Genes, isolation

Taq DNA polymerase from Thermus aquaticus) has made it unnecessary to add fresh enzyme for each round of synthesis. Because the amount of target DNA theoretically doubles each round, 25 rounds would increase its concentration about 33 million times. In practice, the increase is actually more like a million times, which is more than ample for gene isolation. Thus, starting with a tiny... 

Grunstein, M., and Hogness, D. S., 1975. Colony hybridization A specific method for the isolation of cloned DNAs tliat contain a specific gene. Proceedings of the National Academy of Sciences U.S.A. 72 3961—3965. Article describing die colony hybridization technique for specific gene isolation. 

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. 

Gene isolation is rendered most simple if there is available a corresponding gene product (protein) for which we have some sequence information or an antibody. The approach is further facilitated if the product is abundant and is therefore represented by an abundant mRNA... 

Fig. 6. The potential of, and problems with, gene isolation by linkage.

Krynetski EY, Fessing MY, Yates CR et al. Promoter and intronic sequences of the human thiopurine S-methyltransfer-ase (TPMT) gene isolated from a human PAC1 genomic library. Pharm Res 1997 14 1672-1678. 

BISHOP, G.J., HARRISON, K., JONES, J.D., The tomato Dwarf gene isolated by heterologous transposon tagging encodes the first member of a new cytochrome P450 family, Plant Cell, 1996, 8, 959-969. 

Cahilly-Snyder, L., et ah. Molecular analysis and chromosomal mapping of amplified genes isolated from a transformed mouse 3T3 cell line. Somat Cell Mol Genet, 1987, 13(3), 235-44. 

Mizuno, D., Higuchi, K., Sakamoto, T., Nakanishi, H., Mori, S. and Nishizawa, N. 2003. Three nicotianamine synthase genes isolated from maize are differentially regulated by iron nutritional stams. Plant and Cell Physiology, 44 S148-S148. 

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