Selective enrichment of cDNAs from salt-stress-induced genes in the wheatgrass, Lophopyrum elongatum, by the formamide-phenol emulsion reassociation technique.
We present a novel technique for the enrichment of cDNA libraries to enhance the abundance of clones of differentially expressed genes. The technique is relatively simple, requires moderate quantities of poly(A) + RNA and results in preferential enrichment of clones derived from mRNAs that were of low abundance in their original population. This method was used to isolate cDNA clones of salt-stress-induced genes in the roots of Lophopyrum elongatum, a highly salt-tolerant wheatgrass. An excess of sonicated plasmid DNA from a cDNA library from nonstressed roots was hybridized in a formamide-phenol emulsion with inserts from a cDNA library of stressed roots. Clones that were more abundant in, or were unique to, the library of the stressed roots were recovered as double-stranded fragments by virtue of reconstituted restriction-enzyme-digested ends by ligating them to a plasmid vector. The resulting enriched library was screened by differential colony hybridization and clones of eleven different genes that were more strongly expressed in stressed roots than in controls were selected.
Gulick
P J
PJ
Department of Agronomy and Range Science, University of California, Davis 95616.
Dvorák
J
J
D015152:Blotting, Northern; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D004655:Emulsions; D005559:Formamides; D015723:Gene Library; D017343:Genes, Plant; D005821:Genetic Techniques; D010636:Phenols; D010957:Plasmids; D006109:Poaceae / Q000235:genetics*; D012965:Sodium Chloride / Q000494:pharmacology*; D014170:Transformation, Genetic
D004655:Emulsions; D005559:Formamides; D010636:Phenols; D012965:Sodium Chloride; D004247:DNA