Purification of specific DNA sequences by sulfhydryl-Sepharose chromatography of mercurated polynucleotides.
Recombinant plasmid DNA has been used to purify complementary cDNA by hybridization using a modification of sulfhydryl-Sepharose chromatography described by Dale and Ward ((1975) Biochemistry 14, 2458). Plasmid DNA containing cloned mouse globin or immunoglobulin sequences was mercurated and hybridized in solution to unpurified cDNA. The resulting hybrids were passed over a sulfhydryl-Sepharose column where mercurated polynucleotides are retained. After washing, cDNA hybridized to the mercurated plasmid DNA was melted in situ and eluted while the mercurated plasmid DNA remained bound to the column. The conditions for purification of DNA and RNA sequences are described. The purity of the cDNAs obtained by this method is analyzed by polyacrylamide gel electrophoresis and by hybridization. In addition, this nucleic acid purification procedure has been applied to two problems of general interest: (i) the sensitive titration of specific genes by saturation hybridization; (ii) the purification of DNA fragments bearing specific sequences from restriction endonuclease digests of total cellular DNA. The procedure is generally applicable to the purification by hybridization of any DNA or RNA sequence complementary to an available probe.
D001483:Base Sequence; D002846:Chromatography, Affinity; D004247:DNA / Q000302:isolation & purification*; D007700:Kinetics; D008628:Mercury; D008970:Molecular Weight; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000302:isolation & purification*; D009841:Oligonucleotides / Q000302:isolation & purification*; D010957:Plasmids; D011995:Recombination, Genetic
D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides; D004247:DNA; D008628:Mercury