Cholestyramine as a promising, strong anion exchange resin for direct capture of genetic biomarkers from raw pancreatic fluids

Abstract

The ability to capture cell-free DNA from the gastrointestinal tract, in a minimally invasive manner, could enhance our ability to diagnose gastrointestinal disease, or gain a better understanding of the spatial mapping of the intestinal microbiota. We, therefore, sought to identify a class of capture agents that could directly and efficiently sequester genetic material from intestinal fluids. As a particular case study, we examined the ability to capture DNA from pancreatic secretions, for potential application in enabling the sequestration of early, genetic biomarkers of pancreatic disease. We hypothesized that the cholestyramine series of strong cation exchange resins, which are FDA approved for the treatment of high cholesterol, may be capable of capturing DNA from pancreatic secretions. We identified a particular cholestyramine resin, DOWEX 1 × 2 100-200 mesh, which is able to efficiently capture and purify DNA from pancreatic fluid. Using only 200 μL of pancreatic secretions, we are able to recover 247 ± 182 ng of amplifiable human DNA, giving an estimated pancreatic fluid DNA content of 1.23 ± 0.91 ng/μL. To our knowledge, this is the first demonstration of a material that can effectively capture and purify DNA directly from untreated pancreatic fluids. Thus, our approach could hold high utility for the in vivo capture of DNA and disease biomarkers if incorporated into an appropriate sampling device. Biotechnol. Bioeng. 2017;114: 934-938. © 2016 Wiley Periodicals, Inc.

Keywords: biomarker; cell-free DNA capture; cholestyramine; intestine; pancreatic fluid.

Figure 1

(a) General structure of the cholestyramine resins that were utilized in this work. (b)–(d) Schematic of sample processing protocol for isolating DNA from biological fluid samples. (b) DNA is bound to the capture resin by passing the fluid sample through the resin bed by centrifugation. (c) The bound DNA is then washed 3–5 times using an intermediate ionic strength buffer before (d) eluting the bound DNA with a high ionic strength buffer.

Figure 2

(a) Demonstration of DNA purification using Dowex 1×2 100–200 mesh resin. The flow through during sample application has a dominant protein peak at 280 nm, which is consistent with predominantly protein absorbance, whereas the spectrum of the eluted DNA is shifted toward an absorbance maximum at 260 nm, which is consistent with nucleic acid absorbance. (b) Results of spike-and-recovery experiments using different amounts of WI-38 cell line DNA spiked into simulated intestinal fluid. DNA concentrations for the spike-and-recovery experiment were evaluated using a NanoDrop spectrometer. DOWEX 1 × 2 100–200 mesh resin consistently gave the greatest recoveries across all conditions. Samples were processed in triplicate, and error bars represent +/− 1 SD.

Figure 3

Demonstration of protein elimination and DNA elution using patient-derived pancreatic fluid samples. (a) Analysis of protein content in the original pancreatic fluid sample, as well as the sample flow through and wash steps from four patient samples, evaluated using the absorbance at 280 nm. Five wash steps were used for processing patient-derived samples, since this number of washes reduced the protein absorbance to baseline in the wash flow throughs. (b) Eluted DNA, as evaluated using the PicoGreen dsDNA Assay. DNA remains bound during all wash steps, as demonstrated by the lack of measureable DNA content in the wash step flow throughs, but is eluted upon switching the buffer conditions to 7 M GuHCl. (c)–(d) Evaluation of recovered DNA if multiple elution steps are employed. Over 85% of the bound DNA can be successfully recovered in a single elution step. If desired, additional elutions can improve DNA yields.

Figure 4

(a) Recovered DNA quantities using resin-based capture from endoscopically collected pancreatic fluid samples from 6 patients with suspected chronic pancreatitis at three different time points. (b) Parity plot comparing the amount of DNA recovered using the resin capture approach and DNeasy spin columns. Cholestyramine resin demonstrates a similar performance to the DNeasy spin columns, which represent the state-of-the-art technology for in vitro DNA capture from biological fluids. Samples were processed in duplicate, and error bars represent +/− 1 SD. DNA concentrations were evaluated using the Quantifiler Human DNA Quantification Kit.