Robot printing of reverse dot blot arrays for human mutation detection.
We report on a generally useful, partially automated, human mutation detection method based upon printing moderate density oligonucleotide arrays using a biorobot on activated nylon membranes. The Beckman Biomek 2000 was adapted to this task through fabrication of aluminum membrane filter holders and the development of an addressable Tool Command Language (Tcl) program, which 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, from disposable barrier pipette tips and then to touch the drops on preactivated membranes. Printed arrays consist of alternating rows of oligonucleotides containing normal and mutant sequences. Hybridization of biotin labeled polymerase chain reaction products derived from human patient genomic DNA samples are visualized using chemiluminescent or chromogenic indicators. This technique allows unequivocal genotyping of 32 mutations at the beta-thalassemia locus (11p15.5) and of 34 mutations and one polymorphism at the cystic fibrosis transconductance membrane regulator locus (7p35).
Lappin
S
S
Quest Diagnostics, Van Nuys, and Beckman/Coulter, Fullerton, California, USA.
Cahlik
J
J
Gold
B
B
D002880:Chromosomes, Human, Pair 11 / Q000235:genetics; D002897:Chromosomes, Human, Pair 7 / Q000235:genetics; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D005838:Genotype; D006801:Humans; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods*; D011110:Polymorphism, Genetic; D011327:Printing; D012371:Robotics / Q000379:methods*; D017086:beta-Thalassemia / Q000235:genetics
C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator