Hybrid selection of mRNA with biotinylated DNA.
A novel, convenient, and highly efficient hybrid selection procedure is described. The method utilizes the polymerase chain reaction (PCR) in which one of two primers is biotinylated at the 5'-terminus. The concentration of the biotinylated primer is 100 times that of the other to synthesize biotinylated single-stranded DNA (asymmetric PCR). After hybridization of the biotinylated DNA with mRNA in solution, streptavidin agarose is used to trap the hybrid duplex of mRNA.DNA-biotin onto the solid matrix. The selected mRNA is then eluted from the streptavidin agarose. The quantitative physical recovery of selected mRNA is about 70% with about 33% retention of biological activity.
Soh
J
J
Department of Molecular Genetics and Microbiology, UMDNJ-Robert Wood Johnson Medical School, Piscataway 08854-5635.
Pestka
S
S
D000818:Animals; D001426:Bacterial Proteins; D001483:Base Sequence; D001710:Biotin / Q000378:metabolism; D002847:Chromatography, Agarose; D004277:DNA, Single-Stranded / Q000378:metabolism; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009865:Oocytes; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000302:isolation & purification*; D019809:Streptavidin; D014982:Xenopus laevis / Q000235:genetics*
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D012333:RNA, Messenger; D001710:Biotin; D019809:Streptavidin