POLYNUCLEOTIDE HIGH PERFORMANCE AFFINITY CHROMATOGRAPHY

拟议中的实验将开发出一种强大的方法来 多核苷酸(PN)分离，高效亲和力 层析(PN-HPAC)。PN-HPAC可以提供快速、 遗传性遗传病的临床诊断 在DNA上是已知的。分子生物学及其在人类中的应用 健康和福祉也应受益于 PN-HPAC。该方法包括将定义的DNA序列附加到 大孔二氧化硅载体和制备色谱柱。 连接的DNA将通过碱基配对与 互补的PN链，即使在存在其他PN的情况下 序列。PN杂交的特异度应该很高 选择层析；以此类推，PN-HPAC代表一种 “反向Southern杂交”。提出的计算表明 带有动力学的小柱子(1 X 10 Mm)应该允许快速 要执行的分色。在准备阶段，这些栏目将 用于从粗品中快速分离出信使核糖核酸、质粒和噬菌体 细胞萃取物。从分析上讲，这些列应该既有用又有用 临床和研究；测试选择性的模型研究 PN-HPAC的应用将证明这些应用的可行性。 其他实验和分离的M13噬菌体Pn-1的敏感性 HPAC在重组DNA实验中的应用及在大肠杆菌中的转染 将会建立起来。这些模型研究应建立PN-HPAC 作为分子生物学的宝贵工具。

The proposed experiments will develop a powerful method for polynucleotide (PN) separations, High Performance Affinity Chromatography (PN-HPAC). PN-HPAC may provide the means for rapid, clinical diagnosis for inherited genetic disease where the lesion on DNA is known. Molecular biology, and its applications to human health and well-being should also benefit from the development of PN-HPAC. The method involves attaching a defined DNA sequence to macroporous silica supports and preparing a chromatography column. The attached DNA will specifically hybridize by base pairing with complementary PN strands, even in the presence of other PN sequences. The specificity of PN hybridization should allow highly selective chromatography; by analogy, PN-HPAC represents a sort of "reversed Southern hybridization". Calculations presented suggest that small columns (1 x 10 mm) with kinetics should allow rapid separations to be performed. Preparatively, the columns will be used to rapidly separate mRNA, plasmids, and phages from crude cellular extracts. Analytically, the columns should be useful both clinically and for research; model studies testing the selectivity of PN-HPAC will demonstrate the feasibility of these applications. Other experiments and the susceptibility of isolated M13 phage PN- HPAC in recombinant DNA experiments and transfection of E. coli will be established. These model studies should establish PN-HPAC as a valuable tool for molecular biology.