Batt C A, Wagner P, Wiedmann M, Luo J, Gilbert R
Department of Food Science, Cornell University, Ithaca, NY 14853.
Anim Genet. 1994 Apr;25(2):95-8.
A nonisotopic ligase chain reaction (LCR) assay was developed to detect the mutation (D128G; Shuster et al. (1992) PNAS 89, 9225-9) for bovine leukocyte adhesion deficiency (BLAD). Two sets of diagonally opposed discriminating LCR primers that differentiate the normal and BLAD allele were designed so that the 3' end of each primer overlapped the D128G mutation. These discriminating primers were synthesized with a 5' biotin and could be captured using streptavidin-coated microtitre wells. A common set of primers that abut these discriminating primers were also synthesized and 3'-tailed with digoxigenin-ddUTP. Captured LCR products were then detected using antidigoxigenin antibodies coupled to alkaline phosphatase. The assay readout was a chemiluminescent signal generated by the hydrolysis of Lumi-Phos 530 and the entire assay including DNA isolation can be completed within 8 h.
开发了一种非同位素连接酶链反应（LCR）检测方法，用于检测牛白细胞黏附缺陷（BLAD）的突变（D128G；舒斯特等人（1992年），《美国国家科学院院刊》89卷，9225 - 9页）。设计了两组对角相对的区分性LCR引物，用于区分正常等位基因和BLAD等位基因，使每个引物的3'端与D128G突变重叠。这些区分性引物在5'端合成有生物素，可使用链霉亲和素包被的微量滴定板进行捕获。还合成了一组与这些区分性引物邻接的通用引物，并在3'端用洋地黄毒苷 - ddUTP进行尾端标记。然后使用与碱性磷酸酶偶联的抗洋地黄毒苷抗体检测捕获的LCR产物。检测结果是由Lumi - Phos 530水解产生的化学发光信号，整个检测包括DNA分离可在8小时内完成。
