Generation of full-length cDNA libraries enriched for differentially expressed genes for functional genomics.
Here, we describe the application of a RecA-based cloning technology to generate full-length cDNA libraries enriched for genes that are differentially expressed between tumor and normal tissue samples. First, we show that the RecA-based method can be used to enrich cDNA libraries for several target genes in a single reaction. Then, we demonstrate that this method can be extended to enrich a cDNA library for many full-length cDNA clones using fragments derived from a subtracted cDNA population. The results of these studies show that this RecA-mediated cloning technology can be used to convert subtracted cDNAs or a mixture of several cDNA fragments corresponding to differentially expressed genes into a full-length library in a single reaction. This procedure yields a population of expression-ready clones that can be used for further high-throughput functional screening.
Zhumabayeva
B
B
Clontech Laboratories, Inc., 1020 East Meadow Circle, Palo Alto, CA 94303, USA. bdzhumabayeva@clontech.com
Chang
C
C
McKinley
J
J
Diatchenko
L
L
Siebert
P D
PD
D018076:DNA, Complementary / Q000302:isolation & purification*; D015723:Gene Library; D023281:Genomics; D006801:Humans; D011938:Rec A Recombinases / Q000235:genetics
D018076:DNA, Complementary; D011938:Rec A Recombinases