Walsh M K, Wang X, Weimer B C
Department of Nutrition and Food Sciences, Center for Microbial Detection and Physiology, Utah State University, Logan, UT 84322-8700, USA.
J Biochem Biophys Methods. 2001 Feb 26;47(3):221-31. doi: 10.1016/s0165-022x(00)00146-9.
The attachment of single-stranded DNA to a solid support has many biotechnology and molecular biology applications. This paper compares different immobilization chemistries to covalently link single-stranded DNA (20 base pairs), oligo(1), onto glass beads via a 5'-amino terminal end. Immobilization methods included a one-step 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and a two-step EDC reaction to succinylated and PEG-modified glass beads. The third method used 1,4-phenylene diisothiocyanate to immobilize oligo(1) to aminopropyl glass beads. The influence of coupling buffer, oligo(1) concentration, and EDC concentration was also investigated. The one-step EDC-mediated procedure with succinylated or PEG-modified beads in 0.1 M MES buffer, pH 4.5, resulted in the highest immobilization efficiency, 82-89%. EDC concentrations greater than 50 mM and oligo(1) concentrations of 3 microg/g bead were required for effective immobilization. A complementary oligonucleotide, oligo(2), was able to hybridize to the immobilized oligo(1) with a 58% efficiency. This oligonucleotide was subsequently released at 70 degrees C. The relationship between the surface density of oligo(1) and the hybridization efficiency of the complementary oligonucleotide is described.
单链DNA与固相载体的连接在许多生物技术和分子生物学应用中都有应用。本文比较了不同的固定化学方法，以通过5'-氨基末端将单链DNA（20个碱基对），即寡核苷酸(1)，共价连接到玻璃珠上。固定方法包括一步法使用1-乙基-3-(3-二甲基氨基丙基)碳二亚胺（EDC）以及两步法将EDC与琥珀酰化和聚乙二醇修饰的玻璃珠反应。第三种方法使用1,4-亚苯基二异硫氰酸酯将寡核苷酸(1)固定到氨丙基玻璃珠上。还研究了偶联缓冲液、寡核苷酸(1)浓度和EDC浓度的影响。在pH 4.5的0.1 M MES缓冲液中，用琥珀酰化或聚乙二醇修饰的珠子进行一步法EDC介导的程序，固定效率最高，为82-89%。有效固定需要EDC浓度大于50 mM，寡核苷酸(1)浓度为3 μg/g珠子。互补寡核苷酸寡核苷酸(2)能够以58%的效率与固定的寡核苷酸(1)杂交。该寡核苷酸随后在70℃下释放。描述了寡核苷酸(1)的表面密度与互补寡核苷酸杂交效率之间的关系。
