Recombinant DNA technology, also molecular cloning, or genetic engineering
include the following steps:
1. Restriction digestion from the donor organism of the necessary genes of native DNA (cloned DNA, inserted DNA, target DNA, foreign DNA).
2. Quick decoding of all nucleotides in the purified DNA fragment, which allows to determine the exact boundaries of the gene and the amino acid sequence encoded by the gene.
3. Treatment with restriction endonucleases of a cloning vector that can replicate in the host cell.
4. Crosslinking of two DNA fragments by DNA ligase with the formation of a new recombinant molecule - the “cloning vector - integrated DNA” construct.
5. The introduction of this construct into the host cell (recipient), where it is replicated and transmitted to descendants. This process is called transformation. After transformation, the bacterial cell reproduces a fragment of the cloned DNA by millions of identical cells.
6. Identification and selection of cells carrying recombinant DNA (transformed cells).
7. Obtaining a specific protein product synthesized by host cells