DETECTION OF BOVINE LEUKOCYTE ADHESION DEFICIENCY BY NONISOTOPIC LIGASE CHAIN-REACTION

A nonisotopic ligase chain reaction (LCR) assay was developed to detec t the mutation (D128G; Shuster et al. (1992) PNAS 89, 9225-9) for bovi ne leukocyte adhesion deficiency (BLAD). Two sets of diagonally oppose d discriminating LCR primers that differentiate the normal and BLAD al lele were designed so that the 3' end of each primer overlapped the D1 28G mutation. These discriminating primers were synthesized with a 5' biotin and could be captured using streptavidin-coated microtitre well s. A common set of primers that abut these discriminating primers were also synthesized and 3'-tailed with digoxigenin-ddUTP. Captured LCR p roducts were then detected using antidigoxigenin antibodies coupled to alkaline phosphatase. The assay readout was a chemiluminescent signal generated by the hydrolysis of Lumi-Phos(TM) 530 and the entire assay including DNA isolation can be completed within 8 h.