Dual-Domain Microchip-Based Process for Volume Reduction Solid Phase Extraction of Nucleic Acids from Dilute, Large Volume Biological Samples

• NCJ Number: 308443
• Journal: Analytical Chemistry Volume: 82 Issue: 13 Dated: 2010 Pages: 5669–5678
• Author(s): Carmen R. Reedy; Kristin A. Hagan; Briony C. Strachan; Joshua J. Higginson; Joan M. Bienvenue; Susan A. Greenspoon; Jerome P. Ferrance; James P. Landers
• Date Published: 2010
• Length: 10 pages
• Grant Number(s): 2008-DN-BX-K144
• Agencies: NIJ-Sponsored
• Publication Type: Research (Applied/Empirical)
• Award: Closed System DNA Purification for Degraded and Compromised Evidence in Microfluidic Devices

Annotation

This paper introduces a dual-domain microchip-based process for volume reduction solid phase extraction of nucleic acids.

Abstract

In this study, researchers developed a microfluidic device to carry out integrated volume reduction and purification of nucleic acids from dilute, large volume biological samples commonly encountered in forensic genetic analysis. The dual-phase device seamlessly integrates two orthogonal solid-phase extraction (SPE) processes, a silica solid phase using chaotrope-driven binding and an ion exchange phase using totally aqueous chemistry (chitosan phase), providing the unique capability of removing polymerase chain reaction (PCR) inhibitors used in silica-based extractions (guanidine and isopropanol). Nucleic acids from a large volume sample are shown to undergo a substantial volume reduction on the silica phase, followed by a more stringent extraction on the chitosan phase. The key to interfacing the two steps is mixing of the eluted nucleic acids from the first phase with loading buffer which is facilitated by flow-mediated mixing over a herringbone mixing region in the device. The utility of the device for purifying DNA was demonstrated with dilute whole blood, dilute semen, a semen stain, and a blood sample inhibited with indigo dye, with the resultant DNA from all shown to be PCR amplifiable. The utility of the device for the purification of RNA was also demonstrated by the extraction of RNA from a dilute semen sample, with the resulting RNA amplified using reverse transcription (RT)-PCR. The vrSPE-SPE device reliably yields a volume reduction for DNA and RNA purification on the order of 50- and 14-fold, respectively, both compatible with downstream PCR analysis. In addition, purification of all samples consumed less reagents (2.6-fold) than traditional purification methods, with the added advantage of being a “closed system” that eliminates sample transfer steps, thereby reducing the possible entrance points for contaminants. (Published Abstract Provided)