Search Thermo Fisher Scientific
Type IIS FastDigest Restriction Enzymes |
Type IIS restriction enzymes comprise a specific group of enzymes which recognize asymmetric DNA sequences and cleave at a defined distance outside of their recognition sequence, usually within 1 to 20 nucleotides. This specific mode of action of Type IIS restriction enzymes is widely used for innovative DNA manipulation techniques, such as Golden Gate cloning, enabling sequence-independent cloning of genes without the need to modify them by including compatible restriction sites (scars).
Restriction enzymes are classified into types according to the structure of their cleavage site, i.e., whether the cleavage falls within the recognition site or outside of the recognition site.
| Restriction enzyme type | Cleavage |
|---|---|
| Type I | Type I restriction endonucleases are complex, multifunctional enzymes that both cleave DNA and modify it through methylation. These enzymes recognize specific DNA sequences but cleave the DNA at random sites, often located hundreds to thousands of base pairs away from the recognition site. |
| Type II | Type II restriction enzymes recognize and cleave DNA at specific sequences, typically 4–8 base pairs long, cutting within or near these sites. They require magnesium ions but not ATP. Widely used in molecular biology, they are essential for DNA cloning, mapping, and sequencing due to their precision and reliability. |
| Type III | Type III restriction enzymes recognize specific DNA sequences and cleave at sites a short distance away. They require ATP and S-adenosylmethionine (SAM) for activity. Less commonly used than Type II enzymes, they are important for bacterial defense mechanisms and studying DNA-protein interactions. |
| Type IV | Type IV restriction enzymes specifically target and cleave modified DNA, such as methylated, hydroxymethylated, or glucosylated bases. Unlike other types, they do not recognize specific sequences but instead act on modified nucleotides. These enzymes play a role in bacterial defense systems and are useful for studying DNA methylation patterns. |
The best characterized and most frequently used restriction enzymes are the classical Type II class. Type II restriction enzymes recognize specific 4 to 8 nucleotide sequences that are typically palindromic and cleave within the recognition site, generating either sticky (5′ or 3′ overhangs) or blunt ends.
Type II restriction enzymes are enzymes that recognize specific DNA sequences and cut the DNA at or near these sites. They typically require only magnesium ions (Mg²⁺) as a cofactor for their activity. Upon binding to their recognition site, they introduce double-stranded breaks in the DNA, generating either blunt or sticky ends.
Type IIS restriction enzymes cut DNA at a specific distance from their recognition site, which allows for the creation of custom overhangs that are useful in various molecular cloning techniques. The ligation protocol for Type IIS restriction enzymes can be different because Type IIS enzymes cut outside of their recognition sequences, generating overhangs that are not palindromic. This requires careful design of the DNA fragments to ensure compatible overhangs for ligation. However, the basic steps of the ligation process—using DNA ligase to join compatible ends—remain the same.
Benefits of cloning with Type IIS restriction enzymes
- Single-tube cloning—Digestion and ligation reactions can take place in the same tube at the same time because the restriction site is eliminated from the ligated product.
- Scarless cloning—Scar sequences are not being introduced because the overhang sequence created is not dictated by the restriction enzyme.
- Assembly of multiple fragments—Multiple inserts can be assembled simultaneously by using the right combination of complementary ends.
Thermo Fisher Scientific offers ten Type IIS restriction enzymes within our Thermo Scientific FastDigest restriction enzyme portfolio, which offers:
- One buffer for all restriction and DNA modifying enzymes
- One digestion protocol for all DNA types
- Complete digestion in 15 minutes
- Overnight digestion without star activity
- Direct gel loading for a streamlined protocol
Designing fragments for DNA assembly
There are multiple ways to design sequences for subsequent assembly using Type IIS restriction enzymes. We have outlined the two most popular methods below and in Figure 1.
Generation of fragments by PCR
- The steps for generation of fragments of 150 to 200 bp, or up to 3 kb with GeneArt Strings fragment, are as follows:
- Design primers that contain a Type IIS restriction enzyme recognition site and a 4-nucleotide overhang unique to each insert.
- Amplify sequences of interest using primers with Type IIS restriction enzyme recognition sites.
- GeneratePCR fragments with complementary overhangs to allow assembly and ligation in a user-defined order.
- Ligate fragments into the destination vector.
Click image to enlarge
Figure 1. Generation of fragments for Type IIS restriction enzymes using PCR.
Generation of fragments from synthetic oligos
For cloning short fragments, such as 20 nucleotides with a 4-nucleotide overhang, the steps are as follows (Figure 2):
- Synthesize a pair of complementary oligos for each sequence with a unique set of 4 nucleotide 5′ overhangs corresponding to the desired, adjacent fragment in the final assembly).
- Phosphorylate the 5′ ends of each of the synthetic oligos.
- Anneal synthetic oligos to form the duplexes.
- Finish assembly of fragments by ligation into the destination vector.
Figure 2. Generation of fragments for Type IIS restriction enzymes using synthetic oligos.
Cloning a single fragment using Type IIS restriction enzymes
The DNA fragment to be cloned can be a PCR product, cloned PCR product (for example from TOPO and CloneJET PCR cloning kits), GeneArt String, or synthesized GeneArt clone. Type IIS recognition sites on the fragment’s ends should be oriented such that cleavage leaves the fragment with two sticky ends but removes the enzyme binding sites (Figure 3, shown in red).
The recipient vector must be similarly designed but with the two Type IIS restriction enzymes’ recognition sites oriented so that cleavage leaves the linearized vector with sticky ends compatible with the insert and with the enzyme binding sites removed (Figure 3).
Click image to enlarge
Figure 3. Single fragment cloning using Type IIs restriction enzymes.
Cloning multiple fragments using Type IIS restriction enzymes
Each fragment has a unique set of overhangs which define the order of assembly. Each end is complementary to the end of the fragment it will be adjacent to in the final assembly. The T4 ligase will join the complementary overhangs assembling the fragments into the accepting vector in the desired order (Figure 4).
Figure 4. Multiple fragment cloning using Type IIs restriction enzymes.
Cloning a single fragment using Type IIS restriction enzymes
The DNA fragment to be cloned can be a PCR product, cloned PCR product (for example from TOPO and CloneJET PCR cloning kits), GeneArt String, or synthesized GeneArt clone. Type IIS recognition sites on the fragment’s ends should be oriented such that cleavage leaves the fragment with two sticky ends but removes the enzyme binding sites (Figure 3, shown in red).
The recipient vector must be similarly designed but with the two Type IIS restriction enzymes’ recognition sites oriented so that cleavage leaves the linearized vector with sticky ends compatible with the insert and with the enzyme binding sites removed (Figure 3).
Click image to enlarge
Figure 3. Single fragment cloning using Type IIs restriction enzymes.
Cloning multiple fragments using Type IIS restriction enzymes
Each fragment has a unique set of overhangs which define the order of assembly. Each end is complementary to the end of the fragment it will be adjacent to in the final assembly. The T4 ligase will join the complementary overhangs assembling the fragments into the accepting vector in the desired order (Figure 4).
Figure 4. Multiple fragment cloning using Type IIs restriction enzymes.
Ordering information for FastDigest Type IIS restriction enzymes
Resources for Type IIS restriction enzymes
Resources & support for restriction enzymes
Products related to restriction enzymes
Tools to help your restriction digest
For Research Use Only. Not for use in diagnostic procedures.
Stylesheet for Classic Wide Template adjustments