Preparation of a differentially expressed, full-length cDNA expression library by RecA-mediated triple-strand formation with subtractively enriched cDNA fragments.
We have developed a fast and general method to obtain an enriched, full-length cDNA expression library with subtractively enriched cDNA fragments. The procedure relies on RecA-mediated triple-helix formation of single-stranded cDNA fragments with a double-stranded cDNA plasmid library. The complexes were then captured from the solutions using the digoxigenin-antidigoxigenin paramagnetic beads followed by recovery of the enriched double-stranded cDNA expression library. We have observed a linear relation between the capture of full-length cDNAs in the library and the fold enrichment in the subtracted cDNA population.
Hakvoort
T B
TB
Department of Anatomy and Embryology, University of Amsterdam, The Netherlands.
Spijkers
J A
JA
Vermeulen
J L
JL
Lamers
W H
WH
D000345:Affinity Labels; D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA; D018076:DNA, Complementary / Q000235:genetics*; D004076:Digoxigenin; D015870:Gene Expression; D015723:Gene Library; D008115:Liver Regeneration; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D051381:Rats; D011938:Rec A Recombinases
D000345:Affinity Labels; D018076:DNA, Complementary; C051802:triplex DNA; D004247:DNA; D011938:Rec A Recombinases; D004076:Digoxigenin