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

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).

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 together simultaneously by using the right combination of complementary ends. 
     

Thermo Fisher Scientific offers ten Type IIS restriction enzymes within our Invitrogen Anza Restriction Enzyme Cloning System, a complete system of restriction and DNA modifying enzymes, 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
     

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Type IIS restriction enzymes—background

Restriction enzymes are classified into types according to the structure of their cleavage mode, i.e. whether the cleavage falls within the recognition site or outside of the recognition site.  

The best characterized and most frequently used restriction enzymes are the classical Type II class. These enzymes recognize specific 4 to 8 nucleotide sequences that are typically palindromic and cleave within the recognition site leaving sticky (5′ or 3′ overhangs) or blunt ends. 

In contrast, Type IIS restriction enzymes comprise a special group of enzymes, which cut DNA at a defined distance downstream of the recognition sequence (Figure 1). This is due to the enzyme architecture where the catalytic and recognition domains are separated by a polypeptide linker (Figure 2) [1].

There are no sequence requirements for the identity of bases in the cleavage site; therefore sequences beyond the recognition site can be any combination of nucleotides. With 256 potential overhang sequences possible, multiple fragments of DNA can be assembled using combinations of complementary overhangs. This cloning technique is widely used and is also known as Golden Gate assembly [2].

Figure 1. Comparison of the recognition site and cleavage site of Type II versus Type IIS restriction enzymes.

Figure 2. Computer generated structure of a Type IIS restriction enzyme.

Cloning with Type IIS restriction enzymes

Traditional cloning workflows involve multiple hands-on steps. Typically, a recipient plasmid vector is digested with one or more restriction enzymes, the terminal ends may then be dephosphorylated, and finally the linearized plasmid is gel purified. In parallel, the fragment to be cloned is also digested and gel purified, or alternatively, PCR amplified, digested to create compatible ends, and column purified. The two molecules are then treated with DNA ligase and transformed into E. coli. Traditional restriction enzyme cloning is usually limited to inserting a single DNA fragment into a recipient vector.

In contrast, Golden Gate cloning utilizes Type IIS restriction enzymes, in combination with DNA ligase, in a single reaction tube to drive the insertion of a DNA fragment – or several DNA fragments – into a recipient vector. Typically the reaction, performed in a thermocycler, is cycled repeatedly between the temperatures optimal for the restriction endonuclease (37 ᵒC) and the DNA ligase (23 ᵒC). As a result, Golden Gate DNA assembly reduces (or eliminates) multiple hands-on steps and does not require agarose gel purification [3].

How does Golden Gate cloning work?

The Type IIS restriction enzymes, which cut the DNA downstream from their recognition sites at non-specific sites, can be used to generate DNA fragments with unique overhangs. Assembly of digested fragments then proceeds through the annealing of complementary four-base overhangs on adjacent fragments. The digested fragments and the final assembly no longer contain Type IIS restriction enzyme recognition sites, because these recognition sites where the enzyme had bound was upstream of the cleavage, so no further cutting is possible. The restriction site is eliminated from the ligation product, so digestion and ligation can be carried out simultaneously. The assembly product accumulates with time.

Cloning a single fragment using Type IIS restriction enzymes

The DNA fragment to be cloned can be a PCR product, TOPO cloned PCR product, 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 (shown in orange).

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)

Figure 3. Single fragment cloning.

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.

Cloning a single fragment using Type IIS restriction enzymes

The DNA fragment to be cloned can be a PCR product, TOPO cloned PCR product, 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 (shown in orange).

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)

Figure 3. Single fragment cloning.

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.

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 (Figure 5):

Generation of fragments by PCR (for example, fragments of 150 to 200 bp or up to 3 kb with GeneArt Strings fragments):

  1. Primer design–primers should contain a Type IIS restriction enzyme recognition site and a 4 nucleotide overhang unique to each insert.
  2. PCR amplification–sequences of interest are amplified using primers with Type IIS restriction enzyme recognition sites.
  3. Digestion and ligation–PCR fragments with complementary overhangs are generated allowing assembly and ligation in a user-defined order.
  4. Complete assembly–fragments are ready to ligate into the final destination vector.

Figure 5. Generation of fragments for Type IIS restriction enzymes using PCR.

Generation of fragments from synthetic oligos (ideal for cloning short fragments, for example, 20 nucleotides with a 4 nucleotide overhang) (Figure 6):

  1. 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).
  2. Phosphorylate the 5′ ends of each of the synthetic oligos.
  3. Anneal synthetic oligos to form the duplexes.
  4. Finish assembly of fragments by ligation into the final destination vector.

Figure 6. Generation of fragments for Type IIS restriction enzymes using synthetic oligos.

Reference

  1. Pingoud A, Jeltsch A (2001) Structure and function of type II restriction endonucleases. Nucleic Acids Res 29(18):3705-3727.
  2. Engler C, Kandzia R, Marillonnet S (2008) A One Pot, One Step, Precision Cloning Method with High Throughput Capability. PLoS ONE 3(11): e3647.
  3. Engler C, Gruetzner R, Kandzia R et al. (2009) Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS ONE 4(5):e5553.

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