Posted: May 22nd, 2023

DNA cloning involves the following steps

1. DNA recombination-This involves the identification and isolation of the DNA fragment containing the gene of interest from the chromosomal DNA using restriction enzymes or by using the polymerase chain reaction(PCR),gel electrophoresis and sonication of DNA (Tierney, 2007). The fragment of DNA isolated must be joined to a replicating molecule or vector which acts as a vehicle that transports the DNA into the host cell. Both the isolated DNA fragment and the vector are cut using restriction enzymes at their restriction sites into sizeable fragments suitable for cloning. The desired DNA fragment is inserted into the cut ends of the vector and permanently linked using the enzyme DNA ligase thus forming a recombinant DNA molecule or chimera, but in some instances if processed under in vivo conditions, the enzyme terminal transferase may be added in order to avoid free sticky ends to rejoin instead of forming a chimera since it catalyzes the addition of “tails” of the nucleotide to the 3’ends of the DNA chains.
2. Transformation-This is whereby the recombinant DNA molecule enters the host cell (which is usually a bacterium) and proliferates. The recombinant plasmid molecule also contains color selection markers which show white/blue screening on a media of X-gal (Saiki & Arnheim, 1985).
3. Selective amplification-Within the host bacterium the vector multiplies producing numerous identical copies not only of itself but also of the gene that it carries. After a large number of cell divisions, a colony or a clone of identical host cells is produced.
4. Isolation of desired DNA clones-Culturing of transfected cells is done. The selectable antibiotic resistance markers are used as well as the color selection markers if present in the recombinant plasmid, though further confirmation.