One of the main goals of molecular genetics is to determine the base sequence of the human genome. This is the purpose of the Human Genome Project, an international collaborative research programme aimed at providing a complete analysis of the human genome within the next decade. The first step in such an analysis is to isolate the small sequences of bases in DNA that are transcribed into mRNAs. The information contained in the mRNAs can be isolated and amplified by a technique called cDNA cloning. In this technique, mRNAs from brain tissue, for example, are purified and then treated with reverse transcriptase which converts mRNAs into single complementary strands of DNA. This is called complementary DNA (cDNA). The cDNA provides a template for producing a second strand that is complementary to the first. This double-stranded cDNA is then incubated with bacterial plasmids to produce recombination DNA plasmids. Plasmids may be considered as bacterial viruses that can reproduce themselves when inserted into the appropriate bacteria so that during the process of bacterial cell division multiple copies of the cDNA that had been inserted in the plasmid are formed. As each bacterium is likely to be infected with a plasmid, containing a different type of cDNA, the resulting medium will contain a mixed population of cDNAs from the original brain tissue. This is called a cDNA library.
The individual components of the cDNA library may be obtained by grouping individual bacteria on a culture medium so that they reproduce to form identical clones. This enables a large quantity of specific cDNAs to be produced in a pure form. The cDNA within these plasmid-containing bacteria can then be removed, and the precise nucleotide sequence determined by standard automated analytical procedures.
Since the brain expresses many mRNAs that are also found in non-nervous tissue and are therefore of little interest to the psycho-pharmacologist, it is necessary to isolate only those cDNAs that, for example, encode for a specific enzyme or receptor protein. Several techniques have been developed to achieve this. For example, a specific cDNA plasmid may be inserted into cultured mammalian cells such as fibroblasts that can express the specific receptor or enzyme. Once this has been expressed in the culture medium, the receptor or enzyme can be identified by adding a specific ligand or substrate. This enables those cells that expressed the specific macromolecule of interest to be identified and
Reverse transcriptase transcribes these different mRNAs into single complementary strands of DNA
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