Now we are ready to examine the technical procedures (and some of the theories behind them) that allow molecular biologists to detect, study and clone individual genes--and even produce the proteins coded for by these genes in commercial amounts. These developments have sparked a revolution in the biological sciences and will continue to have far-reaching implications for every area of biology and medicine in the years ahead.
The practice of genetic engineering, or gene cloning, had to await the
development of suitable methods in the early 1970s for cleaving, joining
and manipulating nucleic acids. The basic ideas behind the procedures are
easy to state: genetic information is encoded in genes which are made up
of long polymers of nucleotides in the form of double stranded DNA molecules.
Cleave genomic DNA into large fragments using restriction endonucleases. Restriction endonucleases are used to cleave genomic DNA into large fragments at defined nucleotide sequences.
Insert the DNA into plasmids. These pieces of DNA are inserted into plasmids, which are self-replicating, extrachromosomal genetic elements originally isolated from the bacterium, Escherichia coli. The circular plasmid DNA is opened using the same endonuclease that was used to cleave the genomic DNA.
Join the ends of DNA with the enzyme, DNA ligase. The inserted DNA is joined to the plasmid DNA using another enzyme, DNA ligase, to give a recombinant DNA molecule. The new plasmid vector contains the original genetic information for replication of the plasmid in E. coli plus the inserted DNA which may contain, for example, the human gene for insulin.
Introduce the new vector into E. coli. The new vector is inserted back into E. coli where many copies of the genetic sequence are made as the bacteria grow and divide with the replicating vector inside.
Isolate the newly-synthesized DNA, or the protein coded for by the inserted gene. The E. coli may even transcribe and translate the gene and obligingly produce insulin, if that was the gene incorporated into the vector. Alternatively, many copies of the DNA gene itself may be isolated for sequencing the nucleic acid or for other biochemical studies.
Many variations on the basic methodology have been developed. We will consider only three: the use of vectors made from bacterial viruses (or bacteriophages); cosmid vectors; and the polymerase chain reaction (PCR). Additional methodologies may be found in the reference list.
Figure 16. The general scheme for genetic engineering