POLYNUCLEOTIDE HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

在上一个资助期间，我们开发了将DNA 特别是通过其5 ′-末端连接到大孔HPLC二氧化硅（DNA- 二氧化硅）。 用一种特殊的DNA-二氧化硅，（dT）18-二氧化硅， 显示通过杂交特异性结合寡核苷酸， 以分辨仅由单个碱基对区分的寡核苷酸。 等 色谱柱也用于非常快速和高分辨率的分离， 基于多聚腺苷酸差异的腺苷酸酶长度信使RNA（mRNA） 尾长 最近，这些DNA-二氧化硅已被用于引发 将模板DNA直接（和共价）酶促复制到二氧化硅上 两种不同的方法。 其中之一使用DNA聚合酶（Klenow 片段）以通过引物延伸复制模板DNA。 另一个使用 聚合酶链式反应（PCR）来扩增DNA并将DNA附着到二氧化硅上。 这些令人兴奋的新方法允许复制自然发生的或 在二氧化硅上合成DNA（或RNA）并允许制备DNA-二氧化硅 用化学方法是不可能的。 可以制备单链或双链DNA-二氧化硅， 它们可以可逆地相互转化。 现在我们将推广这些结果。 将开发出更有效的将DNA复制到表面上的方法。 我们还将开发一种简单的方法，直接复制到二氧化硅上 实际上任何已经克隆到质粒中的DNA。 潜在用途 DNA-二氧化硅用于1）合成大量的高纯度单 实际上任何长度的单链DNA和RNA，2）杂交选择 实验，3）DNA结合蛋白分离，和4）DNA的自动化 测序都将被调查。 这些研究可能会有一个 对分子生物学、癌症研究和 病毒复制，并可能导致更有效的方法来确定大 DNA序列，如人类基因组。

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.