Automated DNA fingerprinting analysis of Mycobacterium tuberculosis using fluorescent detection of PCR products

Abstract

DNA fingerprints of Mycobacterium tuberculosis are produced by restriction fragment length polymorphism analysis of the insertion element IS6110. We modified a PCR-based subtyping method, mixed-linker PCR with fluorescent-labeled IS6110-specific oligonucleotides, to demonstrate rapid, automated, and unattended electrophoretic analysis. Variation in band sizing (normally occurring with fragment mobility), an artifact of lane-to-lane and gel-to-gel differences, was controlled with an internal lane standard, resulting in accurate and precise DNA sizing. By using this method, fingerprint analysis can be performed using actual fragment length rather than estimated position analysis.