Gene Patents (Part 4 – the Science, continued)
In Part 3 of this series, we looked at the standard molecular biology tools that can be used to create and manipulate cDNA. In this part, we will look at the ways in which the function of the cDNA may be determined.
Once enough cDNA has been isolated and/or synthesized, we use standard tools to find the sequence of nucleotides in the particular cDNA of interest. Once this is determined, we can determine the chemical structure and characteristics of the cDNA. The particular cDNA could in theory be the subject of a patent application which claims the cDNA as a chemical molecule (more about this in a future post).
However, in addition to its chemical properties, cDNA has informational properties: it contains the genetic code for a particular protein. Knowing the chemical structure will not by itself tell us the protein that it codes for and the function of that protein in the eukaryotic cell from which the cDNA was derived. We will probably have great interest in writing patent claims for the protein and its function (again, more about claiming in a future post).
There are a number of standard methods that can be used to determine the protein that the cDNA codes for. These methods are known as screening methods. Screening methods may be classified as follows.
In silico screening refers to methods that compare the nucleotide sequence of the cDNA of interest or the amino acid sequence of a protein of interest to databases of expressed sequence tags (ESTs) or databases of the complete or partial genome of the organism of interest. This can be done relatively rapidly by computer software such as BLAST (Basic Local Alignment Search Tool). Another variation would be that we have a database of the genome of a related organism (a homologue). Because of the inherent uncertainty of the mathematics, however, we will only have a probability (perhaps quite high) of identifying the protein that the cDNA codes for.
More accurate screening methods are screening by nucleic acid hybridization, screening by expression in vivo, and screening by expression of coding sequences in vitro (Christopher Howe, “Gene Cloning and Manipulation,” Cambridge University Press, 2007).
Screening by nucleic acid hybridization relies on the base pairing (A to T, G to C) through hydrogen bonds between complementary polynucleotides. It uses a probe DNA (labelled in some way such as by radioactivity) to find complementary sequences in the target cDNA. For example, PCR amplification of a known cDNA as described in Part 3 may yield a product that can be used as a probe to find the same sequence in a target cDNA.
Screening by expression in vivo generally relies on selection of a cDNA clone from a library by using a vector to insert the cDNA into a host species (such as E. coli) and then testing for the recombinant host species that expresses the gene of interest, i.e., the genetic phenotype, as described in Part 3.
Screening by expression of coding sequences in vitro relies on the ability of an extract of a bacterial species (e.g. E. coli) to transcribe and translate a plasmid containing the cDNA of interest and detection of the translated product through the incorporation of radiolabelled amino acids into the expressed protein. Transcription and translation were explained in Part 1.
In all of the above methods except the in silico method, the goal is to physically isolate a cDNA that has a high probability of coding for a particular protein of interest, either through expression of the gene in the eukaryotic cell or in another host, or through detection of the protein expressed by the cDNA outside the eukaryotic cell or another living host.