POLYNUCLEOTIDE HIGH PERFORMANCE AFFINITY CHROMATOGRAPHY

多核苷酸高效亲和层析

• 批准号: 3302701
• 负责人: HARRY W JARRETT
• 金额: $ 10.52万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-08-01 至 1992-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3302701
• 关键词: Escherichia coli; affinity chromatography; bacterial virus; clinical biomedical equipment; genetic disorder diagnosis; high performance liquid chromatography; messenger RNA; molecular biology; plasmids; polynucleotides; recombinant DNA; restriction mapping; transfection

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.

拟议的实验将开发一种强有力的方法， 多核苷酸（PN）分离，高效亲和 色谱法（PN-HPAC）。 PN-HPAC可以提供快速， 临床诊断为遗传性遗传病的病变， DNA是已知的。 分子生物学及其在人类 健康和福祉也应该受益于发展 PN-HPAC。 该方法包括将确定的DNA序列连接到 大孔二氧化硅载体和制备色谱柱。 所连接的DNA将通过碱基配对与 互补的PN链，即使在存在其他PN 序列的 PN杂交的特异性应允许高度的 选择性色谱;通过类比，PN-HPAC代表一种 “反向南方杂交”。 计算表明， 具有动力学的小柱（1 x 10 mm）应允许快速 分离要进行。 相应地，这些列将 用于从粗品中快速分离mRNA、质粒和RNA， 细胞提取物 从分析上讲，这些列应该对以下两方面都有用： 临床和研究;测试选择性的模型研究 的PN-HPAC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的易感性 HPAC在重组DNA实验和E.杆菌 将建立。 这些模型研究应建立PN-HPAC 作为分子生物学的宝贵工具。