It uses a single enzyme, making it inexpensive and fuss-free compared to other strategies that use multiple enzymes and steps (Figure 1). Thus, choice of strategy depends on a host of user-defined factors.ĬPEC is one of many sequence-independent cloning techniques used to assemble multi-fragment complex libraries in a one-step PCR reaction. 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. 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). But sequence-independent cloning techniques are best strategies for multi-fragment assembly and construction of complex DNA libraries. Moreover, they are not always strictly sequence independent as some techniques need certain nucleotides in overlap regions. Thus, these strategies are time-consuming and expensive. Generating overhangs takes additional steps, enzymes, and a large amount of starting DNA material. These require complementary single stranded overhangs in both vector and insert. ![]() Sequence-independent strategies are based on homologous recombination. Examples of sequence-dependent cloning strategies include the Univector plasmid vector system and Gateway cloning. Hence, they are inconvenient for multi-fragment cloning, as presence of particular sites at multiple places in the sequence is difficult. They need unique and specific sites present in either insert or vector or both. ![]() Sequence-dependent strategies are based on restriction digestion-ligation techniques or site-specific recombination. Broadly classified, cloning techniques can be divided as sequence dependent and sequence independent strategies. Molecular cloning has come a long way from simple restriction digestion-ligation cloning strategies to a large number of highly efficient alternatives. ![]() Cloning Strategies – a Whole Lot of Options to Choose
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