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

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 employed a primary 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 assembled using adhesive bonding for system assembly. The system produced LDR products from a DNA sample in ~1 h, an 80% reduction in time compared to conventional bench-top 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.

Mutant KRAS Enhances Tumor Cell Fitness by Upregulating Stress Granules

“…This finding has significant implications as it is becoming increasingly apparent that cancer cells within a tumor show extensive genetic and phenotypic heterogeneity, thus rendering the design of effective therapies that target all cell populations within tumors a major challenge. The existence of such heterogeneity in tumors harboring mutant KRAS is indicated by reports demonstrating significant variations in KRAS mutational status in lung, colon, and papillary thyroid carcinomas (Baldus et al, 2010; Dieterle et al, 2004; Han et al, 2011; Perez et al, 2013; Richman et al, 2011). Importantly, it has been shown that mutant KRAS clones can affect the chemosensitivity of non-mutant KRAS clones rendering them resistant to anti-cancer therapies (Hobor et al, 2014; Molinari et al, 2011).…”

Accurate, predictable, repeatable micro-assembly technology for polymer, microfluidic modules

“…In the modular approach, each device or component is developed separately and connected by fluidic interconnects to build a fluidic system [22]. Modular micro- and nanofluidic systems provide flexibility in design and fabrication enabling device-specific material selection and the ability to add additional functions, or extra options, to enhance system performance [23,24]. In addition, each device or component, although designed for one application, may be used for other applications.…”

“…Detection of point mutations in the K-ras gene of the human genome with the ligase detection reaction (LDR) preceded by PCR, shown schematically in the Supplemental Information, was selected as a demonstration of the vertically stacked system [46,47]. Several different configurations of microfluidic modules for executing the assay were assembled with two, one comprised of three steps and the other four, characterized in detail biochemically [23,24].…”

Single‐molecule detection of cancer mutations using a novel PCR‐LDR‐qPCR assay

“…The assays are designed to allow for multiplexing, yet have the convenience of a qPCR readout, which is amenable to detection using commercial instruments, or in the future, automation using microfluidic devices (Dharmasiri et al, 2011; Hashimoto, Barany, & Soper, 2006; Hashimoto, Barany, Xu, & Soper, 2007; Hashimoto et al, 2005; Sinville et al, 2008; Situma et al, 2005; Soper et al, 2005; Wang et al, 2003). Ligation‐based mutation detection technology, such as ligase detection reaction (LDR), has been successfully implemented to detect low‐abundance mutations (Albrecht, Kotani, Lin, Soper, & Barron, 2013; Barany, 1991; Gerry et al, 1999; Han, Lee, Nikitopoulos, Soper, & Murphy, 2011; Hashimoto et al, 2006). One advantage of using PCR‐LDR‐qPCR is the ability to perform proportional PCR amplification of multiple fragments to enrich for low copy targets and subsequently use LDR followed by qPCR detection of the ligation products to identify cancer‐specific mutations.…”