Robot printing of reverse dot blot arrays for human mutation detection

We report on a generally useful, partially automated, human mutation detect ion method based upon printing moderate density oligonucleotide arrays usin g a biorobot on activated nylon membranes. The Beckman Biomek 2000 was adap ted to this task through fabrication of aluminum membrane filter holders an d the development of an addressable Tool Command Language (Tcl) program, wh ich can be invoked through BioScript. During program execution, a robot arm is moved along the x, y, and z axes to expel liquid, without dripping, fro m disposable barrier pipette tips and then to touch the drops on preactivat ed membranes. Printed arrays consist of alternating rows of oligonucleotide s containing normal and mutant sequences. Hybridization of biotin labeled p olymerase chain reaction products derived from human patient genomic DNA sa mples are visualized using chemiluminescent or chromogenic indicators. This technique allows unequivocal genotyping of 32 mutations at the beta -thala ssemia locus (11p15.5) and of 34 mutations and one polymorphism at the cyst ic fibrosis transconductance membrane regulator locus (7p35).