. More recently, the Polymerase Chain Reaction (PCR) has been used as an upstream step in a cloning protocol to introduce the necessary restriction sites for directional cloning, prior to preparation of the vector and insert by restriction digests, followed by fragment purification, fragment ligation, and transformation into an E. coli cloning strain for plasmid amplification. Transformed colonies, now resistant to an antibiotic due to a resistance gene harbored by the plasmid, are screened by colony PCR or restriction digest of plasmid DNA for the correct insert. Direct sequencing of the recombinant plasmid is often performed to verify the sequence integrity of the cloned fragment.
- Low cost
- Versatile
- Main different vector choices
- Directional cloning can be easily done
- Possible sequence constraints due to presence and/or translation of restriction site