The use of R-looping for structural gene identification and mRNA purification.
A method is presented for the purification of mRNAs and the identification of structural gene sequences in recombinant DNA molecules. RNA is hybridized to double-stranded linear DNA such that R-loops are formed between most DNAs and their complementary RNA sequences. These R-loops are purified from unhybridized RNAs by gel filtration chromatography in the presence of a high concentration of salt. The complementary RNAs are released from the R-loops by heating, and are assayed by gel electrophoresis or cell free translation to determine their purity and to identify the proteins for which they code. We have demonstrated that recombinant DNAs containing sequences for abundant or moderately abundant mRNAs of Saccharomyces cerevisiae can be identified by this means.
D001483:Base Sequence; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000032:analysis; D005796:Genes; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D012333:RNA, Messenger / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000032:analysis
D004274:DNA, Recombinant; D012333:RNA, Messenger