Construction of cDNA libraries by blunt-end ligation: high-frequency cloning of long cDNAs from filamentous fungi.
A simplified cDNA synthesis and cloning method, suitable for efficient generation of cDNA libraries at frequencies up to 10(6) clones/micrograms mRNA, is described. Routine synthesis of transcripts of well over 4 kb is facilitated by the use of high-quality RNA template isolated from materials rich in RNases. Laborious cloning steps, like tailing or addition of linkers, can be omitted by the use of efficient blunt-end ligation to plasmid vectors, and rapid verification as well as characterization of the clones is possible by double-stranded plasmid sequencing. Using this method we have constructed several cDNA libraries of different filamentous fungi and show here the synthesis and cloning of cDNA copies larger than 1.8 kb corresponding to three Trichoderma reesei cellulases.
Teeri
T T
TT
VTT, Biotechnical Laboratory, Espoo, Finland.
Kumar
V
V
Lehtovaara
P
P
Knowles
J
J
D010582:Bacteriophage lambda / Q000235:genetics; D002480:Cellulase / Q000235:genetics; D043366:Cellulose 1,4-beta-Cellobiosidase; D002499:Centrifugation, Density Gradient; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000096:biosynthesis / Q000235:genetics*; D011088:DNA Ligases / Q000378:metabolism; D004262:DNA Restriction Enzymes; D006026:Glycoside Hydrolases / Q000235:genetics; D003904:Mitosporic Fungi / Q000235:genetics*; D010957:Plasmids; D012331:RNA, Fungal / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D013698:Templates, Genetic; D014170:Transformation, Genetic; D014242:Trichoderma / Q000235:genetics*
D012331:RNA, Fungal; D012333:RNA, Messenger; D004247:DNA; D004262:DNA Restriction Enzymes; D006026:Glycoside Hydrolases; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase; D011088:DNA Ligases