抗生物素蛋白-生物素基因富集方法在含有特定基因序列的长双链DNA分离中的应用。

Application of the avidin-biotin method of gene enrichment to the isolation of long double-stranded DNA containing specific gene sequences.

作者信息

Pellegrini M, Holmes D S, Manning J

出版信息

Nucleic Acids Res. 1977 Sep;4(9):2961-73. doi: 10.1093/nar/4.9.2961.

DOI:10.1093/nar/4.9.2961

PMID:198749

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC342627/

Abstract

A method of enriching for long double-stranded segments of eukaryotic DNA carrying particular genes is described. A purified RNA coded for by the gene is covalently attached to biotin via the protein, cytochrome c. This modified RNA is hybridized to total nuclear, double-stranded DNA under conditions that allow the formation of R-loops. Avidin, which has a high affinity for biotin, is covalently attached to polymer spheres. The complexes of avidin-spheres with DNA:RNA-biotin R-loop hybrids band in CsCl at a much lower bouyant density than does free DNA. This density is a function of the length of DNA coupled per avidin-sphere. This method was used to prepare very long double-strands of DNA highly enriched in the coding sequences for the large rRNAs of D. melanogaster and L. donovani and the histone mRNAs of S. purpuratus.

摘要

本文描述了一种富集携带特定基因的真核生物DNA长双链片段的方法。由该基因编码的纯化RNA通过细胞色素c蛋白与生物素共价连接。在允许形成R环的条件下，这种修饰的RNA与总的核双链DNA杂交。对生物素有高亲和力的抗生物素蛋白与聚合物球体共价连接。抗生物素蛋白-球体与DNA:RNA-生物素R环杂交体的复合物在CsCl中的浮力密度比游离DNA低得多。这种密度是每个抗生物素蛋白-球体结合的DNA长度的函数。该方法用于制备高度富集黑腹果蝇和杜氏利什曼原虫大rRNA编码序列以及紫球海胆组蛋白mRNA的非常长的双链DNA。