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Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine. [2]
In molecular biology, [1] [2] [3] DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. [4] DNA replication occurs in all living organisms acting as the most essential part of biological inheritance .
There are two major sources of foreign DNA for molecular cloning is genomic DNA (gDNA) and complementary (or copy) DNA (cDNA). cDNA molecules are DNA copies of mRNA molecules, produced in vitro by action of the enzyme reverse transcriptase. In order to obtain the cDNA for a specific gene, it is first necessary to construct a cDNA library.
More than five decades ago, Jacob, Brenner, and Cuzin proposed the replicon hypothesis to explain the regulation of chromosomal DNA synthesis in E. coli. [18] The model postulates that a diffusible, trans-acting factor, a so-called initiator, interacts with a cis-acting DNA element, the replicator, to promote replication onset at a nearby origin.
In nature complementarity is the base principle of DNA replication and transcription as it is a property shared between two DNA or RNA sequences, such that when they are aligned antiparallel to each other, the nucleotide bases at each position in the sequences will be complementary, much like looking in the mirror and seeing the reverse of things.
There are two fundamental differences between the methods. One is that molecular cloning involves replication of the DNA within a living cell, while PCR replicates DNA in the test tube, free of living cells. The other difference is that cloning involves cutting and pasting DNA sequences, while PCR amplifies by copying an existing sequence.
The process of semiconservative replication for the site of DNA replication is a fork-like DNA structure, the replication fork, where the DNA helix is open, or unwound, exposing unpaired DNA nucleotides for recognition and base pairing for the incorporation of free nucleotides into double-stranded DNA.
During replication DNA polymerase begins to copy the DNA. At some point during the replication process, the polymerase dissociates from the DNA and replication stalls. When the polymerase reattaches to the DNA strand, it aligns the replicating strand to an incorrect position and incidentally copies the same section more than once.