ENZYME-LINKED FLUORESCENT DETECTION FOR AUTOMATED MULTIPLEX DNA-SEQUENCING

Initiatives to sequence DNA on a large scale have created a need for i ncreased throughput and decreased costs. One scheme for increasing thr oughput, multiplex sequencing, involves the processing of a mixture of sequencing templates followed by sequential hybridization to reveal t he individual sequence ladders on a membrane. Because multiplex sequen cing has not been fully automated, and has not seemed automatable, few sequencing efforts have attempted to exploit it. We describe here a s cheme for the automation of multiplex sequencing. Probe hybridized to target DNA is detected via spatially localized enzyme-linked fluoresce nce. Light output is high enough that imaging is possible with simple instrumentation. Direct imaging within an automated hybridization appa ratus is made feasible so that the entire process will be automatic on ce a multiplex membrane is produced. The technique has the potential t o increase severalfold the throughput of automated sequencing instrume nts required for sequencing the human genome. (C) 1994 Academic Press, Inc.