A hybridization and enzyme blocking method to enrich minor cDNA sequences.
A method is presented which allows for the enrichment of low frequency cDNA sequences. The crucial step in the procedure is the hybridization of a pool of cDNA to homologous or heterologous RNA to a Rot value which will leave minor sequences in a single strand cDNA form while the major sequences form cDNA:RNA hybrids. This allows subsequent enzymatic differentiation between major and minor sequences resulting ultimately in the degradation of the major sequences. The procedure is general and simple as it requires no column chromatography step. The method is designed to integrate into a widely used cDNA cloning protocol and results either in double-stranded cDNA which can be used for molecular cloning or as a source of probes for hybridization.
D000818:Animals; D001345:Autoradiography; D002645:Chickens; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D004720:Endonucleases; D008722:Methods; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase; D015719:Single-Strand Specific DNA and RNA Endonucleases
D012333:RNA, Messenger; D004247:DNA; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D004720:Endonucleases; D015719:Single-Strand Specific DNA and RNA Endonucleases