Overview Of Recombination Techniques
The basic technique of recombinant DNA involves digesting a vector DNA with a restriction enzyme, which is a molecular scissors that cuts DNA at specific sites. A DNA molecule from the organism of interest is also digested, in a separate tube, with the same restriction enzyme. The two DNAs are then mixed together and joined, this time using an enzyme called DNA ligase, to make an intact, double-stranded DNA molecule. This construct is then put into Escherichia coli cells, where the resulting DNA is copied billions of times. This novel DNA molecule is then isolated from the E. coli cells and analyzed to make sure that the correct construct was produced. This DNA can then be sequenced, used to generate protein from E. coli or another host, or for many other purposes.
There are many variations on this basic method of producing recombinant DNA molecules. For example, sometimes researchers are interested in isolating a whole collection of DNAs from an organism. In this case, they digest the whole genome with restriction enzyme, join many DNA fragments into many different vector molecules, and then transform those molecules into E. coli. The different E. coli cells that contain different DNA molecules are then pooled, resulting in a "library" of E. coli cells that contain, collectively, all of the genes present in the original organism.
Another variation is to make a library of all expressed genes (genes that are used to make proteins) from an organism or tissue. In this case, RNA is isolated. The isolated RNA is converted to DNA using the enzyme called reverse transcriptase. The resulting DNA copy, commonly abbreviated as cDNA, is then joined to vector molecules and put into E. coli. This collection of recombinant cDNAs (a cDNA library) allows researchers to study the expressed genes in an organism, independent from nonexpressed DNA.