POLYNUCLEOTIDE HIGH PERFORMANCE AFFINITY CHROMATOGRAPHY

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During the last funding period, we developed methods for coupling DNA specifically by way of its 5'-terminus to macroporous HPLC silica (DNA- silica). Columns packed with one particular DNA-silica, (dT)18-silica, were shown to bind oligonucleotides specifically by hybridization, and to resolve oligonucleotides differing by a single base pair. Such columns were also used for very rapid and high resolution separations of kilobase length messenger RNAs (mRNA) based upon differences in poly(A) tail length. Recently, these DNA-silicas have been used to prime the enzymatic copying of template DNA directly (and covalently) onto silica by two different methods. One of these uses DNA polymerase (Klenow fragment) to copy a template DNA by primer extension. The other uses the polymerase chain reaction (PCR) to both amplify and attach DNA to silica. These exciting new methods allow the copying of naturally occurring or synthetic DNA (or RNA) onto silica and allow one to prepare DNA-silica with lengths of DNA which would not be feasible by chemical means. either single-stranded or double-stranded DNA-silicas can be made and they can be reversibly interconverted. We will now extend these results. More efficient methods for copying DNA onto surfaces will be developed. We will also develop a simple method to copy directly onto silica virtually any DNA which has been cloned in a plasmid. The potential uses of DNA-silica for 1) the synthesis of large amounts of high purity single stranded DNA and RNA of virtually any length, 2) hybrid selection experiments, 3) DNA-binding protein isolation, and 4) automation of DNA sequencing will all be investigated. These studies will likely have a significant impact on molecular biology, investigations of cancer and viral replication, and may lead to more efficient ways to determine large DNA sequences, such as the human genome.