[26] Purification of nucleic acids by RPC-5 ANALOG chromatography: Peristaltic and gravity-flow applications

RPC-5 column chromatography was established as a powerful technique for the purification (especially preparatively) of nucleic acids. Early versions of RPC column chromatography (RPC-1 to RPC-4) consisted of water-immiscible organic quaternary ammonium compounds coated on diatomaceous earth supports. These were used extensively for the fractionation of transfer RNAs isolated from a wide variety of sources. Later, changing the support to a polychlorotrifluoroethylene resin (RPC-5) led to improved separation of tRNA isoacceptors, shorter analysis times, and easier scale-up for preparative separations. RPC-5 column chromatography is a useful as a tool for both the analytical and preparative fractionation of DNA fragments generated by restriction endonuclease cleavage, of complementary strands of specific DNA restriction fragments, of oligomers of single-stranded DNA and RNA, and of supercoiled plasmid ColE1 DNA. Several difficulties encountered with the originally reported chromatography matrix, however, hindered the widespread use of this technique. The most significant problem was the lack of commercial availability of the matrix. Other difficulties resulted from the composition of the original matrix.