Polymerase chain reaction/ligase detection reaction/hybridization assays using flow-through microfluidic devices for the detection of low-abundant DNA point mutations

Abstract

We have microfabricated a flow-through biochip for the analysis of single base mutations in genomic DNA using two different materials: (1) a polycarbonate (PC) chip for performing a primary polymerase chain reaction (PCR) followed by an allele-specific ligation detection reaction (LDR) and (2) a poly(methyl methacrylate) (PMMA) chip for the detection of the LDR products using a universal array platform. The operation of the device was demonstrated by detecting low-abundant DNA mutations in gene fragments (K-ras) that carry point mutations with high diagnostic value for colorectal cancers. The PC microchip was used for sequential PCR/LDR in a continuous-flow format, in which the following three steps were carried out: (1) exponential amplification of gene fragments from genomic DNA; (2) mixing of the resultant PCR product with a LDR mixture via a Y-shaped passive micromixer and (3) ligation of two primers only when the particular mutation was present in the genomic DNA. A PMMA chip was employed as the microarray device, where zip code sequences (24-mer), which were complementary to sequences present on the discriminating primer, were micro-printed into fluidic channels embossed into the PMMA substrate. We successfully demonstrate the ability to detect one mutant DNA in 80 normal sequences with the integrated microfluidic device. The PCR/LDR/hybridization assay using the microchips performed the entire assay at a relatively fast processing speed: 18.7 min for PCR, 8.1 min for LDR, 5 min for hybridization, 10 min for washing and 2.6 min for fluorescence scanning (total processing time = ca. 50 min) with an order of magnitude reduction in reagents compared to bench-top formats.

Introduction

Colorectal cancers have been determined to possess point mutations in K-ras genes (19 different mutations), which can occur early in the development of the disease in nearly 30–50% of all patients (Andersen et al., 1997, Chiang, 1998, Rothschild et al., 1997). Once acquired, K-ras mutations are conserved throughout the course of tumor development. Therefore, securing information on the presence or absence of these mutations can assist the clinician in managing this disease. There are two potential outcomes of using molecular staging of colorectal cancers from genetic markers acquired either from tissue biopsy samples or circulating DNA: (1) determining clinical prognosis following surgical resection of the affected tissue or (2) development of noninvasive strategies for the early detection of the disease (Diehl et al., 2005).

One major issue that must be addressed for mutation detection is that the mutation of interest (mutant DNA) may be present in a mixed population of higher copy numbers of wild-type DNA. One technique that can distinguish low-abundant mutant DNA from wild-type DNA in a multiplexed format is the ligase detection reaction (LDR) coupled to a primary polymerase chain reaction (PCR) (Barany, 1991, Favis et al., 2000, Hashimoto et al., 2005, Khanna et al., 1999, Wang et al., 2003). A conceptual schematic of the PCR/LDR technique is depicted in Fig. 1.

Attention has focused on developing microfabricated devices for a variety of biomedical applications with a number of devices directed toward DNA amplifications (Burns et al., 1996, Hashimoto et al., 2004, Khandurina et al., 2000, Koh et al., 2003, Kopp et al., 1998, Lagally et al., 2001, Obeid et al., 2003) and dideoxy cycle sequencing (Oda et al., 1998). During the past decade, a number of groups have designed chamber-type PCR microchips (Burns et al., 1996, Khandurina et al., 2000, Koh et al., 2003, Lagally et al., 2001). DNA amplification can also be achieved by shuttling a PCR cocktail in a microchannel repetitively through different isothermal zones using a continuous-flow (CF) format (Hashimoto et al., 2004, Kopp et al., 1998, Obeid et al., 2003). The CFPCR approach can be conducted at relatively high speeds since it is not necessary to heat and cool the large thermal mass associated with most chamber-type devices.

Recently, universal zip code arrays have been developed for monitoring products generated from allele-specific reactions, such as LDR (Favis et al., 2000, Gerry et al., 1999, Hashimoto et al., 2005, Wang et al., 2003). The array format (see Fig. 1) uses small probes that serve as zip codes (24-mer with similar T_m values) that contain unique sequences not found in the sample DNA. The LDR uses discriminating and common primers, with the allele-specific discriminating primer containing on its 5′-end a zip code complement that directs the ligation product to a particular address on the array. The common primer contains a fluorescent dye on its 3′-end. If the mutation is present, LDR ligates the two primers together and generates a fluorescence signal at the appropriate location of the array.

In this paper, we report the development of a polymer flow-through biochip system. The PCR and LDR were operated in a continuous-flow format, which allowed for microarray readout for the detection of low-abundant mutations directly from an input of a small amount of genomic DNA into the biochip. We chose PC as the material for the continuous-flow PCR/LDR (CFPCR/CFLDR) chip due to its high glass transition temperature (145–148 °C) allowing it to withstand the sustained high operating temperatures associated with PCR and LDR (∼95 °C for thermal denaturation). On the other hand, the material for the universal array chip was poly(methylmethacrylate), PMMA, because PMMA has significantly lower amounts of autofluorescence compared to PC (Wabuyele et al., 2001) as well as minimal nonspecific adsorption artifacts between PMMA and DNA (Xu et al., 2003). In this work, the PCR/LDR/hybridization assay was carried out on K-ras genes to detect the presence of point mutations possessing clinical relevance for the diagnosis/prognosis of colorectal cancers.