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Cloning Strategies – a Whole Lot of Options to Choose
Molecular cloning has come a long way from simple restriction digestion-ligation cloning strategies to a large number of highly efficient alternatives. Broadly classified, cloning techniques can be divided as sequence dependent and sequence independent strategies.
Sequence-dependent strategies are based on restriction digestion-ligation techniques or site-specific recombination. They need unique and specific sites present in either insert or vector or both. Hence, they are inconvenient for multi-fragment cloning, as presence of particular sites at multiple places in the sequence is difficult. Examples of sequence-dependent cloning strategies include the Univector plasmid vector system and Gateway cloning.
Sequence-independent strategies are based on homologous recombination. These require complementary single stranded overhangs in both vector and insert. Generating overhangs takes additional steps, enzymes, and a large amount of starting DNA material. Thus, these strategies are time-consuming and expensive. Moreover, they are not always strictly sequence independent as some techniques need certain nucleotides in overlap regions. But sequence-independent cloning techniques are best strategies for multi-fragment assembly and construction of complex DNA libraries. Some examples include: TA cloning, ligation independent cloning (LIC), seamless ligation cloning extract (SLiCE), mating-assisted genetically integrated cloning (MAGIC), sequence and ligation independent cloning (SLIC), In-Fusion, Gibson assembly, polymerase incomplete primer extension (PIPE), and circular polymerase extension cloning (CPEC).
Though all cloning techniques begin with the similar starting material and end with the same products, the protocols differ in time taken, number of steps, cost (depending on number of enzymes used), and error and mutation rates, and therefore accuracy and efficiency of cloning. Thus, choice of strategy depends on a host of user-defined factors.
CPEC – What’s New?
CPEC is one of many sequence-independent cloning techniques used to assemble multi-fragment complex libraries in a one-step PCR reaction. It uses a single enzyme, making it inexpensive and fuss-free compared to other strategies that use multiple enzymes and steps (Figure 1). The technique happens in a single step reaction making it fast and convenient. So how would you go about doing CPEC?
How to CPEC?
- Linearize the vector using restriction digestion or PCR amplification, as would be done for normal cloning.
- Design the insert to have double stranded overlaps to the vector on either side. For this, add overlapping sequences of about 25 bp to both ends of the insert in a PCR reaction. Overlapping regions between vector and insert should have similar Tm (55 to 70° C) for specific annealing.
- Next, mix linearized vector and insert in a typical PCR reaction without primers.
- Carry out PCR reaction using a high-fidelity polymerase (without strand displacement activity), using one cycle for a single insert, and more cycles depending on number of fragments and library complexity (between 2 to 25 cycles). During the denaturation and annealing steps, the insert(s) and vector will hybridize and extend using each other as template to form a complete double stranded plasmid.
- Test a small portion of the PCR product on agarose gel to confirm assembly.
- Transform competent cells with PCR product (a nick is left in each strand after the polymerase extension, which can be filled in vivo by the cellular enzymes).
CPEC is a sequence-independent cloning strategy, making it easy to assemble multiple fragments into any vector and carry out complex library preparations (unlike sequence dependent cloning like Gateway or Univector). It is also flexible as to its use unlike traditional cloning or TA cloning that is restricted in application. It is a single step reaction and uses only a polymerase enzyme (no exonucleases or ligases) which makes it straight-forward, fast, and cost-effective (Gibson is expensive as it uses three separate enzymes, whereas SLIC or PIPE are two-step reactions). Since there is no exonuclease, small fragments can be assembled without worry of chew back (a limitation of Gibson assembly). CPEC occurs at higher temperatures than SLIC or Gibson, therefore non-specific hybridization and stable secondary structure formation is rare. Also, since CPEC is not an amplification process, it will not accumulate mutations. Thus, CPEC shows high cloning accuracy and efficiency.
Compared to SLIC or Gibson assembly, polymerase derived mutations are a concern in CPEC. Mis-priming is possible anywhere along the sequence, not just the termini. It is not possible to clone the same insert multiple times inside a vector.
CPEC seems to offer exciting advantages over standard cloning techniques as well some of the newer versions. Would you try out CPEC in your lab and compare it to your existing techniques? Let us know your experiences!
- Li MZ, Elledge SJ (2005); MAGIC, an in vivo genetic method for the rapid construction of recombinant DNA molecules. Nature Genetics 37, 311–319.
- Quan J, Tian J (2009); Circular Polymerase Extension Cloning of Complex Gene Libraries and Pathways. PLoS ONE 4(7): e6441.
- Quan J, Tian J (2011); Circular polymerase extension cloning for high-throughput cloning of complex and combinatorial DNA libraries. Nature Protocols 6, 242–251.