A vertically stacked, polymer, microfluidic point mutation analyzer: Rapid high accuracy detection of low-abundance K-ras mutations

摘要: Abstract Recognition of point mutations in the K- ras gene can be used for clinical management several types cancers. Unfortunately, assay and hardware concerns must addressed to allow users not well trained performing molecular analyses opportunity undertake these measurements. To provide a larger user base assays, vertically stacked microfluidic analyzer with modular architecture process automation was developed. The employs primary polymerase chain reaction (PCR) coupled an allele-specific ligase detection (LDR). Each functional device, including continuous flow thermal reactors PCR LDR, passive micromixers, ExoSAP-IT purification, designed tested. Individual devices were fabricated polycarbonate using hot embossing assembled adhesive bonding system assembly. produced LDR products from DNA sample approximately 1 h, 80% reduction time compared conventional benchtop instrumentation. Purifying post-PCR enzyme led optimized performance, minimizing false-positive signals producing reliable results. Mutant alleles genomic quantified level 0.25 ng mutant 50 ng wild-type 25-μl sample, equivalent 42 cells.