In-tube cDNA cloning method using a biotinylated RNA probe.
We report a simple, practical method for isolating a particular cDNA from a single-stranded (ss) cDNA library in a tube without using a radioisotope. The method consists of three steps: (i) the capture of a target cDNA by a biotinylated RNA via intermolecular hybrid formation, (ii) the binding of the cDNA-RNA hybrid to an avidin-coated gel, (iii) the recovery of the target cDNA by degradation of the RNA under mild alkaline conditions. The effectiveness of the method was examined by isolating a clone carrying a metapyrocatechase gene from a model library. This model experiment achieved an enrichment of 4800-fold. This method was applied to cloning of a cDNA encoding interleukin 8 (IL-8) from a cDNA library prepared from phorbol myristate acetate-treated U937 cells. Using an RNA probe prepared from a truncated cDNA encoding IL-8, the corresponding full-length cDNA clones were successfully obtained.
Kim
N S
NS
Kanagawa Academy of Science and Technology, Japan.
Kato
S
S
D001710:Biotin; D003001:Cloning, Molecular / Q000379:methods; D016373:DNA, Antisense / Q000235:genetics / Q000302:isolation & purification; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000302:isolation & purification*; D015698:Genomic Library; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics; D009693:Nucleic Acid Hybridization; D012313:RNA / Q000235:genetics; D015347:RNA Probes
D016373:DNA, Antisense; D018076:DNA, Complementary; D004277:DNA, Single-Stranded; D016209:Interleukin-8; D015347:RNA Probes; D012313:RNA; D001710:Biotin