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Good news lab workers! Always hated the tedious work of designing a cloning strategy? Or maybe always dreamed of pooling all the reactions in one tube, just to save time?
Thanks to Mother Nature, and her wonderful type IIS endonucleases, this is now possible!
What is this wonderful enzyme?
Type II enzymes are one of the 4 (I-IV) types of recognized endonucleases (enzymes that cut DNA at a particular recognition site). Type II enzymes cleave within or a short distance from their recognition sites (usually 4–8 nucleotides in length.) They are the better known of all endonucleases and most exploited for recombinant DNA works.
Among them, type IIS are the cool members of the family.
Type IIS enzymes are dimeric enzymes that cleave DNA at a defined distance from their non-palindromic, asymmetric recognition site.1 This means that the target sequence can only be read in one direction and that there is no axis of symmetry between the two DNA strands. The “defined distance” from the recognized motif is the best feature: unlike other endonucleases, like type I that cut at random distance from the recognition site, type IIS cleave the DNA at a fixed distance, usually 1–20 bp away depending on the enzyme.
Ok, but what’s cool about them?
Well, type IIS endonucleases are “special” because they opened the door to sequence-independent cloning: you don’t need to modify your gene of interest to include compatible restriction sites before cloning it.
Several biotech companies have seen the potential of this system, and have recently developed cloning kits based on these enzymes in combination with proprietary vectors featuring the most various attributes (New England Biolabs, DNA2.0, Life Technologies).
How do you do it?
Although different kits have slightly different nuances, the procedure is similar for all:
- Prepare your insert with the proper overhangs using PCR.
- Mix with supplied, linearized vector.
Simple, isn’t it?
Additional perks of cloning with Type II S endonucleases
- One tube cloning. The recognition site is lost after cleavage so digestion and ligation reactions can take place in the same tube at the same time.
- The inserted sequence is always in frame with the functional elements present on the vector. You don’t have to worry about frameshift mutations or unwanted amino acids. Start and stop codons, N- and C-terminal tags or modifications, will be added seamlessly.
- Scarless cloning. The overhang sequence is not dependent on a traditional restriction enzyme, and therefore no extra bases are introduced.
- Assembly of multiple fragments. Depending on the kit, multiple inserts can be assembled together simultaneously by using the right combination complementary ends.
Awesome, what about the downsides?
Despite its versatility and user-friendliness, type IIS-based cloning has also some drawbacks:
Technically speaking, the cloning is not completely sequence independent. In fact, one must make sure that the recognition site is not internal to the gene, or else it will be cut as well. Also, there is only a relatively small number of practical 3-nucleotide combinations when assembling multiple fragments using an enzyme that generates 3-bp overhangs.
Moreover, once the DNA sequence is ligated into a vector, there usually is no possibility of cutting it out, as the restriction sites are lost. To circumvent this problem, kits usually come with two types of vectors: one that serves as an entrance point to the system (and contains the restriction sites) and a wide choice of destination vectors (that don’t contain the sites) in which to re-clone the gene for subsequent expression.
Overall I find the system useful. In my experience, when I need to express a gene in different hosts or fuse it to multiple tags, designing a cloning strategy can be a headache. Having a system that only requires me to PCR in a few nucleotides at the 5’ and 3’ ends of the gene seems like a desirable solution to my troubles.
Therefore ‘thanks’ nature, for offering us such precious tools. And ‘well done’ scientists, for developing kits that harness their power!
- Pingoud A, Jeltsch A (2001). Structure and function of type II restriction endonucleases. Nucleic Acids Res. 29 (18): 3705–27.