Introduction

Detecting rare DNA target molecules in a heterogeneous background of DNA from multiple sources requires the isolation of sufficient DNA targets to exceed the lower limit in sensitivity of the detection assay. Sample aliquots of sufficient size must be processed in order to ensure that the target is present in the aliquot and thus eliminate stochastic sampling error that may lead to false-negative results. The design of a clinical CRC diagnostic assay using stool, an exceedingly complex sample with limited amounts of human DNA, must be able to accommodate large sample aliquots to ensure sufficient target DNA recovery. The most common approach to nucleic acid sample preparation is typically to recover the total DNA or RNA complement of a sample, using ion-exchange supports (1,2,3,4,5), glass beads (6,7), and other solid-phase methods (8,9,10,11), followed by sequence-specific PCR amplification to enrich for the target of interest. Target amplification can be complicated in samples with heterogeneous DNA populations where the target may be mixed with a majority of nucleic acids from other organisms. The PCR may be unable to amplify sufficient rare target for detection. Simply adding more heterogeneous DNA to the reaction can lead to other undesired results such as mis-priming and PCR inhibition.

Sequence-specific hybridization methods are an efficient way of recovering rare targets directly from heterogeneous DNA samples (12,13,14,15). These typically use oligonucleotide capture probes designed to hybridize to the nucleic acid target with subsequent retrieval, such as with the use of a biotinylated capture probe and magnetized beads with immobilized streptavidin (16,17,18). An early application of sequence-specific capture for detecting mycobacterium in heterogeneous clinical samples (16,19) reported an increase in PCR assay sensitivity of 10- to 100-fold. Improved PCR sensitivity results from significant target enrichment and the reduction of nontarget DNA from the reaction, thus providing more robust detection of rare DNA targets.

The reversible electrophoretic capture affinity protocol (RECAP) is a sequence-specific hybridization capture method driven by electrophoresis that was designed for large volumes of minimally purified complex heterogeneous DNA samples. In this report, we use RECAP to recover increased human DNA quantities from the stool samples of colon cancer patients and investigate if the increased recovery improves the detection sensitivity of rare mutated DNA molecules associated with the colon cancers. For comparison, the mutation detection sensitivity of a magnetic bead-based hybridization capture method and a column-based total DNA purification method is also examined.

Materials and Methods

RECAP Hybrid Capture Process

The RECAP apparatus (see the Supplementary Material available online at www.BioTechniques.com) is a multi-channel plate fitted with Spectra/Pro dialysis membrane (MWCO = 3,500; Spectrum, Rancho Dominguez, CA, USA) at both ends of each individual channel ((Figure 1), inset B). The channel plate was placed into a 1× Tris-Borate-Edta Buffer (TBE)-filled A2 Gator electrophoresis gel box (Owl Separation Systems, Portsmouth, NH, USA). The dialysis membranes provided a barrier that retained the DNA samples within each channel while allowing for electrophoresis. For each channel, a DNA capture device ((Figure 1), inset A) was placed in the center of the channel, forming two chambers; one filled with sample and the other with 1× TBE. The capture device contained 100 µl UltraLink Plus streptavidin beads (Pierce, Rockford, IL, USA) sandwiched between two layers of medical grade polyester mesh (pore size = 44 µm; Sefar America, Trenton, NJ, USA), forming a 1.5-mm-thick porous layer of packed beads. The beads were activated by applying 200 pmol of biotin-labeled oligonucleotide capture probes in 100 µl ST buffer (150 mM NaCl, 15 mM Tris-HCl, pH 7.4) across the capture device bead bed with low-speed centrifugation (1900× g) using a capture device wash plate (EXACT Sciences, Marlborough, MA, USA). The probe solution was recovered from the wash plate, reapplied to the bead bed, and recentrifuged for a total of three times. The activated capture device was then serially washed with ST buffer, 0.1 N NaOH followed by ST buffer, using centrifugation as above, to remove nonspecifically bound capture probe. The capture probes (IDT, Coralville, IA, USA) were specific for the human APC gene sequence ((Table 1)).