Genetic approaches reverse genetics

Fig. 4. A comparison of the approaches of reverse genetics and conventional genetics for the analysis of gene function.

As this suggests, the major problem we face is the recognition and isolation of genes which confer stress tolerance. Thereafter, we may use reverse genetics to confirm the adaptive significance of these genes. Other approaches to gene isolation will now be discussed. 

PrPc has a predominantly a-helical conformation 796 Reverse genetics approaches to studying PrPc 797 The function of PrPc remains unknown 797 PrP knockout mice have subtle abnormalities of synaptic transmission 797... 

Reverse genetic approaches start with a sequence and attempt to identify a function for the gene. This can be done by using PCR-based screens on DNA from insertionally mutagenized populations, ectopic misexpression of the gene, and expression studies, e.g., with RT-PCR or microarray analyses. 

Boerjan W, Ralph J, Baucher M (2003) Lignin biosynthesis. Annu Rev Plant Biol 54 519-546 Dixon RA, Reddy MSS (2003) Biosynthesis of monolignols. Genomic and reverse genetic approaches. Phytochem Rev 2 289-306... 

The reverse genetic approach relies on the construction of suitable libraries. The initial step is to homogenize the tissue and isolate the mRNA population. As mRNAs are single stranded and relatively unstable, they are first converted to their complimentary DNA (cDNA) sequence, and then into the more stable double-stranded cDNA molecule. The next step is to find a suitable vector that can carry the cDNA molecule and replicate itself multiple times. This will allow each message to be amplified to the large numbers of copies required for isolation and purification. 

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