Traditional Cloning usually refers to the use of restriction endonucleases to generate DNA fragments with specific complementary end sequences that can be joined together with a DNA ligase, prior to transformation. This typically involves preparing both a DNA fragment to be cloned (insert) and a self-replicating DNA plasmid (vector) by cutting with two unique restriction enzymes that flank the DNA sequence and are present at the preferred site of insertion of the vector, often called the multiple cloning site (MCS). By using two different REs, two non-compatible ends are generated, thus forcing the insert to be cloned directionally, and lowering the transformation background of re-ligated vector alone. Directional cloning is often useful to maintain an open reading frame or another positional requirement with cis-acting regulatory elements. Non-directional cloning can also be performed with compatible ends generated by a single restriction enzyme; in this case, the clones will need to be screened to determine that the gene orientation is correct. Typically, the vector needs to be dephosphorylated to prevent self-ligation, which directly competes with the insert and lowers the efficiency of the cloning reaction.
In the early years of cloning, genomic DNA was often cloned into plasmid vectors using DNA adapters to add the required restriction sites to a gene of interest, prior to ligation. Additionally, genes, or other DNA elements, were swapped between vectors using compatible ends contained by both vectors. More recently, the Polymerase Chain Reaction (PCR) has been used as an upstream step in a cloning protocol to introduce the necessary restriction sites for directional cloning, prior to preparation of the vector and insert by restriction digests, followed by fragment purification, fragment ligation, and transformation into an E. coli cloning strain for plasmid amplification. Transformed colonies, now resistant to an antibiotic due to a resistance gene harbored by the plasmid, are screened by colony PCR or restriction digest of plasmid DNA for the correct insert. Direct sequencing of the recombinant plasmid is often performed to verify the sequence integrity of the cloned fragment.Advantages:
- Low cost
- Main different vector choices
- Directional cloning can be easily done
- Possible sequence constraints due to presence and/or translation of restriction site