Cloning involves isolating, fragmenting, and combining genomic DNA with a vector, followed by introducing the recombinant molecule into a host where it is replicated. The genomic DNA used in cloning is predominantly chromosomal which is large and fragile (i.e., large polymers break). By necessity, the DNA must be broken into manageable pieces, a process normally accomplished by DNA cleaving enzymes called restriction endonucleases. After the DNA is fragmented, the pieces are linked to a vector to form recombined or recombinant molecules. This population of different genomic fragments linked to vector molecules is called a gene bank or gene library.
The typical method of preparing DNA for a gene bank requires that both the genomic and vector DNAs be cleaved with a restriction endonuclease. The restriction endonucleases used for cloning are site specific DNases that recognize four base or longer sequences and cleave the DNA in or near that sequence. Genomic DNA is cleaved randomly to yield an assorted collection of fragments, while the vector is cut only once in a predetermined location. The two sets of molecules are then combined and enzymatically coupled (ligation) by DNA ligase.
Thus far, you have developed a scheme for the cloning process and have isolated both the vector and genomic DNAs. The next step is to construct a gene bank which requires: (1) cleaving the vector site-specifically with a restriction endonuclease; (2) cleaving the genome randomly with a restriction endonuclease; and (3) linking genomic pieces with the vector. In the laboratory you will use restriction endonucleases for the cleavage of genomic and vector DNAs, electrophoretically examine the DNAs, and then recombine DNA molecules through ligation.
KeywordsVortex Migration Phenol Recombination Agarose
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- Perbal B (1984): A Practical Guide to Molecular Cloning. New York: John Wiley & SonsGoogle Scholar
- Sambrook J, Fritsch EF, Maniatis T (1989): Molecular Cloning: A Laboratory Manual. Plainview, NY: John Wiley & SonsGoogle Scholar