GENE ISOLATION PRIOR TO IDENTIFICATION OF PROTEINS

Forward genetics seeks to identify and clone genes that are associated with mutant phenotypes. Such cloning can be accomplished by various strategies whose development has become more sophisticated and efficient with the concurrent developments in sequencing technologies. There is, at the same time, heavy reliance on protein information to identify genes due to the advent of high throughput nucleic acid sequencing technologies and advanced proteomics analysis techniques. These protein sequence-based cloning techniques are sometimes inadequate in biotechnology especially in the absence of comprehensive nucleic acid sequence databases for the target organism. Discovery of specific genes is a key factor in biotechnology because identification of drug targets is a commonplace activity and often a defining step in the discovery process. Forward genetics or other methods of isolation of novel genes that do not rely on previous protein identification (or reverse genetics) is useful and has proved to be successful in finding novel genes and, in some cases, immediately assign them to a pathological condition because of the obvious phenotype of the mutation. This paper gives an overview of currently used techniques in the area of forward genetics and molecular biology-based gene discovery. Very often these techniques are modified and are improved versions of methods whose theoretical bases were laid in the 1970’s when molecular biology took giant leaps. Although mutagenesis predates current technologies, there is a highly beneficial synergy between retroviral mutagenesis strategy and high throughput sequencing.

Keywords: forward genetics; gene discovery; mutagenesis; pyrosequencing, proteomics; subtractive hybridization