Document Type
Article
Publication Date
10-15-2011
Abstract
Recognition of point mutations in the K-ras gene can be used for the clinical management of several types of cancers. Unfortunately, several assay and hardware concerns must be addressed to allow users not well trained in performing molecular analyses the opportunity to undertake these measurements. To provide for a larger user base for these types of molecular assays, a vertically stacked microfluidic analyzer with a modular architecture and process automation was developed. The analyzer employs a primary polymerase chain reaction (PCR) coupled to an allele-specific ligase detection reaction (LDR). Each functional device, including continuous flow thermal reactors for the PCR and LDR, passive micromixers, and ExoSAP-IT purification, was designed and tested. Individual devices were fabricated in polycarbonate using hot embossing and were assembled using adhesive bonding for system assembly. The system produced LDR products from a DNA sample in approximately 1 h, an 80% reduction in time compared with conventional benchtop instrumentation. Purifying the post-PCR products with the ExoSAP-IT enzyme led to optimized LDR performance, minimizing false-positive signals and producing reliable results. Mutant alleles in genomic DNA were quantified to the level of 0.25 ng of mutant DNA in 50 ng of wild-type DNA for a 25-μl sample, equivalent to DNA from 42 mutant cells. © 2011 Elsevier Inc.
Publication Source (Journal or Book title)
Analytical Biochemistry
First Page
211
Last Page
219
Recommended Citation
Han, K., Lee, T., Nikitopoulos, D., Soper, S., & Murphy, M. (2011). A vertically stacked, polymer, microfluidic point mutation analyzer: Rapid high accuracy detection of low-abundance K-ras mutations. Analytical Biochemistry, 417 (2), 211-219. https://doi.org/10.1016/j.ab.2011.06.030
